Journal of Trace Elements in Medicine and Biology 62 (2020) 126573

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Journal of Trace Elements in Medicine and Biology

journal homepage: www.elsevier.com/locate/jtemb

Toxicology

In vitro effects of boric acid on human liver hepatoma cell line (HepG2) at T
the half-maximal inhibitory concentration
Aysegul Tombuloglua, Hulya Copoglub, Yesim Aydin-Sona, N. Tulin Gurayb,*
a
Middle East Technical University, Graduate School of Informatics, Health Informatics Department, Ankara, Turkey
b
Middle East Technical University, Graduate School of Arts and Sciences, Department of Biological Sciences, Ankara, Turkey

ARTICLE INFO ABSTRACT

Keywords: Background: Boron is a prominent part of the human diet and one of the essential trace elements for humans.
Boric acid Dietary boron is mostly transformed into boric acid within the body and has been associated with desirable
IC50 health outcomes. Non-dietary resources of boron, such as boron-based drugs and occupational exposure, might
Cytotoxicity lead to excessive boron levels in the blood and provoke health adversities. The liver might be particularly
DNA damage
sensitive to boron intake with ample evidence suggesting a relation between boron and liver function, although
Microarray
the underlying molecular processes remain largely unknown.
HepG2
Methods: In order to better understand boron-related metabolism and molecular mechanisms associated with a
cytotoxic level of boric acid, the half-maximal inhibitory concentration (IC50) of boric acid for the hepatoma cell
line (HepG2) was determined using the XTT assay. Cellular responses followed by boric acid treatment at this
concentration were investigated using genotoxicity assays and microarray hybridizations. Enrichment analyses
were carried out to find out over-represented biological processes using the list of differentially expressed genes
identified within the gene expression analysis.
Results: DNA breaks were detected in HepG2 cells treated with 24 mM boric acid, the estimated IC50-level of
boric acid. On the other hand, pleiotropic transcriptomic effects, including cell cycle arrest, DNA repair, and
apoptosis as well as altered expression of Phase I and Phase II enzymes, amino acid metabolism, and lipid
metabolism were discerned in microarray analyses.
Conclusion: HepG2 cells treated with a growth-inhibitory concentration of boric acid for 24 h exhibited a se-
nescence-like transcriptomic profile along with DNA damage. Further studies might help in understanding the
concentration-dependent effects and mechanisms of boric acid.

1. Introduction
Boron is one of the rare elements having biological functions in
animals, plants, yeasts, bacteria, and is attributed to evolutionary sig-
nificance dating back to prebiotic ages [1]. As an essential part of
human nutrition, dietary boron is mainly obtained from plant-based
boron chemicals and water dissolved boric acid [2,3]. An average
human diet involves approximately 0.3–4.2 mg Boron daily [4].
Dietary boron has been implicated to be important in bone health,
arthritis, immune response, plasma lipid profiles, and brain function
[3,5]. Higher levels of dietary boron intake have also been associated
with a decreased number of incidences in prostate cancer, lung cancer
[6], cervical cancer [7], and breast cancer [8]. In various animal stu-
dies, dietary boron has been reported to have protective value against
multiple types of liver injuries [15–17], whereas detrimental effects on
the liver were also examined, especially at high levels of boron ad-
ministration [18,19].
Over the last decade, the therapeutic potential of boron-containing
compounds in various diseases has attracted considerable attention.
Approved boron-based drugs are currently being used in the treatment
of multiple myeloma [9], onychomycosis [10], inflammatory skin dis-
orders [11], and boron neutron capture therapy [12]. Boron-based
compounds also hold tremendous potential for various therapeutic
purposes by targeting proteins and exerting antiviral, antibiotic, an-
ticoagulant, antidiabetic, and steroid-like effects [10].
Boron does not accumulate in soft tissues and is maintained at a
fixed concentration range within human plasma, liver, and brain re-
gardless of the extent of boron consumption, which suggests homeo-
static control mechanisms acting on boron levels in the human body
[3]. Many researchers use boric acid or borate to study boron-induced

