Journal of Environmental Science and Health, Part B

Pesticides, Food Contaminants, and Agricultural Wastes

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Cyanogenic glycoside amygdalin influences

functions of human osteoblasts in vitro

Radoslav Omelka, Veronika Kovacova, Vladimira Mondockova, Birgit

Grosskopf, Adriana Kolesarova & Monika Martiniakova

To cite this article: Radoslav Omelka, Veronika Kovacova, Vladimira Mondockova, Birgit
Grosskopf, Adriana Kolesarova & Monika Martiniakova (2021) Cyanogenic glycoside amygdalin
influences functions of human osteoblasts in vitro, Journal of Environmental Science and Health,
Part B, 56:2, 109-116, DOI: 10.1080/03601234.2020.1852054

To link to this article: https://doi.org/10.1080/03601234.2020.1852054

Published online: 27 Jan 2021.

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JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B
2021, VOL. 56, NO. 2, 109–116
https://doi.org/10.1080/03601234.2020.1852054

Cyanogenic glycoside amygdalin influences functions of human

osteoblasts in vitro
Radoslav Omelkaa , Veronika Kovacovab , Vladimira Mondockovaa , Birgit Grosskopfc ,
Adriana Kolesarovad , and Monika Martiniakovab
a
Department of Botany and Genetics, Faculty of Natural Sciences, Constantine the Philosopher University in Nitra, Nitra, Slovak Republic;
b
Department of Zoology and Anthropology, Faculty of Natural Sciences, Constantine the Philosopher University in Nitra, Nitra, Slovak
Republic; cInstitute of Zoology and Anthropology, Georg-August University in Goettingen, Goettingen, Germany; dDepartment of Animal
Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Nitra, Slovak Republic

ABSTRACT ARTICLE HISTORY

Amygdalin has been promoted as an alternative cancer cure. However, it is still unclear how this Received 26 August 2020
cyanogenic glycoside affects non-cancer cells including bone cells. This study first investigated the Accepted 13 November 2020
impact of amygdalin on viability, morphology and expression of important genes in human osteo-
KEYWORDS
blasts in vitro. Primary human osteoblast cultures were exposed to amygdalin at concentrations 0;
Amygdalin; osteoblasts;
0.1; 1 and 10 mg/mL in growth medium for 72 h. Cell viability, osteoblasts morphology and expres- viability; morphology;
sion of 10 genes associated with osteoblast-specific pathways, oxidative stress and cell death were gene expression
determined. Osteoblasts viability was significantly decreased (–27.26%) and their size was reduced
(–23.20%) at the highest concentration of amygdalin (10 mg/mL). This concentration of amygdalin
down-regulated the expression of COL1A1 and ALPL genes, whereas the expression of BGLAP,
TNFSF11 and WNT5A genes was increased. The osteoblast cultivation with 0.1 mg/mL amygdalin
caused down-regulation of COL1A1 gene. No changes in expression were determined for RUNX2,
BAX, CASP1, SOD1 and GPX1 genes among all tested concentrations of amygdalin. In conclusion,
amygdalin in a high concentration negatively affected mineralization of extracellular matrix,
increased bone resorption and decreased osteoblast viability. These changes were accompanied
by modified expression profiles of responsible genes.

Introduction breast cancer,[4] prostate cancer,[10] squamous cell carcin-

oma,[7] colon cancer,[13,17] cervical cancer,[18] bladder can-
Amygdalin (D-mandelonitrile-b-D-gentiobioside), C20H27NO11,
cer[19] by promoting apoptosis in the cancer cells. Yang
a natural product of aromatic cyanogenic glycosides,[1,2] is one
et al.[20] reported that amygdalin caused an increase in the
of the most controversial alternative cancer drugs.[3] This com-
growth of skeletal muscle cell line C2C12 in a concentra-
pound is widely found in the rosaceous plant seeds,[4] e.g. bitter
tion-dependent manner.
almonds,[5] apricot seeds,[6] peaches,[7] apples,[8] plums,[9] black-
Current studies have shown that hydrogen cyanide is also
berries and other plants.[1] Amygdalin is composed of two mole-
released in normal cells; therefore, it may not be safe for human
cules of glucose, one molecule of benzaldehyde and one of
organism.[12] Our previous in vivo studies revealed evident
hydrocyanic acid.[10,11] Amygdalin itself is nontoxic, but highly
changes in compact bone microstructure of amygdalin-exposed
poisonous cyanide from its molecule can be released by the
rabbits consistent with a different vascularization and changed
action of some enzymes (e.g. b-glucosidase, rhodanese).[12]
biomechanical properties,[21,22] which support aforementioned
For many years, amygdalin (also known as vitamin B
fact. Therefore, this in vitro study was aimed to investigate for
17)[13] belonged to pharmacological components for the
the first time the impact of amygdalin on human osteoblasts
treatment of various diseases including asthma, bronchitis,[6]
viability, morphology and gene expression with an emphasis to
emphysema, leprosy, pain[10] and diabetes.[14] Several clinical
explain changes that were observed in our in vivo studies.
trials have documented its beneficial and therapeutic effects
in preventing and/or treating chronic gastritis,[15] and ath-
erosclerosis.[14] There are some studies describing the cyto- Materials and methods
toxic and antiproliferative activity of amygdalin (because of
Osteoblast culture
hydrogen cyanide production[16]) on cancer cells. According
to these studies, amygdalin can inhibit in vitro proliferation, Primary human osteoblasts obtained from PromoCell
invasion and migration of non-small-cell lung cancer,[1] (Heidelberg, Germany; C-12760) were used. This type of cell

CONTACT Monika Martiniakova mmartiniakova@ukf.sk; mmartiniakova@pobox.sk Department of Zoology and Anthropology, Faculty of Natural Sciences,
Constantine the Philosopher University in Nitra, Trieda A. Hlinku 1, 94974 Nitra, Slovak Republic.
ß 2020 Taylor & Francis Group, LLC
110 OMELKA ET AL.

Table 1. Description of the genes used in the current study.

