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Toxicity of binary mixtures of Al2O3 and ZnO

nanoparticles toward fibroblast and bronchial
epithelium cells

Jéssica Schveitzer Köerich , Diego José Nogueira , Vitor Pereira Vaz , Carmen
Simioni , Marlon Luiz Neves Da Silva , Luciane Cristina Ouriques , Denice
Schulz Vicentini & William Gerson Matias

To cite this article: Jéssica Schveitzer Köerich , Diego José Nogueira , Vitor Pereira Vaz , Carmen
Simioni , Marlon Luiz Neves Da Silva , Luciane Cristina Ouriques , Denice Schulz Vicentini &
William Gerson Matias (2020): Toxicity of binary mixtures of Al2O3 and ZnO nanoparticles toward
fibroblast and bronchial epithelium cells, Journal of Toxicology and Environmental Health, Part A,
DOI: 10.1080/15287394.2020.1761496

To link to this article: https://doi.org/10.1080/15287394.2020.1761496

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JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A
https://doi.org/10.1080/15287394.2020.1761496

Toxicity of binary mixtures of Al2O3 and ZnO nanoparticles toward fibroblast

and bronchial epithelium cells
Jéssica Schveitzer Köerich a, Diego José Nogueira a, Vitor Pereira Vaz a, Carmen Simioni a
,
Marlon Luiz Neves Da Silva a, Luciane Cristina Ouriques b, Denice Schulz Vicentini a,
and William Gerson Matias a
a
Department of Sanitary and Environmental Engineering, Federal University of Santa Catarina, Florianópolis, Brazil; bDepartment Of Cell
Biology, Embryology and Genetics, Federal University of Santa Catarina, Florianópolis, Brazil

ABSTRACT KEYWORDS
The objective of this study was to examine the cytotoxic effects of binary mixtures of Al2O3 and Binary toxicity;
ZnO NPs using mouse fibroblast cells (L929) and human bronchial epithelial cells (BEAS-2B) as nanoparticles; aluminum
biological test systems. The synergistic, additive, or antagonistic behavior of the binary mixture oxide; zinc oxide; in vitro
cytotoxicity
was also investigated. In toxicity experiments, cellular morphology, mitochondrial function (MTT
assay), apoptosis, nuclear size and shape, clonogenic assays, and damage based upon oxidative
stress parameters were assessed under control and NPs exposure conditions. Although Abbott
modeling results provided no clear evidence of the binary mixture of Al2O3 and ZnO NPs
exhibiting synergistic toxicity, some specific assays such as apoptosis, nuclear size and shape,
clonogenic assay, activities of antioxidant enzymatic enzymes catalase, superoxide dismutase, and
levels of glutathione resulted in enhanced toxicity for the mixtures with 1 and 1.75 toxic units (TU)
toward both cell types. Data demonstrated that co-presence of Al2O3 and ZnO NPs in the same
environment might lead to more realistic environmental conditions. Our findings indicate cyto-
toxicity of binary mixtures of Al2O3 and ZnO NPs produced greater effects compared to toxicity of
either individual compound.

Introduction exposed to NPs during production processes or

through ingestion of contaminated water and food
The global market for nanoparticles (NPs) has
or exposure to dermal products (Wiesner et al.
experienced growth over the past two decades in
2006). Further, Donaldson et al. (2001) reported
diverse fields including aerospace, automotive,
that ultrathin metal-containing particles present in
energy, cosmetics, electronics and optics industries,
the air exerted harmful effects on health. While
biosensors, and diagnostics (Alim et al. 2018; Angell
incorporating NPs into products improves our qual-
et al. 2018; Kaweeteerawat et al. 2017). In addition,
ity of daily life, concerns regarding the consequences
these substances are used in electrochemistry to
for human health and environment are growing
obtain catalysts, coatings, paints, pigments, compo-
(Alaraby et al. 2016; Niska et al. 2018).
sites, filtration and purification systems, textiles, and
Among the most important NPs in production
many other products (Alim et al. 2018; Kengar et al.
are those comprised of zinc oxide (ZnO) and alu-
2019; Wang and Nowack 2018). Due to unique char-
minum oxide (Al2O3) (Balasubramanyam et al.
acteristics associated with reduced particle size NPs
2009; Kermanizadeh et al. 2016). Al2O3 NP pos-
exhibit a wide range of potential applications in
sesses important properties including good ther-
nanomedicine, oral care, and restorative dentistry
mal conductivity, reduced size and shape
and as antimicrobials NPs (Callister et al. 2020;
capability, and high strength and stiffness (Kar
Duran et al. 2016; Kaweeteerawat et al. 2017;
et al. 2008). Thus, Al2O3 NPs are used in the
Kermanizadeh et al. 2016; Su et al. 2018; Zhao and
production of abrasives, refractories, ceramics,
Castranova 2011). Consequently, humans may be
electrical insulators, catalysts, paper, spark plugs,