⁎
Corresponding author at: METU Biological Sciences, Universiteler Mah. Dumlupinar Blv. No:1, 06800 Cankaya Ankara, Turkey.
E-mail address: guray@metu.edu.tr (N.T. Guray).

https://doi.org/10.1016/j.jtemb.2020.126573
Received 17 March 2020; Received in revised form 18 May 2020; Accepted 26 May 2020
0946-672X/ © 2020 Elsevier GmbH. All rights reserved.
A. Tombuloglu, et al.
bioprocesses since the most abundant physiological form of boron is
expected to be boric acid due to the excessive energy required to break
the Boron-Oxygen bond [2,11,12].
In this study, the antiproliferative and genotoxic effects of boric acid
on HepG2 cell line, a commonly used cell line in toxicology studies,
have been investigated through in vitro assays. For better understanding
the mechanisms elicited by boric acid at the IC50 level, gene expression
changes in response to boric acid were also studied via microarray
technology.
2. Materials and methods
2.1. Materials
DMEM low glucose with L-glutamine&sodium pyruvate, and 10x
trypsin-EDTA were purchased from Biowest (Nuaillé, France). Pen-strep
solution, heat-inactivated fetal bovine serum, phosphate-buffered saline
without Mg and Ca, trypan blue, and XTT cell proliferation kit were
obtained from Biological Industries (Beit-Haemek, Israel). Dimethyl
sulfoxide (DMSO) and formaldehyde were obtained from Applichem
(Darmstadt, Germany).
Low melting agarose, NaCl and Trizma Base, Na2EDTA propidium
iodine, 99.8% methanol, and Giemsa were obtained from Sigma (USA).
Hydrogen peroxide, 30%, TritonX-100, NaOH, KCl, and acetic acid
were obtained from Merck Millipore (Darmstadt, Germany).
Other reagents used in the study were Mitomycin C, obtained from
Sigma (USA), and Cytochalasin B obtained from Serva (Heidelberg,
Germany). For total RNA isolation, an RNeasy mini kit was obtained
from Qiagen (Germany). In microarray experiments, GeneChip® Human
Gene 1.0-st Array, GeneChip IVT Plus Kit, GeneChip Hybridization
Wash, and Stain Kit were purchased from Affymetrix (Thermo Fisher
Scientific). Cell culture grade boric acid (Sigma, USA) was kindly pro-
vided by Dr. Serap Kolukisa from Boren Institute.
2.2. Cell culture
HepG2 cell line, which is a well-established in vitro model ex-
tensively used in drug metabolism and toxicity research [13], was
purchased from ATCC (USA). HepG2 cells were cultured with DMEM
low glucose with L-glutamine with sodium pyruvate in 25 mM HEPES
supplemented with 10 % FBS and 1 % pen-strep solution and main-
tained at 37 °C temperature, 95 % air, and 5 % CO2. Cells were seeded
in six-well plates at a density of 3 × 105 cells/mL and passaged at
around every two days when the confluence level reached approxi-
mately 80 %.
Boric acid was dissolved in either 0.1% DMSO or complete cell
culture medium at room temperature, and the stock solution was di-
luted to the desired concentration for various assays.
2.3. XTT assay
The XTT assay was carried out according to the manufacturer’s in-
structions. HepG2 cells were seeded at a density of 1 × 105 cells/mL in
96 well plates and incubated for one day before boric acid treatment.
Boric acid was supplemented to the media at final concentrations ran-
ging between 0.5 mM and 40 mM, and cells were incubated for 24 h
with boric acid. XTT solution (0.1 mL activator and 5 mL XTT reagent)
was added to the wells and incubated for 4 h. Absorbances at 450 nm
and 630 nm were measured with an ELISA reader (Thermo Fisher
Scientific) at 450 nm. The cell viability curve was plotted from the
calculated cell viability percentage values for each boric acid con-
centration. IC25 and IC50 concentrations were determined from the cell
viability curve.
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Journal of Trace Elements in Medicine and Biology 62 (2020) 126573
2.4. Genotoxicity assays
In order to study possible genotoxic effects and DNA damage of
boric acid treatment, Cytokinesis-blocked micronucleus (CBMN) assay
and single cell gel electrophoresis (Comet) assay were performed.
2.5. Cytokinesis-blocked micronucleus (CBMN) assay
In vitro cytokinesis-blocked micronucleus assay was carried out ac-
cording to the protocol of Fenech et al. [14]. HepG2 cells were seeded
at a density of 3 × 105 cells/mL within six-well plates and cultured for
24 h. The cells were treated with boric acid at both IC25 and IC50
concentrations and as a positive control with 1 μg/mL mitomycin C
(MMC) for 24 h.
2.6. Single cell gel electrophoresis (Comet) assay
The single cell gel electrophoresis assay (SCGE) was performed
based on the procedure of Singh et al. [15] with minor modifications.
HepG2 cells were seeded in six-well plates at a density of 3 × 105