Gene Comment
COL1A1 Encodes the pro-alpha1 chains of type I collagen, the major protein component of the bone extracellular matrix
ALPL Alkaline phosphatase tissue-nonspecific, important for mineralization of bone matrix and differentiation of osteoblasts
BGLAP Osteocalcin or bone gamma-carboxyglutamate protein, a highly abundant bone protein that regulates bone remodeling and energy metabolism
TNFSF11 TNF Superfamily Member 11, essential for osteoclastogenesis, it codes receptor activator of nuclear factor kappa B ligand (RANKL)
WNT5A Wnt Family Member 5 A, signal protein stimulating proliferation and differentiation of osteoblasts
RUNX2 Runt-related transcription factor 2 (RUNX2), key transcription factor associated with osteoblast differentiation
BAX BCL2 associated X, apoptosis regulator – plays a role in the mitochondrial apoptotic process
CASP1 Caspase 1, involved in inflammatory cytokine signaling and other types of cell death than apoptosis
SOD1 Superoxide dismutase 1, involved in reactive oxygen species (ROS) metabolism
GPX1 Glutathione peroxidase 1, involved in protecting cells against oxidative damage

culture was isolated from femoral trabecular bone tissue of
the knee or hip joint. Cells were suspended in an osteoblast
growth medium and seeded in a 25 cm2 (PromoCell
Osteoblast Growth Medium, C-27001; 20 000 cells per cm2).
Media were supplemented with Supplement Mix
(PromoCell, C-39615), containing all supplements necessary
for the optimal growth of human osteoblasts, and antibiotics
Biomyc 1 (PromoKine, PK-CC03-036-1C) and Biomyc 2
(PromoKine, PK-CC03-037-1C) according to the manufac-
turer’s protocol. Cells were cultured at 37
C in air contain-
ing 5% CO2 in a humidified atmosphere. Cell confluence
and morphology were daily observed under an inverted
phase-contrast microscope (Motic AE2000 Binocular, Motic
China Group Co., Ltd., Nanjing, China). The culture
medium was changed every 2 to 3 days. Osteoblasts were
subcultured when reaching 80%90% confluence using
PromoCell DetachKit (C-41210). Experiments were per-
formed during the exponential growth phase at the
5th passage.
Amygdalin treatment
Amygdalin from apricot seeds ( 99% purity, from apricot
seeds, Sigma-Aldrich, St. Louis, MO, USA) was freshly dis-
solved in cell culture medium and then added to human
osteoblasts at concentrations 0.1, 1 and 10 mg/mL for 72 h.
The cells of the control group were left untreated. In all
experiments, amygdalin-treated osteoblasts were compared
to control ones. The doses used in our experiment were
based on previous in vitro studies.[3,23,24]
Viability assay
The proliferation and viability were measured by the Cell
Counting Kit-8 (CCK-8; Sigma-Aldrich Co. Ltd.), according
to the manufacturer’s protocol. Osteoblasts were cultured at
1.0  1.2  104 cells/well in 96-well plates during 24 h for
adhesion. The cells were subsequently treated with amyg-
dalin at concentrations mentioned above. After 72-h cultiva-
tion, 10 lL of CCK-8 solution was added to each well
containing 100 lL of medium and incubated for 4 h. The
absorbance was measured with a microtiter plate reader
(Varioskan Flash, Thermo Scientific, Waltham, MA, USA) at
a wavelength of 450 nm. Cell viability was expressed as the
proportion of optical density (OD) to the control. Three
parallel experiments in three technical replicates
were performed.
Osteoblast morphology
Primary human osteoblasts were seeded in 96-well culture
plates (1.0  1.2  104 cells/well). After adhesion (24 h) and
amygdalin treatment (72 h), the cell morphology was
observed under an inverted optical microscope (Motic
AE2000 Binocular). The quantitative parameters of the cells,
including cell area (lm2) and perimeter (lm), were assessed
by ImageJ software (NIH Image, Public Domain).
Gene expression
Cells were seeded in 12-well culture plates (1.2  105 cells/
well). After incubation for 24 h to allow attachment, the cells
were treated with amygdalin for 72 h in referred concentra-
tions and conditions. RNeasy Plus Mini Kit (Qiagen, cat. no.
74134) was used to isolate mRNA from cell cultures.
Isolated mRNA was purified from genomic DNA contamin-
ation and quantified by Quantus Fluorometer (Promega,
Madison, WI, USA). The cDNA library was obtained from
the RNA using the RT2 First Strand Kit (Qiagen, Hilden,
Germany, cat. no. 330404). Expression of 10 selected genes
(COL1A1, ALPL, BGLAP, TNFSF11, WNT5A, RUNX2, BAX,
CASP1, SOD1, and GPX1; Table 1) was determined by real-
time PCR on the Custom RT2 profiler PCR Array (Qiagen)
using 96-well plates according to the manufacturer’s instruc-
tions. These genes encode products of osteoblast activity
linked with bone formation and remodeling, as well as those
associated with cell death and response to oxidative stress.
Raw Ct values were analyzed using the GeneGlobe Data
Analysis Center (Qiagen), the software recommended for
the processing of the data. Three genes (GAPDH, RPLP0,
and ACTB) were chosen as reference ones, from which
RPLP0 and ACTB were selected by the software as the most
suitable for normalization.
Statistical analysis
General statistics and comparisons were performed using
SPSS 17.0 software. The results were expressed as mean-
± standard deviation (SD) or mean ± standard error of the
mean (SE) of independent experiments measured as sixtipli-
cate (evaluation of morphology, viability) or in triplicate (for
the gene expression). The data were determined using one-
way analysis of variance (ANOVA) followed by Games-
Howell and/or Tukey’s post hoc tests or t-test, depending on
the type of the experiment. Gene expression data were