CONTACT William Gerson Matias william.g.matias@ufsc.br Laboratory of Environmental Toxicology, Department of Sanitary and Environmental
Engineering, Federal University of Santa Catarina, Florianópolis, Brazil
Supplemental data for this article can be accessed on publisher’s website.
© 2020 Taylor & Francis
2 J. S. KÖERICH ET AL.
light bulbs, artificial gems, alloys, glass, and heat
resistant fibers (Krewski et al. 2007). ZnO NPs
have received wide attention due to their effective
performance as antimicrobial agents, pesticides,
fungicides, herbicides, fertilizer, soil enhancers,
nanobiosensors, and in water purification systems
(Cure et al. 2018; Rajput et al. 2018).
Studies on the effects of Al2O3 and ZnO NPs
individually, through in vitro assessments, were
widely reported (Chen et al. 2019; Demir, Creus,
and Marcos 2014; Hashimoto, Sasaki, and Imazato
2016; Lin et al. 2008; Mancuso et al. 2018; Ng et al.
2017; Nogueira et al. 2019). However, since the rapid
development of nanotechnology and widespread
application of various types of NPs is well established
it is conceivable that a complex scenario might be
due to co-exposure to these materials in the environ-
ment (Yu et al. 2016). It has been documented that
the combined effects of different types of NPs may
lead to additive, synergistic, or antagonistic impacts
on aquatic organisms as compared to their indivi-
dual effects (Iswarya et al. 2016; Ye et al. 2017; Yu
et al. 2016). Dávila-Grana et al. (2018) found that
incubation of a lymphocytic cell line to a mixture of
metal oxide NPs such as CeO2 with ZnO resulted in
a synergistic effect at low and medium concentra-
tions as evidenced by greater cell mortality and acti-
vation of MAP kinases and increased NFκB levels. Li
et al. (2015) noted that the toxicity of Cu NPs toward
human hepatoma (HepG2) cells was higher in
a mixture containing a nontoxic ZnO concentration.
Previously Benavides et al. (2016) examined
exposure of Carassius auratus to binary metal
mixtures and noted that Al2O3 combined with
ZnO may be more toxic than compared to each
NP individually. An increase in catalase and
superoxide dismutase activities was found in
gills and livers of fish, especially after 14 days
exposure, indicating synergistic response. The
interaction between Al2O3 and ZnO NPs may
be therefore an issue of concern and the adverse
effects of this NPs mixture need to be verified
using in vitro tests. Previous investigators
demonstrated that individually Al2O3 and ZnO
NPs produced a reduction in cell viability,
altered cell morphology, increased number of
apoptotic cells, and enhanced generation of reac-
tive oxygen species (ROS) (Lin et al. 2008;
Nogueira et al. 2019; Srikanth et al. 2015; Wang
et al. 2015). In addition, ZnO NPs appear to be
more toxic than Al2O3 NPs in different cell lines
(Jain et al. 2015; Jeng and Swanson 2006; Zhang
et al. 2011; SoSong et al. 2017; Ivask et al. 2015).
In this study, the mouse fibroblast (L929) cell line
was selected to simulate dermal exposure as the
model to investigate potential effects on skin, since
previous studies showed n that NPs enter the skin
barrier (Crosera et al. 2016, 2009). The human
bronchial epithelial (BEAS-2B) cell line was also
used in the toxicity tests since the respiratory tract
is another potential route of exposure for airborne
NPs (Ferreira, Cemlyn-Jones, and Robalo Cordeiro
2013). Therefore, the aim of this study was to deter-
mine if the actions of binary mixtures of these NPs
were additive, synergistic, or antagonistic.
Materials and methods
Chemicals
The powder sample of ZnO NPs was synthesized by
the polymeric precursor method according to
Vicentini, Smania, and Laranjeira (2010). The sample
of Al2O3 NPs (nanopowder, particle size 13 nm, 99.8%
purity), RPMI-1640 Medium, fetal bovine serum
(FBS), penicillin, L-glutamine, phosphate buffer saline
(PBS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H
-tetrazolium bromide (MTT), trypan blue, parafor-
maldehyde, crystal violet, 4-(1,1,3,3-tetramethylbu-
tyl)phenyl-polyethylene glycol, t-octylphenoxyp
olyethoxyethanol (Triton X-100), methanol, 2′,7′-
dichlorofluorescein diacetate (DCFH-DA), acridine
orange, and propidium iodide (AO/PI), 2-(4-amidi-
nophenyl)-6-indolecarbamidine dihydrochloride, 4′,
6-diamidino-2-phenylindole dihydrochloride (DAPI)
was purchased from Aldrich–Sigma (St. Louis, USA).
Trypsin–EDTA was purchased from Vitrocell
(Campinas, Brazil), reverse osmosis (RO) water
(0.05 S/cm), and ultrapure (UP) water (18.2 mΩ)
were used in the experiments.
Characterization of zinc oxide and aluminum
oxide
The samples (1 g/L) were dispersed in ultrapure
(UP) water and RPMI supplemented with 10% FBS
by sonication (10% – 50 W power in a Q500 W
Qsonica sonicator). Through transmission electron
 JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A
microscopy (TEM) (JEOL JEM-1011 microscope,
100 kV) size and morphology of particles were deter-
mined. The images from the TEM were analyzed
using software of particle analyzer, ImageJ (version
1.46 r – http://rsb.info.nih.gov/ij/). Through
NanoBrook 90Plus Zeta analyzer (Brookhaven
Instruments Corp.) were determined the effective
diameter (ED) by dynamic light scattering (DLS)
and zeta potential (ZP) by phase analysis light scat-
tering (PALS). Using an elemental analyzer
(Quantachrome Instrument, model Nova-BET
1200 E), it was possible to determine the surface
area by the Brunauer–Emmett–Teller method
(BET) (Brunauer, Emmett, and Teller 1938).
In vitro toxicological evaluation, cell lines,
culture, and maintenance
The L929 and BEAS-2B cells were cultured in
RPMI-1640 medium supplemented with 2% (v/v)
L-glutamine, 1% (v/v) penicillin, 1% (v/v) pyru-
vate, and 10% (v/v) FBS in an incubator at 37°C
with 5% CO2. Cells were seeded onto 96-well
plates at a density of 5 × 104 cells/well for the