cell/mL and cultured for 24 h. Cells were treated with boric acid at the
IC50 concentration, no treatment (medium control), and 0.1% DMSO
treatment conditions as the negative control and 40 μM and 100 μM
H2O2 as the positive control for 24 h. Tail length, tail moment, and the
percentage of DNA in the tail were calculated with OpenComet soft-
ware, which provided fully automated analysis and plugins in ImageJ
software.
2.7. Target cDNA preparation and microarray hybridization
HepG2 cells were seeded at a density of 3 × 105 cells/mL within
35 mm cell culture dishes. Cells were treated with 0.1% DMSO as the
control and boric acid at the IC50 concentration as the test sample and
left for 24 -h incubation. Total RNAs from HepG2 cells were isolated by
Qiagen RNeasy total RNA isolation kit (Qiagen, Germany) according to
the manufacturer’s protocol. The quality and quantity of isolated RNA
were monitored with Nanodrop (Thermo, USA). RNA samples were
used for obtaining single-stranded cDNA, which was then fragmented
labeled and hybridized to GeneChip® Human Gene-1.0-st. Affymetrix
arrays were washed, stained, and scanned according to the supplier’s
protocols.
The raw microarray data files were submitted to the GEO repository
(accession number GSE1442264).
2.8. Pre-processing and statistical analysis of microarrays
The quality of the microarray data was screened on arrayanalysi-
s.org online platform [16]. Any noteworthy problem was not detected
in the quality of the microarray dataset. Hence, the raw dataset was
imported into the R environment for further pre-processing and statis-
tical analysis steps. The raw microarray dataset was RMA-normalized
and re-mapped to Ensembl Transcript IDs using custom cdf, which was
described by Dai [17]. E-bayes moderated t-test of the limma package
was used to find statistically significant genes (BH adjusted p-value <
0.05). Statistically significant transcripts expressed over a 2-fold
change ratio were selected as the differentially expressed transcript list.
2.9. Pathway and gene ontology enrichment analysis
The DAVID Bioinformatics platform functional enrichment tool [18]
was used to identify pathways and GO categories enriched in statisti-
cally significant transcripts. Lists of down-regulated transcripts and up-
regulated transcripts were submitted to DAVID separately. Pathways
and GO categories with Benjamini-Hochberg corrected FDR < 0.05
were considered as significantly enriched functional annotations.
A secondary enrichment analysis was carried out by submitting the
A. Tombuloglu, et al.
Fig. 1. Percent cell viability quantified by XTT assay demonstrated inhibition of
cell growth in response to boric acid in a dose-dependent manner. Bars re-
present % cell viability as means ± SEM.
whole list of differentially expressed genes to the DAVID functional
enrichment tool. Significantly enriched functional annotations for
KEGG and Reactome pathways, GO MF and GO BP terms were
remapped onto a more extensive integrated network with
EnrichmentMap [19], which were clustered into functional annotation
groups and re-annotated using WordCloud [20].
3. Results
3.1. Inhibition of HepG2 cell growth at various boric acid concentrations
The relative viability of HepG2 cells in response to boric acid
treatment for 24 h at concentrations varying between 0.5 and 40 mM
was monitored via XTT cell proliferation assay. At concentrations
higher than 1 mM, the XTT assay demonstrated a general trend for a
decrease in % cell viability with increasing boric acid concentration,
indicating a dose-dependent cytotoxic effect for the given concentration
range. When the cells were treated with boric acid at a concentration
lower than 1 mM, any significant change in cell growth was not ob-
served (Fig. 1).
The IC50 concentration was calculated as 24 mM from the curve.
Similarly, IC75 and IC25 concentrations shown by 75% and 25% de-
crease in HepG2 cell proliferation were estimated as 36 mM and 12 mM,
respectively.
3.2. Genotoxic effects of boric acid at inhibitory concentrations
Micronucleus and single cell gel electrophoresis (Comet) assay were
used to investigate the genotoxic effect of boric acid treatment for 24 h
at IC25 and IC50 concentrations on HepG2 cells.
Single cell gel electrophoresis assay was used for a quantitative
assessment of DNA damage occurring within boric acid-treated cells
with respect to negative and positive controls. The tail moment quan-
tified for boric acid-treated HepG2 cells at the IC50 level was sig-
nificantly (p < 0.05) higher than 40 μM H2O2 treated cells.
DNA damage was examined in the presence of boric acid treatment