analyzed by The GeneGlobe Data Analysis Center (Qiagen)
software. The p value less than .05 was considered to be
statistically significant.
Results
Effects of amygdalin on viability of human osteoblasts
Amygdalin administration had a dose-dependent effect on
human osteoblast viability counting 99.22%, 96.26%, and
72.74%, for 0.1, 1, and 10 mg/mL, respectively. Significant
inhibition of cell viability was observed only at concentra-
tion of 10 mg/mL (Figure 1).
Effects of amygdalin on morphology of human
osteoblasts
In all experimental groups, polygonal-shaped or flattened
spindle-shaped cells with short dendrites were observed after
72 h of cultivation (Figure 2). Treatment of the cells with
amygdalin caused limited visible morphological changes.
However, morphometrical measurements revealed consider-
ably decreased size (area and perimeter) of the cells
(–23.2%; p < .05) at 10 mg/mL of amygdalin (Table 2).
Effects of amygdalin on gene expression in human
osteoblasts
The gene expression profiles of primary osteoblasts after
amygdalin treatment are summarized in Table 3 and Figure
3. Amygdalin at 10 mg/mL concentration significantly
Figure 1. The impact of amygdalin on viability of human osteoblasts.
The values are shown as mean ± SD (standard deviation), p < .05 ().
Figure 2. Representative images of human osteoblasts in vitro.
(a) Morphology of the cells after amygdalin treatment at concentration 0 lg/mL.
(b) Morphology of the cells after amygdalin treatment at concentration 0.1 mg/mL.
(c) Morphology of the cells after amygdalin treatment at concentration 1 mg/mL.
(d) Morphology of the cells after amygdalin treatment at concentration 10 mg/mL.
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B 111
down-regulated the expression of COL1A1 (–1.21 in fold
regulation, 0.82 fold change), and ALPL genes (–2.14 in fold
regulation, 0.47 fold change; p < .05), whereas the expression
of BGLAP, TNFSF11 and WNT5A genes was significantly
increased (1.31, 1.30, and 1.25 in fold change, respectively).
The osteoblast cultivation with 0.1 mg/mL amygdalin caused
down-regulation of COL1A1 gene (–1.12 in fold regulation,
0.89 fold change; p < .01). No changes in expression were
determined for RUNX2, BAX, CASP1, SOD1, and GPX1
genes among all tested concentrations of amygdalin.
Discussion
Despite the quantity of research studies that have been con-
ducted on the antitumor effects of amygdalin, very limited
information is available regarding the action of this com-
pound on non-cancer cells. The action of amygdalin in can-
cer cells is based on the fact that b-glucosidase enzyme,
which can release cyanide anion from the amygdalin mol-
ecule, shows higher activity in cancer tissues than in non-
cancer ones. On the contrary, there is a cyanide detoxifying
enzyme rhodanese present in normal cells but with negli-
gible activity in cancer cells.[18] It was proposed that a com-
bination of these enzymes in a cancer-specific manner could
provide a selective advantage for the killing of cancer cells
by amygdalin without having harmful effects on normal
cells. However, the rhodanese is also present in cancer cells
and b-glucosidase has been found (in lower amounts) in
physiologically unchanged tissues.[12] Thus, amygdalin treat-
ment could also affect non-cancer cells.
Our results demonstrate that the highest concentration of
amygdalin (10 mg/mL) negatively affected osteoblast viabil-
ity. A decrease in cell viability and proliferation has been
observed in several studies focusing on cancer lines. Chen
et al.[18] analyzed the effects of amygdalin (at doses 1.25,
2.5, 5, 10, and 20 mg/mL) on the viability of human cervical
cancer HeLa cell line and human embryonic amnion FL cell
line. Amygdalin significantly inhibited the viability of HeLa
cells; however, the authors did not observe any differences
in the viability of FL cells. According to Syrigos et al.,[25]
amygdalin was cytotoxic to HT1376 bladder cancer cells
only at the high concentration (40.4 mM). The exposure to
lower dose of amygdalin (10 mg/mL) resulted in a lower cell
number, suppression of growth, and proliferation of bladder
cancer cell lines.[19] In addition, significant reduction of
112 OMELKA ET AL.

growth and proliferation was revealed in kidney carcinoma
cell lines and kidney fibroblast cells after amygdalin admin-
istration.[8,26] Amygdalin also reduced the viability of COLO
201 and SNU-C4 human colon cancer cells,[13,17] and
Hs578T breast cancer cells.[4] The aforementioned studies
have mostly shown a dose-dependent cytotoxicity of amyg-
dalin on the cells. On the other hand, amygdalin at doses
equal to those used in our study (100; 1 000 lg/mL)
increased the viability of porcine ovarian granulosa cells, but
the highest concentration (10,000 lg/mL) had no effect.[23]
Amygdalin treatment of another type of non-cancer cells –
cultured renal interstitial fibroblasts – inhibited their
proliferation.[26]
Although osteoblasts showed no apparent differences in
cell shape in all experimental groups in our study, their size
decreased as the concentration of amygdalin increased.
Current studies have found that amygdalin could negatively
affect morphology of various cancer cells. In COLO 201
human colon cancer cells, cell rounding, irregularity in
Table 2. Data of cell size parameters after amygdalin treatment.
Average cell
Amygdalin (mg/mL) area (lm2)
0 (1) 2994.81 ± 865.82
0.1 (2) 2619.17 ± 515.00
1 (3) 2376.52 ± 684.73
10 (4) 2300.24 ± 568.77
Tukey / Games-Howell test 1:4
Results are mean of 30 parallel measurements. The values are shown as
mean ± SD (standard deviation). p < .05 ().
shape, cytoplasmic blebbing, nuclear condensations, and
DNA fragmentation were observed.[13] Amygdalin-treated
HeLa cells became round in shape, with nuclear condensa-
tion and fragmentation.[18]
According to our findings, amygdalin administration was
associated with decreased expression of COL1A1 and ALPL
genes and increased expression of BGLAP gene. Osteoblasts
start to acquire alkaline phosphatase (ALP), secrete collagens
(mainly type 1 collagen; COL1A1), and non-collagenous
proteins including osteocalcin (OC, BGLAP) during differ-
entiation and transition to mature status.[27,28] Type I colla-
gen is a major structural component of the extracellular
matrix;[29,30] therefore, it plays an important role in bio-
mechanical properties of bone (bearing, torsional stiffness,
tensile strength).[31] ALP, enzyme specific for osteoblasts,[27]
is involved in the skeletal mineralization[32] and calcification
process.[33] Osteocalcin is a bone-derived multifunctional
hormone, directly secreted and expressed by mature osteo-
blasts.[27,34] Consistent with our results, but using the
immunohistochemistry, Zhang et al.[35,36] demonstrated that
amygdalin (at doses 50, 100, and 200 lM) increased expres-
sion of osteocalcin and decreased expression of type 1 colla-
Average cell
perimeter (lm)
gen in bone marrow mesenchymal stem cells of rabbits.
552.74 ± 119.78 Thus, our results clearly showed that amygdalin treatment
486.29 ± 84.23 negatively affected mineralization of extracellular matrix by
502.84 ± 120.87 osteoblasts. A possible mechanism of amygdalin-dependent
450.43 ± 89.87
1:4 production of ALP and collagen may include a reduction of
pH. Some studies revealed that cyanide intoxication resulted
in severe metabolic acidosis[37–39] associated with decreased
ALP activity and inhibited collagen synthesis.[40,41] On the

Table 3. The effect of amygdalin treatment on gene expression of human osteoblasts.

