Article

Toxicology and Industrial Health

2021, Vol. 37(12) 752–762
Accelerated apoptosis, oxidative stress, and © The Author(s) 2021
Article reuse guidelines:
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cholinergic inflammation in blood of DOI: 10.1177/07482337211053164
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metalworkers

Gabriela Bonfanti-Azzolin1,2,3, Camila P Capelleti1, Kelly S Rodrigues1,

Suellen Da R Abdallah1, Ana P Frielink1, Giovana Rupphental1, Bianca B Kuhn1,
Roberta Cattaneo2,3, Patricia Wolkmer4, Josiane W Bortolotto1 and
Mariana M Parisi1,2,3 

Abstract
Metalworkers are exposed to numerous chemicals in their workplace environment, such as solvents, heavy
metals, and metalworking fluids, that have a negative impact on their health. Furthermore, there is an increase
in the prevalence of chronic diseases among metalworkers; however, the molecular mechanisms involved in
this increased predisposition to chronic diseases are unclear. Considering that occupational exposure
represents a potential risk for metalworkers, the aim of this study was to measure biomarkers of oxidative
stress, inflammation, and cytotoxicity in the peripheral blood of metalworkers from Southern Brazil. The
study included 40 metalworkers and 20 individuals who did not perform activities with any recognized
exposure to chemical substances, such as those working in administration, commerce, and education, as
controls. Cellular and molecular biomarkers as leukocyte viability, intracellular production of reactive species,
mitochondrial mass and membrane potential and plasma lipid peroxidation, sulfhydryl groups, total anti-
oxidant capacity, and butyrylcholinesterase activity were evaluated in the blood of metalworkers and controls.
Metalworkers were found to have higher rates of apoptosis, increased production of reactive species, and
increased mitochondrial potential and mass in leukocytes associated with decreased antioxidant defenses and
increased activity of the butyrylcholinesterase enzyme in their plasma. It can be concluded that cytotoxicity,
oxidative stress, and inflammation are involved in the multiplicity of health outcomes related to chemical
exposure in the metalworking industry.