viability assay, in 24-well plates at a density of
1 × 105 cells/well for ROS measurements and for
nuclear morphometry (NM) and acridine orange
and propidium iodide (AO/PI) staining. In the
clonogenic assay, 6-well plates were seeded at
a density of 5 × 105 cells/well and TEM culture
flasks (25 cm2) seeded at a density of 2.5 × 106
cells/ml were used to evaluate cell membrane
integrity (lipid peroxidation – LPO).
MTT assay
In assay to determine cell viability, as proposed by
Mosmann (1983), the cells were treated with dif-
ferent concentrations of Al2O3 NPs (15.62; 31.50;
62.50; 125; 250 or 500 mg/L) and ZnO NPs (2.50;
5; 10; 25; 50 or 100 mg/L). Cell viability was
determined by measuring the optical absorbance
at 570 nm on BioTek ELx800 equipment through
a microplate reader after 24 h for treatment and
3 h for incubation with MTT.
Oxidative stress biomarkers assay
Prior to oxidative stress biomarkers assays, cells
were treated for 24 h. Using an excitation wave-
length of 485 nm and emission wavelengths of
3
530 nm, intracellular ROS content was determined
as previously described (Keston and Brandt 1965),
with the aid of a microplate reader (SpectraMax
Paradigm, Molecular Devices). The activity of
CAT and SOD as well as GSH levels in cells was
determined. The antioxidant enzyme CAT was
measured based upon the decrease of the H2O2
absorbance at 240 nm (Aebi 1984). The SOD
enzyme employed XOD/cytochrome c method at
absorbance at 550 nm (McCord and Fridovich
1969). Levels of GSH were measured following
reaction with DTNB at absorbance at 412 nm
(Beutler, Duron, and Kelly 1963). For thiobarbitu-
ric acid-reactive species (TBARS) the absorbance
at 532 nm was determined by estimating malon-
dialdehyde (MDA) according to Draper and
Hadley (1990). All enzymatic/non-enzymatic
activity data were calculated as specific activity in
U/mg protein. It was used The Bradford (1976)
method was employed to determine total protein
mass (mg) in order to normalize the CAT, SOD,
GSH, and MDA results.
AO/PI staining assay
Prior to the AO/PI staining assay, cells were trea-
ted for 24 h with NPs or control. Briefly, AO/PI
staining was used to assess the number of viable,
apoptotic, and necrotic cells as previously
described (Bank 1988). Cells were then rinsed
twice with PBS and centrifuged for 4 min at
2000 g. Finally, cells were stained for 5 min with
the staining mixture (100 μg/ml AO and 100 μg/ml
PI) and then observed under a fluorescence micro-
scope (Olympus BX41) with an excitation filter of
480/430 nm. The viable, apoptotic, and necrotic
cells were quantified in a population of 200 cells.
Nuclear morphometric assay
The cells treated with NPS for 24 h were fixed
using a cold paraformaldehyde solution (4%), fol-
lowed by washing with PBS, then permeabilized
with 0.1% Triton X-100 for 10 min and incubated
with 300 nM DAPI for 30 min. The nuclear mor-
phometry was determined as previously described
(Filippi-Chiela et al. 2012), based upon photo-
graphs taken under the fluorescence microscope
(Olympus BX41), using the ImageJ software (ver-
sion 1.46 r – http://rsb.info.nih.govij/) coupled
with NII_Plugin.
4 J. S. KÖERICH ET AL.
Cell uptake of NPs
At the end of incubation with NPs for 24 h, cells
were removed, washed with PBS, and prefixed in
2.5% glutaraldehyde solution in 0.1 M sodium
cacodylate buffer and post-fixed in 1% osmium
tetroxide (pH 7.4), dehydrated in a graded series
of acetone (30%, 50%, 70%, 90%, and 100%) and
submerged in Spurr’s resin using a graded series of
acetone-Spurr’s resin. Thin sections (70 nm) were
obtained by ultramicrotome (LEICA UC7) before
observation by TEM (JEOL JEM-1011, 100 kV).
Clonogenic assay
The clonogenic assay used in this study was per-
formed according to Franken et al. (2006). Briefly,
after exposure to NPs for 24 h cells were trypsi-
nized, a specific number of cells counted (2000
cells/well) and then seeded in 6-well plates. After
10 days, the plates were fixed with methanol and
stained with 0.1% crystal violet and rinsed with
water, air-dried, photographed (Olympus SZX16)
and the colonies containing more than 50 cells
counted.
Toxicity tests with mixtures
Binary mixtures of Al2O3 NPs with ZnO NPs were
obtained following the toxic units (TU – control,
0.25, 0.5, 0.75, 1, 1.5, and 1.75) design proposed by
Sprague and Ramsay (1965). Their equitoxic pro-
portions were calculated (Cao et al. 2007) in order
to acquire as much information as possible on the
Figure 1. TEM images for (a) Al2O3 NPs, (b) ZnO NPs, and (c) binary mixture. Size frequency distribution histograms showing
Gaussian curve (n=100 particles) for (d) Al2O3 NPs and (e) ZnO NPs.
concentration–response relationships of the mix-
tures (Appendices 2 and 3). Using the Abbott
model (Abbott 1925) as modified by Colby
(1967), the expected toxicity values for the binary
nanoparticle mixtures were calculated. The mix-
ture may be characterized according to the ratio
between observed and expected toxicity as follows:
less than 1 = antagonist, equal to 1 = additive, and
greater than 1 = synergistic.
Statistical analysis
Statistical analysis was performed using software
GraphPad Prism® v6.0. ANOVA was carried out
and assumptions of normality (Shapiro-Wilk’s
test) and homogeneity of variances (Bartlett’s test)
were tested. Grubbs Test was applied to detect out-
liers. Data were analyzed by one or two-way
ANOVA followed by the Tukey’s post hoc test for
parametric data or the Kruskal–Wallis test, fol-
lowed by Dunn’s post hoc test for non-parametric
data. Results are presented as mean ± standard
deviation (SD) and differences were considered sta-
tistically significant at p < .05.
Results
Characterization of NPs
The TEM images illustrate the morphology of the
Al2O3 NPs (Figure 1a), ZnO (Figure 1B), and the
 JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 5