0.1 mg/mL 1 mg/mL 10 mg/mL
Amygdalin
Gene p value Fold change p value Fold change p value Fold change
COL1A1 0.009 0.89 0.758 1.20 0.047 0.82
ALPL 0.176 0.77 0.379 0.86 0.022 0.47
BGLAP 0.424 0.92 0.399 0.90 0.035 1.31
TNFSF11 0.968 1.00 0.358 0.90 0.029 1.30
WNT5A 0.213 0.83 0.878 1.20 0.037 1.25
RUNX2 0.713 0.97 0.673 0.96 0.271 1.20
BAX 0.669 0.96 0.678 0.97 0.192 1.26
CASP1 0.308 1.90 0.989 0.98 0.910 0.98
SOD1 0.633 1.40 0.983 1.00 0.183 1.19
GPX1 0.102 0.90 0.251 0.96 0.867 1.10
Results are expressed as fold change to RPLP0 and ACTB normalized mRNA values. p < .05 (), p < .01 ()

Figure 3. Changes in gene expression of human osteoblasts after amygdalin treatment.

The values are shown as mean ± SE (standard error of the mean), p<.05 (), p<.01 ().

other hand, up-regulated BGLAP gene may be beneficial for
bone where OC influences and regulates mineralization of
bone matrix.[42,43] In addition, OC modulates energy metab-
olism via enhancement of glucose uptake in muscle, insulin
production in the pancreas, insulin sensitivity in the liver
and adipose tissue, up-regulation of adiponectin expression
in adipose tissue, promotion of b-cell proliferation in the
pancreas, it affects male fertility and cognitive function of
the brain.[44]
The TNFSF11, WNT5A, and RUNX2 belong to other key
genes regulating bone formation, remodeling, and homeostasis.
In our study, TNFSF11 and WNT5A genes were up-regulated
by 10 mg/mL amygdalin. In general, TNSFS11 gene encodes
RANKL (receptor activator of nuclear factor kappa B ligand),
which is expressed by osteoblasts and is indispensable for
osteoclastogenesis.[45] In addition, WNT5A enhances RANKL-
induced osteoclastogenesis through the receptor Ror2.[46] Thus,
increased levels of RANKL and WNT5A indicate a significant
effect of amygdalin on osteoclast formation and production,
and thereby on bone resorption. The process could be sup-
ported by cyanide-induced metabolic acidosis, which increases
RANKL expression.[47] In addition, metabolic acidosis induces
bone resorption through an increased function of osteo-
clasts.[41,48] WNT5A glycoprotein is also produced during an
inflammatory state or under pathophysiological conditions,
which is often accompanied by a reduction of extracellular
pH.[49,50] However, we did not observe a pro-inflammatory cell
death (pyroptosis) in all analyzed groups, since the expression
of CASP1 gene remained unchanged. Moreover, amygdalin
was found to inhibit NF-jB, an important factor of inflamma-
tory response.[35] It is also known that cyanide causes intracel-
lular hypoxia by reversibly binding to mitochondrial
cytochrome-a3 oxidase. Hypoxia can negatively influence
osteoblast activity together with formation of bigger osteoclasts,
which may contribute to a higher bone resorption.[51]
RUNX2 is required for the proliferation of osteoblast pro-
genitors[52] and it is closely associated with the regulation of
osteoblasts differentiation and their function.[53] RUNX2 has
the ability to up-regulate the expression of bone matrix pro-
tein genes including COL1A1 and BGLAP. The expression of
RUNX2 increases in immature osteoblasts and decreases dur-
ing osteoblast maturation.[54] Our data suggest that amygdalin
treatment does not interfere with RUNX2 gene regulation.
Programmed cell death is an essential part of the devel-
opment and tissue homeostasis making possible the removal
of unwanted cells.[55] There are a number of different forms
of cell death, which may potentially be affected by amyg-
dalin treatment. Apoptosis is regulated by two of the most
significant genes, BCL-2 (anti-apoptotic) and BAX (pro-
apoptotic).[56] While BCL-2 is responsible for the regulation
of apoptotic pathways and protects against cell death,[10]
BAX is expressed abundantly during apoptosis and its pro-
duction results in promoted cell death.[13] Amygdalin is able
to regulate apoptotic proteins and signaling molecules in dif-
ferent cancer lines.[12] Numerous studies have confirmed
that amygdalin increased BAX level and caspase-3 activity
and inhibited expression of BLC-2 gene in colon, prostate,
HeLa, breast, bladder, and lung cancer cells.[4,10,13,18]
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B 113
However, in our study no changes in BAX expression were
found after amygdalin exposure. On the other hand, it can-
not be ruled out that several forms of cell death may be
induced within the same tissue. Although apoptosis is the
fastest, other forms only become visible when apoptosis is
inhibited. Intracellular ATP levels may affect the form of
cell death where high ATP levels lead to apoptosis, while a
low ATP level results in necrosis. It is possible that ATP lev-
els decreased in amygdalin-treated cells as a result of cyan-
ide-induced inhibition of cytochrome-c oxidase in the
respiratory electron transport chain of the mitochondria.
This could lead to weakening both oxidative metabolism
and the associated process of oxidative phosphorylation.[57]
Finally, energy-dependent apoptotic cell death could be
switched to necrosis. Moreover, over-expression of WNT5A
was reported to be able to block apoptosis in mature osteo-
blasts via b-catenin pathway.[58] Application of Annexin V
and propidium iodide labeling could more precisely distin-
guish between apoptosis and necrosis and detect their pres-
ence after amygdalin administration. Some recent studies
point to pyroptosis as another possible mechanism of cell
death. Pyroptosis is a pro-inflammatory form of cell death,
which can be induced by both infectious and noninfectious
stimuli. Caspase-1 is activated during pyroptosis by a large
complex – inflammasome, and subsequently mediates the
maturation and secretion of proinflammatory mediators (IL-
1b and IL-18).[55] Glycosides like amygdalin are able to
modulate pyroptosis in neurons.[59] We did not find any
effect of amygdalin administration on CASP1 gene expres-
sion, suggesting that amygdalin does not contribute to the
CASP1 stock. However, the activation of CASP1 is ultim-
ately essential for its function during pyroptosis. The detec-
tion of caspase (–1, 4, 5) activation together with further
analysis of other markers including NLRP3 (NOD-like
receptors protein 3), membrane Gasdermin D or production
of proinflammatory mediators, could more clearly indicate
the presence of pyroptosis.
Oxidative stress is consistent with inhibition of bone for-
mation by modulating the differentiation, function, and sur-
vival of osteoblasts and by stimulating bone resorption.[60]
On the contrary, the reactive oxygen species (ROS) are of
great importance for cancer therapy because they have a