at a concentration required to inhibit half of the growth of HepG2 cells
(Fig. 2). Any genotoxic effect, however, was not detected within cells
treated at the concentration inhibiting quarter of the cell growth (IC25).
The micronucleus test was also used in order to inspect the geno-
toxic effect of boric acid at IC50 concentration. Mitomycin C (1 μg/ml),
a chemical that inhibits DNA synthesis and disrupts DNA structure by
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Journal of Trace Elements in Medicine and Biology 62 (2020) 126573
cross-linking the nucleobases, was used as the positive control. A sig-
nificant increase in the number of micronuclei (p < 0.05) was noted in
boric acid-treated HepG2 cells at IC50-level, whereas any significant
change in the amount of micronuclei formation was not examined at
IC25 concentration of (12 mM) boric acid (Fig. 3).
3.3. Microarray data analysis
In order to find out the molecular mechanisms and responses as-
sociated with cytotoxicity at the IC50 level (24 mM) boric acid treat-
ment, which had both growth inhibitory and genotoxic effects on
HepG2 cell line, microarray analyses were carried out.
Transcripts varying in expression level in boric acid-treated HepG2
cells at IC50 concentration were identified using e-bayes moderated t-
test. A total of 6841 transcripts significantly (BH adjusted p < 0.05)
differing in their expression level had more than two-fold change ex-
pression in treated cells. Among the differentially expressed transcripts,
2685 were upregulated, 4156 were downregulated, which are visua-
lized in the volcano plot and heatmap (Fig. 4).
3.4. GO & pathway enrichment analysis
To gain insight into the molecular functions and processes of dif-
ferentially expressed transcripts, enrichment analyses were carried out.
Pathways annotated within the KEGG database, Reactome database,
and gene ontology categories significantly over-represented in the lists
of upregulated transcripts or downregulated transcripts were found
(Fig. 5).
Downregulated transcripts were enriched in 250 gene ontology terms
and 58 pathways, whereas 116 gene ontology terms and 11 pathways were
found to be significantly enriched in upregulated transcripts. DNA re-
plication and cell cycle are among the most significantly enriched biolo-
gical processes in the downregulated transcript set. Functional and com-
partmental terms most significantly enriched in downregulated transcripts
involve nucleoside binding and chromosome-related terms. Top sig-
nificantly enriched GO terms associated with upregulation involve xeno-
biotic glucuronidation, regulation of molecular function, regulation of
phosphorous metabolism, and intracellular signaling as biological pro-
cesses, glucuronosyltransferase activity, protein heterodimerization ac-
tivity, enzyme inhibitor activity, enzyme binding, and small molecule
binding terms as molecular function, cell-substrate junction and focal ad-
hesion as cellular compartment.
Pathway analysis uncovered similar processes with significant gene
ontology terms. The top 20 results from Reactome and KEGG analysis
are presented to summarize the enriched biological pathways (Fig. 6).
In conjunction with DNA replication and cell cycle, DNA repair me-
chanisms, such as homologous DNA repair, nucleotide excision repair,
mismatch repair, DNA synthesis-related pathways like activation, and
assembly of the pre-replicative complex, removal of licensing factors
from the origin are among enriched pathways in downregulated tran-
scriptional response. Metabolic pathways, steroid and cholesterol bio-
synthesis, and transcription factor SREBF regulated pathway are also
primary downregulated responses.
According to pathway enrichment analysis, many metabolic path-
ways, including drug metabolism, chemical carcinogenesis pathway,
and p53 signaling pathway, are found as enriched upregulated path-
ways with the highest significance.
In order to obtain a comprehensive view of biological processes in
response to boric acid treatment and to better understand the inter-
relations among enrichment results, a second functional enrichment
analysis was carried out with the whole list of differentially expressed
transcripts, and results were visualized in a network reconstructed
using EnrichmentMap [19]. The reconstructed network consisted of
significantly enriched terms from the whole list of differentially ex-
pressed transcripts as nodes which are connected by edges representing
the similarity between the transcript content of the nodes.
A. Tombuloglu, et al. Journal of Trace Elements in Medicine and Biology 62 (2020) 126573