mixture (Figure 1c). Data demonstrate that NPs
used were spherical and slightly agglomerated.
Further, it can be clearly seen from Figure 1c
that by mixing the two types of NPs there was
a greater amount of Al2O3 NPs than ZnO NPs.
This difference occurred because the relative sur-
face areas estimated by BET equation were
682.7 m2/g for Al2O3 NPs and 7.76 m2/g for
ZnO NPs (Table 1). Based upon the histograms
exhibiting Gaussian curves for frequency distribu-
tion, the particle diameters were 11.19 ± 2.50 nm
for Al2O3 NPs and 39.34 ± 14.97 nm for ZnO NPs
(Figure 1d,e). To evaluate the dispersion stability
of the NP suspensions, the effective diameter (ED)
and surface charges of Al2O3 NPs and ZnO NPs
individually and in binary mixtures were deter-
mined in the UP water and RPMI medium con-
taining 10% FBS (Table 1). The ED results for the
Al2O3 NPs and ZnO NPs individually and in the
binary mixtures dispersed in ultrapure water (UP)
or RPMI medium confirmed the presence of par-
ticle agglomerates greater than 100 nm in dia-
meter. In all cases, the ED was smaller when NPs
were dissolved in UP water compared to RPMI
medium. At the same time, the zeta potential
(ZP) values for NPs, determined individually and
in the binary mixture (Al2O3/ZnO NPs) remained
stable in UP water but displayed only numerical
decreases in RPMI medium over time.
Viability assay for NPs individually and in binary
mixtures
Using the MTT assay to measure cell viabilityAl2O3
and ZnO NPs individually produced a concentration-
dependent cytotoxic effect with concentration–
response curves presented in Appendix 1. After 24 h
exposure for the two types of cells ZnO NPs were
found to be more toxic with lower IC50 values than
Al2O3 NPs (Appendix 1). The L929 cells were more
sensitive than BEAS-2B cells with IC50 values of
approximately 280.4 and 42.9 µg/ml for Al2O3 NPs
and ZnO NPs, respectively. The IC50 values for BEAS-
2B cells were approximately 378.1 and 75.2 µg/ml for
Al2O3 NPs and ZnO NPs, respectively.
Considering the potential simultaneous occur-
rence of Al2O3+ ZnO NPs and based upon the
IC50 values obtained for the individual NPs, toxi-
city tests were conducted for binary mixtures with
concentrations of NPs corresponding to the toxic
units (TU) (Tables 1 and 2). Consequently, the
equipotential concentration of each type of NP
was obtained to prepare the binary mixtures, as
presented in Figure 2 and Table 2. The Ratio of