potential to provide an effective approach for a selective kill-
ing of cancer cells.[61] Evidences for the amygdalin potenti-
ation of oxidative stress were provided in breast cell lines.
The likely mechanism may involve cyanide-induced inhib-
ition of cytochrome-c oxidase activity followed by a stimula-
tion of ROS formation.[62] The increased oxidative stress
and ROS accumulation usually results in increased expres-
sion of antioxidant enzymes. Gene expression of two anti-
oxidant enzymes was also analyzed in our study. Superoxide
dismutase (SOD) is the first detoxification enzyme and the
most powerful antioxidant in the cell. Cu/Zn-SOD, one of
the SOD forms encoded by the SOD1 gene, is predominant
in eukaryotes.[63] It converts the superoxide radicals to
hydrogen peroxide, which is subsequently broken down into
water by glutathione peroxidase. In our study, no changes in
114 OMELKA ET AL.
SOD1 and GPX genes expression were found suggesting that
ROS was not induced in amygdalin-treated osteoblasts.
The data of this study clearly explain microstructural
alterations in the bone, which were observed in our previous
in vivo studies.[21,22] Subacute exposure to amygdalin, as
well as long-term amygdalin supplementation, caused evi-
dent changes in bone microstructure where a decreased
density and sizes of secondary osteons in the middle part of
substantia compacta (due to a replacement of Haversian
bone tissue by plexiform bone tissue) and altered biomech-
anical properties were identified. These differences are
closely related to dissimilar bone remodeling with an accel-
erated bone resorption, and also with collagen fiber altera-
tions in secondary osteons.[21,22] We have demonstrated an
up-regulation of TNFSF11, WNT5A, and down-regulation of
ALPL, which directly point to a higher bone resorption and
insufficient matrix mineralization. In addition, decreased
COL1A1 as a template for mineral deposition supports
aforementioned alterations in bone mineralization and pro-
vides basis for collagen damage. This, together with the dir-
ect action of cyanide, creates an overall picture of
amygdalin-induced changes in the bone.
Conclusions
Our study provides the initial information on the effect of
amygdalin on viability, morphology, and expression of
important genes in cultivated human osteoblasts. The high-
est concentration of amygdalin decreased osteoblasts viabil-
ity and their size. We revealed that amygdalin in a high
concentration directly affected mineralization of extracellular
matrix by down-regulation of COL1A1 and ALPL genes and
it increased osteoclast production and bone resorption by
up-regulation of TNFSF11 and WNT5A gene expression.
Amygdalin also increased expression of BGLAP gene.
Amygdalin-dependent apoptosis, pyroptosis, and production
of oxidative stress in human osteoblasts were not confirmed
by expression profiles of the genes encoding enzymes and
regulatory proteins involved. These results support the
amygdalin-dependent microstructural findings of our previ-
ous in vivo studies where alterations in collagen synthesis,
bone mineralization, and enhanced bone resorption were
identified. We can conclude that amygdalin treatment can
also affect non-cancer cells with negative consequences on
their functions.
Disclosure statement
All authors declare that they have no competing interests concerning
this article.
Funding
This study was supported by the projects VEGA 1/0505/18 and KEGA
041UKF-4/2020. The funders had no role in the study design, data col-
lection and analysis, writing the manuscript, and decision to submit
the article for publication.
ORCID
Radoslav Omelka http://orcid.org/0000-0002-6493-9880
Veronika Kovacova http://orcid.org/0000-0002-8491-0399
Vladimira Mondockova http://orcid.org/0000-0002-7186-4282
Birgit Grosskopf http://orcid.org/0000-0003-0815-5077
Adriana Kolesarova http://orcid.org/0000-0002-1272-9099
Monika Martiniakova http://orcid.org/0000-0003-1889-026X
References
[1] Qian, L.; Xie, B.; Wang, Y.; Qian, J. Amygdalin-Mediated
Inhibition of Non-Small Cell Lung Cancer Cell Invasion
In Vitro. Int. J. Clin. Exp. Pathol. 2015, 8, 5363–5370.
[2] Kopcekova, J.; Kolesarova, A.; Kovacik, A.; Kovacikova, E.;
Gazarova, M.; Chlebo, P.; Valuch, J.; Kolesarova, A. Influence
of Long-Term Consumption of Bitter Apricot Seeds on Risk
Factors for Cardiovascular Diseases. J. Environ. Sci. Health B
2018, 53, 298–303. doi:10.1080/03601234.2017.1421841.
[3] Halenar, M.; Medvedova, M.; Maruniakova, N.; Kolesarova, A.
Ovarian Hormone Production Affected by Amygdalin Addition
In Vitro. J. Microbiol. Biotech. Food Sci. 2015, 4, 19–22. doi:10.
15414/jmbfs.2015.4.special2.19-22.
[4] Lee, H. M.; Moon, A. Amygdalin Regulates Apoptosis and
Adhesion in hs578t Triple-Negative Breast Cancer Cells.
Biomol. Ther. (Seoul) 2016, 24, 62–66. doi:10.4062/biomolther.
2015.172.
[5] Juengel, E.; Afschar, M.; Makarevic, J.; Rutz, J.; Tsaur, I.; Mani,
J.; Nelson, K.; Haferkamp, A.; Blaheta, R. A. Amygdalin Blocks
the In Vitro Adhesion and Invasion of Renal Cell Carcinoma
Cells by an Integrin-Dependent Mechanism. Int. J. Mol. Med.
2016, 37, 843–850. doi:10.3892/ijmm.2016.2454.
[6] Kovacikova, E.; Kovacik, A.; Halenar, M.; Tokarova, K.;
Chrastinova, L.; Ondruska, L.; Jurcik, R.; Kolesar, E.; Valuch, J.;
Kolesarova, A. Potential Toxicity of Cyanogenic Glycoside
Amygdalin and Bitter Apricot Seed in Rabbits-Health Status
Evaluation. J. Anim. Physiol. Anim. Nutr. (Berl) 2019, 103,
695–703. doi:10.1111/jpn.13055.
[7] Nour, A.; Azar, B.; Rabata, A.; Manadili, A. The Effect of
Amygdalin in the Treatment of Squamous Cell Carcinoma
Induced in the Buccal Pouch of Golden Syrian Hamster. Iosr-
Jdms. 2016, 15, 75–79. doi:10.9790/0853-15297579.
[8] Juengel, E.; Thomas, A.; Rutz, J.; Makarevic, J.; Tsaur, I.;
Nelson, K.; Haferkamp, A.; Blaheta, R. A. Amygdalin Inhibits
the Growth of Renal Cell Carcinoma Cells In Vitro. Int. J. Mol.
Med. 2016, 37, 526–532. doi:10.3892/ijmm.2015.2439.
[9] Song, Z.; Xu, X. Advanced Research on Anti-Tumor Effects of
Amygdalin. J. Cancer Res. Ther. 2014, 10, 3–7. doi:10.4103/
0973-1482.139743.
[10] Chang, H. K.; Shin, M. S.; Yang, H. Y.; Lee, J. W.; Kim, Y. S.;
Lee, M. H.; Kim, J.; Kim, K. H.; Kim, C. J. Amygdalin Induces
Apoptosis through Regulation of Bax and Bcl-2 Expressions in
Human DU145 and LNCaP Prostate Cancer Cells. Biol. Pharm.
Bull. 2006, 29, 1597–1602. doi:10.1248/bpb.29.1597.
[11] Sireesha, D.; Reddy, B. S.; Reginald, B. A.; Samatha, M.; Kamal,
F. Effect of Amygdalin on Oral Cancer Cell Line: An In Vitro
Study. J. Oral Maxillofac Pathol. 2019, 23, 104–107. doi:10.
4103/jomfp.JOMFP_281_18.
[12] Liczbi