Fig. 2. Incubation of HepG2 cells with boric acid at the IC50 level (24 mM) for 24 h led to a boosted tail moment % quantified via SCGE assay, indicating DNA
fragmentation similar to hydrogen peroxide (40 and 100 μM). Tail moment values are represented as the mean ± SEM (*p < 0.05).

Fig. 3. Boric acid treatment of HepG2 at the IC50 level for 24 h significantly increased the frequency of micronuclei with respect to control groups. Frequency values
are represented as the mean ± SEM. (***p < 0.001).

The network was clustered using WordCloud [20]. Nine main
clusters of functional transcript sets were identified. Four of the clusters
labeled as DNA repair, cell cycle, nucleotide-binding, and DNA orga-
nization are closely connected, indicating a high degree of transcrip-
tional commonality among these functional groups. The majority of the
functional transcript sets in these four groups consist of downregulated
transcripts.
Lipid metabolism and amino acid metabolism are highly inter-
connected, implying that shared transcripts function in both processes.
Transcript sets in xenobiotic metabolism have relatively higher overall
betweenness sharing connections with all clusters of the enrichment-
terms network. At the heart of the Xenobiotic metabolism cluster, there
are sets mainly comprised of upregulated glucuronyltransferase tran-
scripts, representing glucuronidation-related terms.
4. Discussion
Boron acts on a wide range of physiological processes of which
molecular mechanisms are not very well known. In this study, the effect
of boric acid, the physiologically available form of boron, was in-
vestigated in HepG2 cell line at half-maximal inhibitory (IC50) con-
centration in order to gain insight into boric acid-dependent molecular
mechanisms by analyzing the differential gene expression profiles.
In the present study, growth inhibition and a gradual decrease in the
viability of HepG2 cells were observed at 5 mM and higher concentra-
tions, with an estimated IC50 value of 24 mM. Several in vitro studies
performed with different cell lines presented similar or comparable
growth inhibition curves upon treatment with boric acid. Hek and HeLa
cell lines had a significant decrease in cell proliferation at concentra-
tions exceeding 5 mM boric acid [21]. Four different dermal cell lines
ceased to grow at about 8 mM boric acid treatment [22] without any
signs of cytotoxicity at lower concentration ranges. For DU-145 cell
line, 6.25 mM was the initial concentration of boric acid in which a
significant reduction in cell growth was noticed, and the IC50 value was
estimated as 10.77 mM [23]. Boron treatment might also trigger the
proliferation of cell lines depending on the concentration [21,24,25]. In

4
A. Tombuloglu, et al. Journal of Trace Elements in Medicine and Biology 62 (2020) 126573

Fig. 4. Transcripts with log fold-change < -2, > 2, and with q < 0.05, were determined as the downregulated transcript list and the upregulated transcript list,
respectively. Differentially expressed transcripts are highlighted in blue in the volcano plot (A), and visualized as clusters in a heatmap with red color showing higher
expression levels, yellow color lower expression levels (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article).