Table 1. Data for Al2O3 and ZnO NPs characterized in UP water and RPMI medium (1 g/L).
NPs Size±SD (nm) Surface area (m2/g) Diluent ED ± SD (nm) PZ ± SD (mV) pH
Al2O3 11.19 ± 2.50 682.7 UP water 187.05 ± 0.80 −51.13 ± 0.57 7.65
RPMI 226.32 ± 0.50 5.88 ± 1.34 8.10
ZnO −51.13 ± 0.57 7.76a UP water 276.35 ± 0.55 −34.18 ± 0.10 7.52
RPMI 887.56 ± 3.30 6.77 ± 2.51 8.09
Al2O3+ ZnO - - UP water 240.33 ± 3.05 −32.11 ± 0.36 7.43
- - RPMI 173.82 ± 0.55 7.87 ± 5.15 9.74
a
The surface area of ZnO NPs used in this study was reported previously described in the literature (Melegari et al. 2019).

Table 2. Abbott modeling representing the type of interaction between the Al2O3 and ZnO NPs in a binary mixture.
Cells TU Observed inhibition (%) Expected inhibition (%) Ratio of Inhibition (RI) Relation
L929 0.25 5.3 ± 3.80 7.25 ± 7.56 0.78 ± 0.82 Additive
0.50 12.25 ± 2.89 29.35 ± 16.05 0.46 ± 0.15 Additive
1 16.08 ± 5.12 65.40 ± 7.63 0.24 ± 0.04 Additive
1.50 25.70 ± 4.95 73.20 ± 2.54 0.35 ± 0.07* Antagonistic
1.75 34.29 ± 4.11 72.35 ± 2.89 0.47 ± 0.03* Antagonistic
2 43.50 ± 0.99 76.30 ± 3.67 0.57 ± 0.01* Antagonistic
BEAS-2B 0.25 21.9 ± 0.14 29.70 ± 3.81 0.74 ± 0.10 Additive
0.50 22.61 ± 1.69 28.15 ± 0.49 0.80 ± 0.04 Additive
1 39.46 ± 3.89 46.15 ± 1.48 0.85 ± 0.05 Additive
1.50 64.22 ± 2.85 67.50 ± 3.53 0.95 ± 0.09 Additive
1.75 64.83 ± 5.12 66.35 ± 3.74 0.98 ± 0.12 Additive
2 71.44 ± 3.91 76.60 ± 3.25 0.93 ± 0.01 Additive
*represent significative difference (p < 0.05) between toxicity induced by the binary mixture and expected toxicity.
6 J. S. KÖERICH ET AL.

Figure 2. Inhibitory effects of Al2O3 NPs, ZnO NPs, and a binary mixture on cells, measured by the MTT assay with L929 (a) and BEAS-
2B (b) cell lines.

Inhibition (RI) was calculated for the binary mix-
tures using both cell types. The RI values were less
than 1 and not significant from 1 in both cell types
(Table 2) indicating an additive effect produced by
the binary mixtures at all TU conditions for BEAS-
2B cells and for 0.25 − 1 TU for L929 cells.
However, for TU from 1.5 to 2 for L929 cells,
there was a significant antagonistic response.
Oxidative stress biomarkers
The ability of the mixture to induce oxidative stress
was determined by measuring activities of CAT and
SOD as well as levels of ROS, GSH, and MDA in L929
and BEAS-2B cells incubated for 24 h (Figure 3).
A significant increase in ROS generation was noted
at the toxic units (TU) group 1 and 1.75 for both cells

Figure 3. Oxidative stress biomarkers (a) ROS, (b) CAT, (c) SOD, (d) GSH, and (e) MDA for L929 and BEAS-2B cells exposed to the
mixture (Al2O3+ZnO NPs) for 24 h. Values are mean ± SD (n=2). Different letters indicate significant differences between toxic units
for each cell type according to 2-way ANOVA results (p < 0.05).
 JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 7

compared to control and 0.25 TU (Figure 3a). Further,
the mixture induced a significant elevation in CAT
activity and reduction SOD activity, particularly in the
case of 1.75 TU in BEAS-2B cells (Figure 3b,c). In
contrast, there was no marked difference in the activ-
ities of CAT and SOD in L929 cells. GSH levels were
significantly lower in both cell types from 1 TU for
BEAS-2B cells and 1.75 TU for L929 cells (Figure 3d).
Notably, there was a rise in lipoperoxidation (LPO) for
1 and 1.75 TU groups in BEAS-2B cells and 1.75 TU in
L929 cells attributed to enhanced levels of MDA, the
by-product of LPO (Figure 3e).
Apoptosis and nuclear morphometry
Figure 4 illustrates the morphological changes char-
acteristic of viable cells (early apoptotic cells with
irregularly structured green nuclei and chromatin
condensation visible as bright green patches, along
with fragments or apoptotic bodies), apoptotic cells
(late apoptotic cells with orange to red nuclei with
highly fragmented chromatin), and necrotic cells (uni-
formly orange to red nuclei with organized structure,
ascribable to necrotic cells).
As shown in Figures 4a,b, in the case of the
BEAS-2B cells there was a significant rise in apop-
totic cell number and diminished viable cells for 1
and 1.75 TU compared to control. With respect to
L929 cells, a significant elevation in apoptotic cell
number and a significant decrease in viable cells
was found at 0.25 TU, indicating that this cell type
may be more sensitive than BEAS-2B.
Based on the ImageJ results (Figure 4c,d), the
cells showed a higher nuclear irregularity index
and more apoptotic cells for 1 and 1.75 TU,
which is consistent with the results of the AO/IP