nski, P.; Bukowska, B. Molecular Mechanism of
Amygdalin Action In Vitro: Review of the Latest Research.
Immunopharmacol. Immunotoxicol. 2018, 40, 212–218. doi:10.
1080/08923973.2018.1441301.
[13] Kim, J.-Y.; Song, Y.-K.; Lim, H.-H. Amygdalin Extract from
Armeniaceae Semen Induces Apoptosis in Human COLO 201
Colon Cancer Cells. Korean J. Intern. Med. 2005, 26, 108–121.
doi:10.3904/kjim.2011.26.1.108.
[14] Jiagang, D.; Li, C.; Wang, H.; Hao, E.; Du, Z.; Bao, C.; Lv, J.;
Wang, Y. Amygdalin Mediates Relieved Atherosclerosis in
Apolipoprotein E Deficient Mice through the Induction of

Regulatory T Cells. Biochem. Biophys. Res. Commun. 2011,
411, 523–529. doi:10.1016/j.bbrc.2011.06.162.
[15] Wei, Y.; Xie, Q.; Ito, Y. Preparative Separation of Axifolin-3-
Glucoside, Hyperoside and Amygdalin from Plant Extracts by
High-Speed Countercurrent Chromatography. J. Liq.
Chromatogr. Relat. Technol. 2009, 32, 1010–1022. doi:10.1080/
10826070902790983.
[16] Moon, J. Y.; Kim, S. W.; Yun, G. M.; Lee, H. S.; Kim, Y. D.;
Jeong, G. J.; Ullah, I.; Rho, G. J.; Jeon, B. G. Inhibition of Cell
Growth and Down-Regulation of Telomerase Activity by
Amygdalin in Human Cancer Cell Lines. Anim. Cells Syst.
2015, 19, 295–304. doi:10.1080/19768354.2015.1060261.
[17] Park, H. J.; Yoon, S. H.; Han, L. S.; Zheng, L. T.; Jung, K. H.;
Uhm, Y. K.; Lee, J. H.; Jeong, J. S.; Joo, W. S.; Yim, S. V.; et al.
Amygdalin Inhibits Genes Related to Cell Cycle in SNU-C4
Human Colon Cancer Cells. World J. Gastroenterol. 2005, 11,
5156–5161. doi:10.3748/wjg.v11.i33.5156.
[18] Chen, Y.; Ma, J.; Wang, F.; Hu, J.; Cui, A.; Wei, C.; Yang, Q.;
Li, F. Amygdalin Induces Apoptosis in Human Cervical Cancer
Cell Line HeLa Cells. Immunopharmacol. Immunotoxicol.
2013, 35, 43–51. doi:10.3109/08923973.2012.738688.
[19] Makarevic, J.; Rutz, J.; Juengel, E.; Kaulfuss, S.; Reiter, M.;
Tsaur, I.; Bartsch, G.; Haferkamp, A.; Blaheta, R. A. Amygdalin
Blocks Bladder Cancer Cell Growth in Vitro by Diminishing
Cyclin a and cdk2. PLoS One. 2014, 9, e105590. doi:10.1371/
journal.pone.0105590.
[20] Yang, C.; Li, X.; Rong, J. Amygdalin Isolated from Semen
Persicae (Tao Ren) Extracts Induces the Expression of
Follistatin in HepG2 and C2C12 Cell lines. Chin. Med. 2014, 9,
23. doi:10.1186/1749-8546-9-23.
[21] Kovacova, V.; Sarocka, A.; Omelka, R.; Bauerova, M.;
Grosskopf, B.; Formicki, G.; Kolesarova, A.; Martiniakova, M.
Subacute Exposure to Amygdalin Influences Compact Bone
Remodelling of Rabbits. J. Physiol. Pharmacol. 2019, 70,
641–648. doi:10.26402/jpp.2019.4.15.
[22] Kovacova, V.; Sarocka, A.; Blahova, J.; Sranko, P.; Omelka, R.;
Galbavy, D.; Kolesarova, A.; Martiniakova, M. Long-Term
Peroral Administration of Bitter Apricot Seeds influences
Cortical Bone Microstructure of Rabbits. J. Anim. Physiol.
Anim. Nutr. (Berl) 2020, 104, 362–370. doi:10.1111/jpn.13229.
[23] Halenar, M.; Tusimova, E.; Nynca, A.; Sadowska, A.; Ciereszko,
R.; Kolesarova, A. Stimulatory Effect of Amygdalin on the
Viability and Steroid Hormone Secretion by Porcine Ovarian
Granulosa Cells In Vitro. J. Microbiol. Biotech. Food Sci. 2016,
5, 44–46. doi:10.15414/jmbfs.2016.5.special1.44-46.
[24] Halenar, M.; Medvedova, M.; Baldovska, S.; Michalcova, K.;
Kolesarova, A. Co-Administration of Amygdalin and
Deoxynivalenol Disrupted Regulatory Proteins Linked to
Proliferation of Porcine Ovarian Cells in Vitro. Potravinarstvo
Slovak J. Food Sci. 2017, 11, 503–509. doi:10.5219/791.
[25] Syrigos, K. N.; Rowlinson-Busza, G.; Epenetos, A. A. In Vitro
Cytotoxicity following Specific Activation of Amygdalin by
b-Glucosidase Conjugated to a Bladder Cancer-Associated
Monoclonal Antibody. Int. J. Cancer 1998, 78, 712–719. doi:10.
1002/(sici)1097-0215(19981209(78:6 < 712::aid-ijc8 > 3.0.co;2-d.
[26] Guo, J.; Wu, W.; Sheng, M.; Yang, S.; Tan, J. Amygdalin
Inhibits Renal Fibrosis in Chronic Kidney Disease. Mol. Med.
Rep. 2013, 7, 1453–1457. doi:10.3892/mmr.2013.1391.
[27] Roeder, E.; Matthews, B. G.; Kalajzic, I. Visual Reporters for
Study of the Osteoblast Lineage. Bone 2016, 92, 189–195. doi:
10.1016/j.bone.2016.09.004.