general, studies indicate a trend in which boric acid supports cell pro-
liferation at micromolar doses while inhibiting cell growth after passing
a certain concentration threshold.
The study revealed that DNA damage and transcriptional changes
accompanying inhibited cell growth of boric acid treatment with gen-
otoxicity assays and microarray hybridizations. Severe DNA damage
was detected in HepG2 cells, at half maximal inhibitory concentration,
in two independent genotoxicity assays. In addition to the decline in
cell growth and impairment in DNA integrity, a substantial change in
the transcriptomic profile was noted. Specifically, massive down-
regulation of transcripts functioning in the cell cycle, DNA repair,
chromosomal organization, and metabolic pathways and upregulation
of apoptotic transcripts are noticeable.
Like the concentration-dependent effects of boron on cell growth,
boron-associated genotoxicity and apoptotic markers might also be
highly dependent on the doses of boron exposure. Although, genotoxic
effects of boric acid have been reported in zebrafish [26], plants
[27,28] and cultured HeLa cell line [29], in many studies performed
with lower concentration ranges, boric acid emerged as an anti-geno-
toxic agent, reducing the effects of DNA damage-inducing chemicals
[30–32]. Boric acid supplementation in drinking water and food may
have both anti-apoptotic and apoptotic effects on cells of ostrich chick

Fig. 5. Top 10 significantly enriched GO categories of A) Biological Process B) Molecular Function C) Cellular Component are graphed for their –log Benjamini values
as gray bars for downregulated transcript list and as black bars for upregulated transcript list.