Figure 4. Induction of apoptosis in L929 and BEAS-2B cells exposed to the mixture (Al2O3 + ZnO NPs) for 24 h: (a) Percentage of
viable, apoptotic, and necrotic cells for L929 and (b) percentage of viable, apoptotic, and necrotic cells for BEAS-2B. Morphometric
analysis of nuclei of L929 and BEAS-2B cells exposed to the mixture for 24 h: (c) percentage of nuclei normal, apoptotic, and irregular
for L929 and (d) percentage of normal, apoptotic, and irregular nuclei for BEAS-2B. Scale bar, 200 µm. Values are mean ± SD (n=2).
Different letters indicate significant differences among toxic units of each cell according to 2-way ANOVA results (p<0.05).
8 J. S. KÖERICH ET AL.

staining assay. Once again, the L929 cells appeared
to be more sensitive than BEAS-2B cells, due to
the higher number of apoptotic nuclei and lower
number of normal nuclei when compared to
BEAS-2B, this confirms cell death occurred at 1
and 1.75 TU.
Clonogenic assay
(Figure 6b,c,i,j) associated with invaginations of
the cell surface.
After 24 h treatment, the TEM images showed
the NP mixture within the endocytic vesicles in
different stages of maturation (Figure 6d,k) and
large lysosomes with undigested metallic material.
The non-dissolution of NPs led to formation of
many residual bodies containing partially undi-
gested material to be excreted (Figure 6c,e,f,h-L).

The colony-forming ability was used to determine

the long-term cytotoxic potential of the mixture
(Al2O3+ ZnO NPs) in L929 and BEAS-2B cells
(Figure 5). A significant inhibition of colonies
was observed in L929 cells following a 24 h incu-
bation to 1 or 1.75 TU, equating to a 61.5% and
98.5%, respectively, reduction in survival. For
BEAS-2B cells, only exposure to 1.75 TU was
found to significantly diminish colonies, to
a 90.8% reduction in survival.
Cell uptake of NPs
TEM was used to determine the distribution and
localization of the mixture in L929 and BEAS-2B
cells (Figure 6). The results obtained illustrate that
cells are able to internalize the mixture since
aggregates of various sizes interacted with the cell
membrane and were internalized by the cells
Discussion
The study reported here is the first to assess the
characterization of the binary mixture of Al2O3
and ZnO NP and their respective effect on cells
related to human exposure. In the mixture, there
was a greater agglomeration of Al2O3 than ZnO
due to its larger estimated surface area and conse-
quently its smaller diameter. In all cases, the EDs
were smaller when NPs were dissolved in UP
water compared to the RPMI medium since in
water there are more free ions on the NP surface
and, consequently, NPs presented greater stability
and smaller agglomerations (Melegari et al. 2019;
Vicentini et al. 2017). However, some components
such as serum albumin, amino acids, and ionic
salts present in RPMI medium may influence ED
and stability of the NPs, resulting in the formation
of aggregates that allow them to disperse in larger

Figure 5. Clonogenic assay shows the long-term effects of the mixture (Al2O3 + ZnO NPs) on L929 and BEAS-2B cells. Representative
images of the colony formation (containing ?50 cells) after treatment with different TUs of the mixture for 10 d. (a–b) Graph shows
results for the percentage of colony forming units. Values are mean ± SEM (n=3). Different letters indicate significant differences in
the same groups after the exposure according to one-way ANOVA (p<0.05).
 JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 9

Figure 6. TEM cross-section images of randomly selected L929 and BEAS-2B cells after exposure to the mixture (Al2O3 + ZnO NPs) for
24 h, showing the location of the NPs. The ultrastructural morphology of plasma membrane invaginations/protrusions (d and k)
suggests clathrin-mediated endocytosis. (b, c, i and l) The NP aggregates can be seen inside vesicles (yellow arrow). Internalized NPs
took the same intracellular trafficking route in L929 and BEAS-2B cells: Endosomes with the mixture can be found in different stages
of maturation, (c and j) early endosome (EE), (e, h, and j) late endosome (LE) and (f and l) lysosome (l). (g) shows a more general
overview of control cell (untreated).