[28] Rutkovskiy, A.; Stensløkken, K. O.; Vaage, I. J. Osteoblast
Differentiation at a Glance. Med. Sci. Monit. Basic Res. 2016,
22, 95–106. doi:10.12659/msmbr.901142.
[29] Shi, S.; Kirk, M.; Kahn, A. J. The Role of Type I Collagen in
the Regulation of the Osteoblast Phenotype. J. Bone Miner. Res.
1996, 11, 1139–1145. doi:10.1002/jbmr.5650110813.
[30] Komori, T. Regulation of Proliferation, Differentiation and
Functions of Osteoblasts by Runx2. IJMS. 2019, 20, 1694. doi:
10.3390/ijms20071694.
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B 115
[31] Gelse, K.; P€oschl, E.; Aigner, T. Collagens-Structure, Function,
and Biosynthesis. Adv. Drug Deliv. Rev. 2003, 55, 1531–1546.
doi:10.1016/j.addr.2003.08.002.
[32] Millan, J. L. Alkaline Phosphatases: Structure, Substrate
Specificity and Functional Relatedness to Other Members of a
Large Superfamily of Enzymes. Puronergic Signal 2006, 2,
335–341. doi:10.1007/s11302-005-5435-6.
[33] Golub, E. E.; Boesze-Battaglia, K. The Role of Alkaline
Phosphatase in Mineralization. Curr. Opin. Orthop. 2007, 18,
444–448. doi:10.1097/BCO.0b013e3282630851.
[34] Xu, Z.; Dai, F.; Chen, J.; Lv, M.; Cheng, J.; Zhang, X.; Lin, B.
Experimental Research into the Potential Therapeutic Effect of
GYY4137 on Ovariectomy-Induced Osteoporosis. Cell Mol.
Biol. Lett. 2018, 23, 47. doi:10.1186/s11658-018-0114-0.
[35] Zhang, A.; Pan, W.; Lv, J.; Wu, H. Protective Effect of
Amygdalin on LPS-Induced Acute Lung Injury by Inhibiting
NF-jB and NLRP3 Signaling Pathways. Inflammation 2017, 40,
745–751. doi:10.1007/s10753-017-0518-4.
[36] Zhang, J.; Cai, H.; Guo, X.; Yin, J.; Yang, B.; Feng, Y.; Jin, H.
Effects of Amygdalin on Alcohol-Induced Adipogenic
Differentiation of Rabbit Bone Marrow Mesenchymal Stem
Cells. Int. J. Clin. Exp. Pathol. 2016, 9, 12439–12445.
[37] Aitken, D.; West, D.; Smith, F.; Poznanski, W.; Cowan, J.;
Hurtig, J.; Peterson, E.; Benoit, B. Cyanide Toxicity following
Nitroprusside Induced Hypotension. Can. Anaesth. Soc. J.
1977, 24, 651–660. doi:10.1007/bf03006709.
[38] Yen, D.; Tsai, J.; Wang, L. M.; Kao, W. F.; Hu, S. C.; Lee,
C. H.; Deng, J. F. The Clinical Experience of Acute Cyanide
Poisoning. Am. J. Emerg. Med. 1995, 13, 524–528. doi:10.1016/
0735-6757(95)90162-0.
[39] Park, J.; Lee, S.; Choi, H.; Choi, Y. H.; Lee, Y. Management of
Cyanide Intoxication with Extracorporeal Membrane
Oxygenation and Continuous Renal Replacement Therapy.
Korean J. Crit. Care Med. 2015, 30, 218–221. doi:10.4266/kjccm.
2015.30.3.218.
[40] Krieger, N. S.; Sessler, N. E.; Bushinsky, D. A. Acidosis Inhibits
Osteoblastic and Stimulates Osteoclastic Activity In Vitro. Am.
J. Physiol. 1992, 262, F442–F448. doi:10.1152/ajprenal.1992.262.
3.F442.
[41] Frick, K. K.; Jiang, L.; Bushinsky, D. A. Acute Metabolic
Acidosis Inhibits the Induction of Osteoblastic Egr-1 and Type
1 Collagen. Am. J. Physiol. 1997, 272, C1450–1456. doi:10.
1152/ajpcell.1997.272.5.C1450.
[42] Zoch, M. L.; Clemens, T. L.; Riddle, R. C. New Insights into the
Biology of Osteocalcin. Bone 2016, 82, 42–49. doi:10.1016/j.
bone.2015.05.046.
[43] Blair, H. C.; Larrouture, Q. C.; Li, Y.; Lin, H.; Beer-Stoltz, D.;
Liu, L.; Tuan, R. S.; Robinson, L. J.; Schlesinger, P. H.; Nelson,
D. J. Osteoblast Differentiation and Bone Matrix Formation
In Vivo and In Vitro. Tissue Eng. Part B Rev. 2017, 23,
268–280. doi:10.1089/ten.TEB.2016.0454.
[44] Han, Y.; You, X.; Xing, W.; Zhang, Z.; Zou, W. Paracrine and
Endocrine Actions of Bone-the Functions of Secretory Proteins
from Osteoblasts, Osteocytes, and Osteoclasts. Bone Res. 2018,
6, 1–11. doi:10.1038/s41413-018-0019-6.
[45] Takahashi, N.; Maeda, K.; Ishihara, A.; Uehara, S.; Kobayashi,
Y. Regulatory Mechanism of Osteoclastogenesis by RANKL and
Wnt Signals. Front Biosci. (Landmark Ed) 2011, 16, 21–30. doi:
10.2741/3673.
[46] Maeda, K.; Kobayashi, Y.; Udagawa, N.; Uehara, S.; Ishihara,
A.; Mizoguchi, T.; Kikuchi, Y.; Takada, I.; Kato, S.; Kani, S.;
et al. Wnt5a-Ror2 Signaling between Osteoblast-Lineage Cells
and Osteoclast Precursors Enhances Osteoclastogenesis. Nat.