5
A. Tombuloglu, et al.
Fig. 6. The top 20 enriched pathways for upregulated and downregulated transcripts are graphed for their –log Benjamini values as gray bars for downregulated
transcript list and as black bars for upregulated transcript list.
spleen [33], brain [34], and kidney [33], depending on the dose of
boric acid. Below 1 mM, boric acid causes growth inhibition in several
prostate cancer cell lines without any apparent signs of apoptosis
[11,35], along with a decrease in the mRNA and protein levels of
CHOP, a pro-apoptotic gene [35]. Morphological characteristics of
HepG2 cells and molecular markers at this concentration range in-
dicated the induction of a senescent-like phenotype by boric acid [36].
DU-145 cells, however, exhibited increased amounts of apoptotic
markers, CASP3, CYCS, and Bax when cultured with boric acid at
6.15 mM and higher concentrations [23]. Treatment of HepG2 cell line
with borax, the mineral salt form of boron, at a concentration range of
0.5–16 mM up to 72 h led to a concentration-dependent increase in the
proportion of apoptotic cells. At the estimated IC50 level of borax, up-
regulation of p53 and Bax and downregulation of Bcl2 were reported
[37]. According to our results, treatment of HepG2 cells with IC50 level
of boric acid for 24 h was accompanied by downregulation of a high
number of transcripts in DNA synthesis and cell cycle and upregulation
of relatively few transcripts involved in the p53 signaling pathway
(Fig. 6), including markers of cellular senescence-like p21 and Gadd45
and markers of early apoptosis-like FAS, PMAIP1, TP53I3, EI24, and
ZMAT3. Moreover, the pathway term “Activation of ATR in response to
replication stress” was significantly enriched. ATR signaling mediates
DNA damage response characterized by stalling in cell cycle progres-
sion or apoptotic induction in response to DNA double-strand breaks,
and might drive the cell towards senescence if the damage cannot be
repaired [38].
From these transcriptional changes, senescence-linked cell cycle
arrest might be inferred as a likely mechanism of boric acid-induced
growth inhibition in HepG2 cells, although upregulated apoptotic genes
indicate induction of early-stage apoptosis. According to a novel model,
downregulated DNA repair pathways might be a common feature of
senescent cells and might cause or exacerbate DNA damage. The model
postulates that E2F controls the transcription of DNA repair genes as a
key regulator in senescence [39]. In the transcriptional profile of boric
acid-treated cells, downregulated DNA damage pathways form a large
cluster among enriched terms (Fig. 7). Moreover, E2F activated tran-
scription pathway was found to be significantly enriched in the list of
downregulated transcripts. These transcriptional patterns might be a
further indicator of senescence in boric acid-treated HepG2 cells and
also a causative role for the DNA damage.
Although, there have been few attempts to gain molecular insight
[36,37], our knowledge about the molecular mechanisms of boron-re-
lated toxic effects at higher concentrations is limited. Most often, di-
verse biological outcomes of boric acid treatment are hypothetically
linked to the ability of boric acid to form complexes with key
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Journal of Trace Elements in Medicine and Biology 62 (2020) 126573
biomolecules and inhibit a vast range of enzymes, including serine
proteases, histone deacetylases, and NAD-dependent oxidoreductases
[40,41].
Recently, inhibition of histone deacetylases and subsequent dis-
organization of the chromosomal structure has been suggested as a
common molecular mechanism among eukaryotes lying behind double-
strand DNA breaks in response to high-boron stress. In Arabidopsis
thaliana, at a toxic level, boric acid led to histone hyperacetylation,
which is essential for preserving the chromosomal integrity and pro-
tecting DNA from reactive molecules. Functional 26S proteasome was
necessary for counteracting the high-boron induced hyperacetylation
and genotoxic outcome [29]. Histone deacetylase inhibitors could lead
to transcriptional repression of DNA damage response/repair pathways
through the decreased activity of E2F transcription factor, resulting in
increased susceptibility to DNA damage [42]. Moreover, healthy cells
were found to be capable of fixing DNA damage caused by histone
deacetylase inhibitors while cancer cells were not, which confers an
advantage for HDACs in tumor-targeted therapies [43]. When similar
transcriptional responses noted in this study are considered, inhibition
of histone deacetylases might be the primary mechanism that underlies
the DNA damage and senescent-like transcriptional profile of boric
acid-treated HepG2 cells. Furthermore, if histone deacetylation is a
universal mechanism among eukaryotes fundamental to B-induced ef-
fects, the cancer-targeted action of histone deacetylases might explain
the anti-carcinogenic effects of dietary boron and the higher sensitivity
of carcinogenic cell lines for cytotoxic effects of boron with respect to
healthy cell lines [36,44].
Many of the cytotoxic mechanisms of bortezomib, a boron-con-
taining peptide drug used as a proteasome inhibitor in cancer therapy,
show overlap with those of boric acid treatment at the IC50 con-
centration (Fig. 7). In HepG2 cell line, bortezomib was reported to lead
to cell cycle arrest depending on the duration and concentration of
bortezomib treatment and invoke pro-apoptotic genes. Bortezomib-in-
duced cell cycle arrest was mediated via downregulation of E2F1
transcription factor and its targets involved in the cell cycle and upre-
gulation of p21. In a similar way with the repressed DNA repair path-
ways identified for IC50-level boric acid-treated HepG2 cells, borte-
zomib leads to suppression of DNA repair associated pathways, thereby
reinforcing the susceptibility of the cells to DNA damage [45,46].
Sensitization of cells to DNA damage by bortezomib might be through
downregulation of genes in DNA repair pathways and also inhibition of
the ubiquitin-proteasome system, and subsequent enrolment of DNA
repair proteins like BRCA1 and RAD51 to DNA damage sites, leading to
double-stranded DNA breaks and cell death [47]. Treatment with the