sizes, decreasing the exposed surface area
(Maiorano et al. 2010; Nogueira et al. 2019; Wells
et al. 2012). At the same time, the zeta potential
(ZP) values remained virtually unchanged indicat-
ing that there was no marked alteration in the
physicochemical properties of the individual NPs
and their binary mixtures.
Several investigators demonstrated that cyto-
toxicity is independent of the size of NPs (Lin
et al. 2008; Yuan et al. 2010). Therefore, it is
essential to assess the toxicity of each specific
material. After 24 h incubation, for the two types
of cells ZnO NPs were found to be induced greater
toxicity. Previously, data reported that Al2O3 NPs
10 J. S. KÖERICH ET AL.
produced low toxicity toward mammalian cells
(Alshatwi et al. 2013; Nogueira et al. 2019;
Oesterling et al. 2008; Radziun et al. 2011). In
contrast, several in vitro studies demonstrated
that ZnO NPs exhibited significant cytotoxic
effects, probably due to their solubility, leading to
an increase in the intracellular levels of Zn2+
(Pandurangan and Kim 2015; Zhang et al. 2018).
With respect to the binary mixture, an antagonis-
tic effect was observed for L929 cell for the 1.5 to 2
TU suggesting interaction between Al2O3 and
ZnO NPs diminished the cytotoxic potency of
the mixture.
Although the mechanisms underlying NPs-
mediated toxicity is not yet clear, one of the pos-
sible reasons for the high level of stress exerted by
ZnO NPs, rather than the interaction of the NPs
within the mixture, might be adsorption of Zn2+
by the Al2O3 NPs (Sen and Sarzali 2008). Al2O3
NPs possess a relatively large surface area (88-fold
higher than of ZnO NPs), thereby providing sig-
nificant potential for sorption of various metal
ions, inorganic anions, and organic ligands
(Mahdavi, Jalali, and Afkhami 2013; Sen and
Sarzali 2008). Previously Hua, Peijnenburg, and
Vijver (2016) noted that the presence of TiO2
NPs decreased Zn2+ toxicity toward zebrafish
embryos. Dalai et al. (2014) showed that the pre-
sence of Al2O3 NPs affected Cr(VI) toxicity, since
the absorption of Cr(VI) by Al2O3 NPs was found
to reduce Cr(VI) mediated toxicity toward the
freshwater microalgae Scenedesmus obliquus.
Therefore, the addition of Al2O3 NPs may affect
the toxicity of ZnO NPs, by decreasing the toxicity
of ZnO NPs via a protective effect at higher con-
centrations (Benavides et al. 2016; Dalai et al.
2014; Dávila-Grana et al. 2018).
The MTT assay was conducted to evaluate the
cytotoxicity of the mixture (Al2O3+ ZnO NPs)
compared with each type of NP alone. However,
no synergistic effect was observed, probably
because the MTT assay is a standard metabolic
assay based upon a simple colorimetric reaction
but limited by possible interference of the absor-
bance of particles (Almutary and Sanderson 2016).
For this reason, the ability of the mixture to induce
oxidative stress was assessed by measuring levels of
ROS, GSH, and MDA as well as activities of CAT
and SOD in L929 and BEAS-2B cells exposed for
24 h. An increase in the ROS generation was noted
at TU 1 and 1.75 for both cell types. When the
generation of ROS exceeds the ability of the cell to
neutralize the effects of the radicals, the accumula-
tion of pro-oxidants occurs in the cell, leading to
a state of oxidative stress (Wu et al. 2014). As the
oxidative stress in the cell rises, different biological
outcomes occur, such as a change in the activity of
antioxidant enzymes CAT and SOD and levels of
GSH (Dayem et al. 2017). The mixture induced
a significant elevation in CAT activity levels
accompanied by a decrease SOD activity and
GSH levels in BEAS-2B cells. In contrast, there
was no marked difference in the activity of CAT
and SOD in the L929 cells; however, the levels of
GSH were significantly reduced in L929 cells. The
oxidation of GSH indicates the inability of cells to
scavenge ROS generated in response to the mix-
ture, leading to the development of oxidative stress
in these cells, which may lead to oxidative damage
of the lipids (Li, Xia, and Nel 2008). In agreement
our findings showed an increase in LPO in both
cells, as evidenced by elevated MDA levels, the by-
product of LPO, indicative of damage the cell
membranes (Birben et al. 2012). In several other
studies, LPO due to ROS generation was found to
be associated with exposure to Al2O3 and ZnO
NPs, resulting in cellular toxicity (Alshatwi et al.
2013; Daoud Ali, Alkahtani, and Alarifi 2015; Król
et al. 2017; Li, Zhou, and Fan 2016; Nogueira et al.
2019; Roy, Das, and Dwivedi 2015; Srikanth et al.
2015).
Wang et al. (2015) indicated that increased ROS
levels may induce apoptosis. Our findings demon-
strated a significant increase in apoptotic cell
numbers concomitant with a marked reduction
in viable cells in both cell types. Various investi-
gators noted that cellular apoptosis was initiated
by Al2O3 or ZnO NPs alone (Akhtar et al. 2012;
Alshatwi et al. 2013; Nogueira et al. 2019; Wang
et al. 2015). In both types of cells, there was an
elevation in ROS and MDA levels accompanied by
a fall in SOD activity and GSH levels, which may