Med. 2012, 18, 405–412. doi:10.1038/nm.2653.
[47] Frick, K. K.; Bushinsky, D. A. Metabolic Acidosis Stimulates
RANKL RNA Expression in Bone through a Cyclo-Oxygenase-
Dependent Mechanism. J. Bone Miner. Res. 2003, 18,
1317–1325. doi:10.1359/jbmr.2003.18.7.1317.
[48] Krieger, N. S.; Bushinsky, D. A.; Frick, K. K. Cellular
Mechanisms of Bone Resorption Induced by Metabolic
116 OMELKA ET AL.
Acidosis. Semin. Dial. 2003, 16, 463–466. doi:10.1046/j.1525-
139x.2003.16100.x.
[49] Valencia, J.; Martinez, V. G.; Hidalgo, L.; Hernandez-Lopez, C.;
Canseco, N. M.; Vicente, A.; Varas, A.; Sacedon, R. Wnt5a
Signaling increases IL-12 Secretion by Human Dendritic Cells
and enhances IFN-c Production by CD4þ T cells. Immunol.
Lett. 2014, 162, 188–199. doi:10.1016/j.imlet.2014.08.015.
[50] Riemann, A.; Wußling, H.; Loppnow, H.; Fu, H.; Reime, S.;
Thews, O. Acidosis Differently Modulates the Inflammatory
Program in Monocytes and Macrophages. Biochim. Biophys.
Acta. 2016, 1862, 72–81. doi:10.1016/j.bbadis.2015.10.017.
[51] Chang, J.; Jackson, S. G.; Wardale, J.; Jones, S. W. Hypoxia
Modulates the Phenotype of Osteoblasts Isolated from Knee
Osteoarthritis Patients, Leading to Undermineralized Bone
Nodule Formation. Arthritis Rheumatol. 2014, 66, 1789–1799.
doi:10.1002/art.38403.
[52] Kawane, T.; Qin, X.; Jiang, Q.; Miyazaki, T.; Komori, H.;
Yoshida, C. A.; Matsuura-Kawata, V. K. D. S.; Sakane, C.;
Matsuo, Y.; Nagai, K.; et al. Runx2 is Required for the
Proliferation of Osteoblast Progenitors and Induces
Proliferation by Regulating Fgfr2 and Fgfr3. Sci. Rep. 2018, 8,
13551. doi:10.1038/s41598-018-31853-0.
[53] Bruderer, M.; Richards, R. G.; Alini, M.; Stoddart, M. J. Role
and Regulation of RUNX2 in osteogenesis. Eur. Cell. Mater.
2014, 28, 269–286. doi:10.22203/ecm.v028a19.
[54] Komori, T. Runx2, an Inducer of Osteoblast and chondrocyte
differentiation. Histochem. Cell Biol. 2018, 149, 313–323. doi:10.
1007/s00418-018-1640-6.
[55] Miao, E. A.; Rajan, J. V.; Aderem, A. Caspase-1-Induced
Pyroptotic Cell Death. Immunol. Rev. 2011, 243, 206–214. doi:
10.1111/j.1600-065X.2011.01044.x.
[56] Gokalp-Ozkorkmaz, E.; Asir, F.; Basaran, S. O.; Agacayak, E.;
Sahin, F.; Kaya, S.; Erdogan, G.; Deveci, E. Examination of Bcl-
2 and Bax Protein Levels for Determining the Apoptotic
Changes in Placentas with Gestational Diabetes and
Preeclampsia. Proceedings 2018, 2, 1548. doi:10.3390/
proceedings2251548.
[57] Cassiem, W.; de Kock, M. The anti-Proliferative Effect of
Apricot and Peach Kernel Extracts on Human Colon Cancer
Cells In Vitro. BMC Complement Altern. Med. 2019, 19, 32.
doi:10.1186/s12906-019-2437-4.
[58] Kikuchi, A.; Yamamoto, H.; Sato, A.; Matsumoto, S. Wnt5a: its
Signalling, Functions and Implication in Diseases. Acta Physiol.
(Oxf.) 2012, 204, 17–33. doi:10.1111/j.1748-1716.2011.02294.x.
[59] She, Y.; Shao, L.; Zhang, Y.; Hao, Y.; Cai, Y.; Cheng, Z.; Deng,
C.; Liu, X. Neuroprotective Effect of Glycosides in Buyang
Huanwu Decoction on Pyroptosis following Cerebral Ischemia-
Reperfusion Injury in Rats. J. Ethnopharmacol. 2019, 242,
112051. doi:10.1016/j.jep.2019.112051.
[60] Domazetovic, V.; Marcucci, G.; Iantomasi, T.; Brandi, M. L.;
Vincenzini, M. T. Oxidative Stress in Bone Remodeling: Role of
Antioxidants. Clin. Cases Miner. Bone Metab. 2017, 14,
209–216. doi:10.11138/ccmbm/2017.14.1.209.
[61] Perillo, B.; Di Donato, M.; Pezone, A.; Di Zazzo, E.;
Giovannelli, P.; Galasso, G.; Castoria, G.; Migliaccio, A. ROS in
Cancer Therapy: The Bright Side of the Moon. Exp. Mol. Med.
2020, 52, 192–203. doi:10.1038/s12276-020-0384-2.
[62] Abboud, M. M.; Al Awaida, W.; Alkhateeb, H. H.; Abu-Ayyad,
A. N. Antitumor Action of Amygdalin on Human Breast
Cancer Cells by Selective Sensitization to Oxidative Stress. Nutr.
Cancer 2019, 71, 483–490. doi:10.1080/01635581.2018.1508731.
[63] Ighodaro, O. M.; Akinloye, O. A. First Line Defence
Antioxidants-Superoxide Dismutase (SOD), Catalase (CAT) and
Glutathione Peroxidase (GPX): Their Fundamental Role in the
Entire Antioxidant Defence Grid. Alexandria J. Med. 2018, 54,
287–293. doi:10.1016/j.ajme.2017.09.001.