proteasome inhibitor drug, bortezomib, resulted in a decline in alcohol-
A. Tombuloglu, et al.
induced lipid deposition in the livers of rats accompanied by down-
regulated genes in fatty acid, triglyceride, and cholesterol synthesis
[48], which further indicates similarity with boric acid treatment due to
induced downregulation of lipid metabolic pathways. It is also inter-
esting to note that bortezomib-induced cytotoxic effects take place via
histone deacetylases. Unlike histone deacetylase inhibitors, however,
bortezomib targets histone deacetylases via transcriptional mechan-
isms. Boric acid might inhibit serine proteases, which are the major
subunits making up the 26S proteasome [41], although most probably,
in a much weaker fashion than bortezomib, which is one of the most
potent inhibitors of serine proteases. Therefore, proteasome inhibition
activity might also be affecting cellular processes after boric acid
treatment.
Along with transcriptional changes that could be associated with boric
acid-related cytotoxicity, transcriptional reprogramming of metabolic pro-
cesses was also remarkable with boric acid treatment, mainly involving
downregulated amino acid metabolism, lipid metabolism transcripts, and
upregulated xenobiotic transcripts. Boron is considered to be a metabolic
regulator for a variety of organisms [41]. In yeast, a link between boron
stress and amino acid metabolism was shown, and a dysregulated amino
acid control system was proposed as a contributor to boron toxicity [49,50].
As shown in this study, boric acid-related downregulation of steroid and
lipid metabolism pathways is particularly intriguing due to the growing
interest in the role of boron as a regulator of lipid metabolism. Dietary
boron has been implicated in enhancing the plasma lipid profile [51] and
preventing fatty liver [52]. Moreover, Boron is known to be involved in the
metabolism of steroid hormones. Plasma levels of estrogen and testosterone
have been shown to be sensitive to boron intake. [8,53]. Hypothetically,
boron might be modulating steroid metabolism by getting involved in the
catalysis of hydroxyl group addition to hormones or binding to hormones,
preventing them from getting degraded [8]. According to another hypoth-
esis, boron might interact with transport proteins and interfere with the
binding of steroid hormones [54]. In our study, the gene expression profile
revealed SREBF regulated expression as a significant pathway enriched
among downregulated genes (Fig. 6). Furthermore, transcripts encoding
SREBF1 and SREBF2 were found to be significantly downregulated. SREBP
proteins are transcription factors involved in regulating the transcription of
enzymes and other proteins in lipid metabolism [55]. Downregulated
SREBF and its downstream targets have also been observed in in vitro ex-
periments carried out with other boron-containing compounds [48,56].
These results suggest that the downregulation of SREBF-mediated metabolic
pathways as an important mechanism in altered lipid or steroid metabolism
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Journal of Trace Elements in Medicine and Biology 62 (2020) 126573
Fig. 7. An enrichment-terms network was built
to visualize and understand the interconnec-
tions of GO terms and pathways enriched
within differentially expressed transcripts.
Each transcript set for any significant GO term
and pathway is represented as a node. The
color and color intensity of the nodes denote
the mean of the log fold-change scores of
transcripts involved in the set. In general, sets
with a higher count of downregulated tran-
scripts are represented as nodes of darker blue
color and sets with a higher count of upregu-
lated transcripts are represented as nodes of
darker red color. The size of the nodes is de-
termined by the number of transcripts in any
transcript set. (For interpretation of the refer-
ences to colour in this figure legend, the reader
is referred to the web version of this article).
as a result of boron exposure. Upregulation of genes encoding Phase I and
Phase II metabolic enzymes in response to high boric acid levels is also
another notable result of the analysis, which might reflect how boron is
detoxified in the liver at high concentrations.
In conclusion, DNA damage and transcriptional reprogramming
towards a senescence-like profile were observed in HepG2 cells cultured
with boric acid at half-maximal inhibitory concentration. As in many
cases in the literature [39,42,45], the DNA damage might be through
sensitization of cells as a result of downregulated DNA repair associated
response that is interrelated with chromosome organization and cell
cycle processes (Fig. 7), while direct binding of boric acid to DNA could
still be influential, as boric acid was shown to interact with DNA, re-
sulting in denaturation of the strands in a concentration-dependent
manner [57]. Similarities in gene expression patterns of histone dea-
cetylase inhibitors and bortezomib, point toward enzyme-inhibition as
the main mode of action for the cytotoxic profile of boric acid.
The results of transcriptomic analysis shown here could be useful in
understanding the molecular mechanisms of the in vitro effects of boric
acid and might potentially provide further clues about the molecular
basis of Boron based genotoxicity [26,29] as well as antiproliferative
and apoptotic effects [44,58] examined in other studies. Further in-
vestigations are needed in order to see how this vast range of effects
changes in response to time and dose, and also to find out the details of
the underlying regulatory mechanisms of transcriptional changes.
Author contributions
Authors A. Tombuloglu and H. Copoglu contributed equally to this
study.
Funding
This work was supported by the Research Fund of the Middle East
Technical University [project numbers BAP-07-02-2017-004-310, BAP-
01-08-2017-002].
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
A. Tombuloglu, et al.
Acknowledgements
The authors would like to thank Dr. Serap Kolukisa for initiating this
study, and Dr. Nurcan Tuncbag for the valuable comments during the
research.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jtemb.2020.126573.
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