have contributed to the observed apoptosis.
Based upon the ImageJ results, the cells exhibited
a higher nuclear irregularity index and more apop-
totic cells after exposure to the binary mixture. The
nuclear irregularity index, besides assessing apopto-
sis, is an indicator of defective activation or
 JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A
inactivation of the cell cycle checkpoint signaling
processes (Filippi-Chiela et al. 2012). The L929
cells appeared to be more sensitive than BEAS-2B
cells, due to the higher number of apoptotic nuclei,
lower number of normal nuclei and decrease in
viable cells for a low UT compared to BEAS-2B.
This confirms the presence of cell death for 1 and
1.75 TU, as apoptotic cells showed nuclear conden-
sation and fragmentation (Filippi-Chiela et al. 2012).
Colony formation was inhibited in L929 cells
exposed at 1 and 1.75 TU and for BEAS-2B calls at
1.75 TU possibly due to enhanced intracellular uptake
of the NPs through the endocytosis process. Taccola
et al. (2011) reported that endocytosis may occur
when ZnO NPs came into contact with the growth
medium proteins that adhere to their surfaces. These
proteins are then identified by cell surface receptors
(Boucrot and Kirchhausen 2007). The interaction of
NPs with cellular proteins is dependent upon cell
cycle phases. Patel et al. (2016) found higher ZnO
uptake occurs in the G2/M cell phase and NPs induce
cell cycle arrest by activating the ROS. For Al2O3, the
reduction of cells in the G2/M cell phase might induce
the cell cycle to stop in the G0-G1 cell phases
(Periasamy, Athinarayanan, and Alshatwi 2016),
where cell growth occurs. According to the prolifera-
tion results, the cells were no longer able to undergo
normal cell division, which indicates that the cell cycle
was affected following treatment with the NPs.
One of the most probable causes for the observed
decrease in proliferation of L929 and BEAS-2B cells is
the high uptake of aggregates of NPs of various sizes
that was evident in the TEM results. The dynamics of
uptake of Al2O3 NPs and ZnO NPs by cells was
comparable to that previously observed for various
cell lines (Jeng and Swanson 2006; Kim, Baek, and
Choi 2010; Nogueira et al. 2019; Roy, Das, and
Dwivedi 2015; Zhang et al. 2013), suggesting that the
uptake process may be induced by different mechan-
isms such as phagocytosis, pinocytosis, macropinocyto-
sis, or receptor-mediated endocytosis. In addition, most
uptake processes involve lysosomes, where a variety of
macromolecules are degraded (Böhme et al. 2014).
However, the mixture that accumulates in endosomes
and lysosomes may undergo dissolution in lysosomes
and initiate lysosomal destabilization, which enables
NPs to enter the cytosol (Stern, Adiseshaiah, and
Crist 2012; Zhang et al. 2013). Finally, the TEM images
displayed that the cytotoxic effect may be due to
11
propagation of inflammation or induction of oxidative
stress in the cells. As shown by Il et al. (2015), who
examined different particle shapes and sizes of Al2O3
NPs, cytotoxicity occurred via a mechanism involving
lysosomal destabilization and generation of ROS, which
leads to mitochondrial perturbation resulting in cellular
death by apoptosis (Fink and Cookson 2005).
Conclusions
The mixture at 1 and 1.75 toxic units (TU) was
more toxic toward both types of cells, even though
it presented antagonism at 1.75 TU toward the
L929 cell line. Antagonism may be attributed to
the protective effect of Al2O3, due to its large sur-
face area and ion sorption potential, reducing the
toxicity of ZnO. In addition, the L929 cells were
found to be more sensitive than BEAS-2B cells due
to the higher number of apoptotic cells, larger
apoptotic nuclei, and greater colony growth inhi-
bition predominantly at 1 and 1.75 TU. In sum-
mary, the results reported herein highlight the
need to better understand how mixtures of differ-
ent NPs affect specific cells in distinct ways.
Acknowledgments
We would like to thank the staff of the Central Laboratory of
Electron Microscopy (LCME) and Multiuser Laboratory of Biology
Studies (LAMEB) at the Federal University of Santa Catarina,
Florianópolis, SC, Brazil for assistance with microscopy analysis.
Declaration of interest
The authors report no conflicts of interest. The authors are
solely responsible for the content and writing of this article.
Funding
This work was funded by the National Council for Scientific
and Technological Development (CNPq) and Coordination
of Improvement of Higher Education Personnel (CAPES).
ORCID
Jéssica Schveitzer Köerich http://orcid.org/0000-0002-
8862-1105
Diego José Nogueira http://orcid.org/0000-0001-5831-
4043
Vitor Pereira Vaz http://orcid.org/0000-0003-3689-2656
Carmen Simioni http://orcid.org/0000-0001-5629-9361
12 J. S. KÖERICH ET AL.
Marlon Luiz Neves Da Silva http://orcid.org/0000-0002-
6602-2910
Luciane Cristina Ouriques http://orcid.org/0000-0003-
2964-6772
Denice Schulz Vicentini http://orcid.org/0000-0001-7080-
9911
William Gerson Matias http://orcid.org/0000-0002-2386-
0578
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