Keywords
apoptosis, biomarkers, leukocytes, workplace, oxidative

Received 13 June 2021; Revised 11 September 2021; Accepted 24 September 2021

Introduction
The metalworking industry is responsible for trans-
forming metals into products, such as machinery,
equipment, vehicles, and transportation materials and
supplying these to several sectors of the economy,
including agribusiness (Vöth et al., 2019). Brazil is a
strategic point in the production of agricultural im-
plements. Its metal-mechanic sector extends throughout
the South and Southeast regions of the country and
represents approximately one-third of the industrial
segment and national industrial gross domestic product
(Goncalves et al., 2018).
1
Group of Integral Attention to Health, Center for Health and
Rural Sciences, University of Cruz Alta, Brazil
2
Postgraduate Program in Integral Attention to Health (PPGAIS),
University of Cruz Alta, Brazil
3
Interdisciplinary Health Research Group, Center for Health and
Rural Sciences, University of Cruz Alta, Brazil
4
Group of Animal Health, Centre for Health and Rural Sciences,
University of Cruz Alta, Brazil
Corresponding author:
Corresponding author: Mariana Migliorini Parisi, Center for Health
and Rural Sciences, University of Cruz Alta, Rodovia Jacob Della
Mea Km 3.6, Cruz Alta, 98005-972, Brazil.
Email: mariana_parisi@yahoo.com.br
Bonfanti-Azzolin et al.
Production processes in the metalworking indus-
tries involve sectors such as painting, welding, casting,
and machining (Lillienberg et al., 2008). In this
context, metalworkers are exposed to a wide range of
compounds, such as heavy metals, solvents, and
metalworking fluids (MWF), that can negatively affect
their health (Fornander et al., 2013; Hopf et al., 2019).
Metals such as lead, cadmium, arsenic, and manganese
are commonly related to metallurgy and are recognized
as agents that can damage many target organs in the
human body (Chen et al., 2019). In addition, a study
conducted by Hamzah et al. (2016) showed that cobalt
and chromium levels were above the allowed exposure
limit and that they were related to decreased lung
function in workers exposed.
The MWFs are complex mixtures of eight or more
chemicals (Al-Humadi et al., 2000; Fornander et al., 2013)
that generate aerosols which can remain in a suspension
for several hours and reach the metalworkers’ breathing
zone (Lillienberg et al., 2008; Hopf et al., 2019). In ad-
dition, these aerosols may contain volatile organochlorine
compounds or aromatic hydrocarbons such as toluene and
xylene, which are used for cleaning or as solvents (Jabbar,
2018). Acute and chronic exposure to aerosols can cause
coughs, rhinitis, and wheezing (Fornander et al., 2013),
besides being related to the appearance of health problems
and chronic pathologies such as metabolic syndromes
(Jeong, 2018), diabetes (Yang et al., 2015), neoplasms
(mainly rectal, pharynx, esophagus, and skin) (Malloy
et al., 2007; Park, 2018), chronic bronchitis and rhinitis
(Lillienberg et al., 2010), and chronic obstructive pul-
monary disease and asthma (Park, 2019).
Occupational exposure to chemicals in the workplace
may be related to increased apoptosis in leukocytes
(Lopez-Vanegas et al., 2020). The main mechanisms
proposed to explain the cytotoxicity caused by exposure
to heavy metals and MWFs involve the induction of
oxidative stress and inflammation (Huang et al., 2017;
Chen et al., 2019). Cellular apoptosis can be initiated by
extracellular signals that activate death receptors, or by
intracellular signals mediated by mitochondria. The in-
crease in reactive oxygen species or decrease in antiox-
idant defenses caused by exposure to xenobiotics may be
the inducer of alterations in the mitochondria that lead to
cell death (Circu and Aw, 2010). In this way, dermal
exposure to MWFs has been reported to generate oxi-
dative stress in rats (Al-Humadi et al., 2000; Shvedova
et al., 2002), and chronic respiratory exposure to high
concentrations of MWFs has been reported to result in
lung inflammation in rats and mice (Hopf et al., 2019),
which can be the trigger for the induction of apoptosis.
753
Despite these evidences, there are no previous studies
describing oxidative citotoxicity and inflammatoy
changes in the peripheral blood of metalworkers.
In fact, oxidative stress has been significantly as-
sociated with systemic chronic low-grade inflamma-
tion, mitochondrial dysfunction, and apoptosis
(Hussain et al., 2016; Leon-Pedroza et al., 2015),
which are factors associated with the development
and/or progression of several chronic pathologies
(Kudryavtseva et al., 2016; Bhatti et al., 2017; Jha
et al., 2017; Lejri et al., 2019; Vona et al., 2019; Luc
et al., 2019). Mitochondrial dysfunction increases the
production of reactive oxygen species, which can
induce the oxidation of biomolecules, leading to loss
of homeostasis and cell death (Bhatti et al., 2017). In
addition, oxidative stress can induce inflammation
through several pathways, mainly by activating nu-
clear factor kappa B (Mishra et al., 2018). Therefore,
our hypothesis is that the detection of alterations in
cytotoxic, oxidative and inflammatory biomarkers in
metalworkers could partially explain the increased
prevalence of chronic diseases in this population.
Considering that occupational exposure represents a
potential risk for metalworkers, we established as a
research question “Do metalworkers have higher rates
of cytotoxicity, oxidative stress, and inflammation in
peripheral blood compared to unexposed controls?”
Thus, in this study, we describe apoptosis, mito-
chondrial dysfunction, production of reactive species,
antioxidant defenses, lipid peroxidation, and butyr-
ylcholinesterase enzyme activity as biomarkers of
cellular and molecular alterations in metalworkers.
Materials and methods
Subjects
This was a cross-sectional analytical study that in-
cluded 60 participants. In order to recruit metal-
workers, researchers contacted three metalworking
industries that produce agricultural implements in the
Southern Region of Brazil, two of which agreed to
participate in the study. Metalworkers aged between 18
and 60 years and with at least 12 months of activity in
metalworking environment in painting, welding,
casting, and machining sectors of the industry were
recruited from these two companies on the day of their
annual periodic examination. A questionnaire cover-
ing demographic and work variables of individuals,
such as gender, age, weight, height, smoking habits,
presence of diseases, use of medications, time, and
sector of work was applied to included metalworkers.
754
From the assessment of the questionnaire, participants
who reported smoking, having a chronic disease or
taking any medication in the month prior to blood
collection were excluded from the sample. Therefore,
the exposed group was composed of 40 metalworkers,
aged between 18 and 60 years, with at least 12 months
of activity in metalwoking indutry in painting,
welding, casting, and machining sectors.
To compose the control group, we recruited from
the community 20 individuals who did not perform
any activity with recognized exposure to chemical
substances, such as those working in administration,
commerce, and education. The control group was
similar in terms of sex, age, and body mass index to the
exposed group and none of the controls reported being
smokers, having chronic illnesses, having had acute
illnesses, or having taken any medication in the month
before blood collection.
This protocol was approved by the Institutional
Review Board (No. 2,442,325), and all participants
signed an informed consent document.
Biological samples
Peripheral blood (20 mL) was drawn from each subject
by venipuncture into EDTA-tubes. Whole blood was
used to determine the total leukocyte count. For the
other experiments, blood was immediately centrifuged
at 400 xg for 15 min at 4°C. Plasma was collected,
aliquoted, and stored at
80°C until biochemical
analysis. The leukocyte layer was transferred to an-
other tube, and the contaminating erythrocytes were
hemolyzed with red blood cell lysis buffer (Roche,
USA), according to the manufacturer’s protocol. En-
riched leukocytes were immediately used for flow
cytometry analysis.
Leukocyte count
Leukocyte counts were performed in whole blood
using a Sysmex KX-21N Automated Hematology
Analyzer, and additional blood smears were prepared
to determine differential leukocyte numbers by mi-
croscopy, according to the method described by
Failace and Fernandes, 2015.
Leukocyte viability assay
Leukocyte cell death was analyzed using the Annexin
Vand propidium iodide assay (BD Biosciences, USA),
which detects the simultaneous occurrence of necrosis
Toxicology and Industrial Health 37(12)
and apoptosis. Annexin-V/PI staining was performed
according to the manufacturer’s instructions. Twenty-
thousand events were acquired and analyzed using a
BD AccuriÔ C6 Plus flow cytometer (BD Biosci-
ences, USA). Data were expressed as percentages of
viable, apoptotic, and necrotic cells.
Leukocyte intracellular production of
reactive species
Intracellular production of reactive species in leuko-
cytes was analyzed using 20,70-dichlorodihydro-
fluorescein diacetate (DCFH-DA) assay. Leukocytes
(1 × 106) were incubated with 10 μM DCFH-DA in
phosphate buffered saline (PBS) for 20 min at 37°C in
the dark. After incubation, the leukocytes were washed
and resuspended in 300 μL PBS. Twenty-thousand
events were acquired and analyzed using a BD Ac-
curiÔ C6 Plus flow cytometer (BD Biosciences,
USA). Data were expressed as the median intensity of
green fluorescence (MFI) in the total leukocyte
population.
Leukocyte mitochondrial mass and
membrane potential
Mitochondrial mass and membrane potential were
assessed using MitoTrackerTM Green FM and Mi-
toTrackerTM Red FM probes (Invitrogen®). Leuko-
cytes (1 × 106) were incubated with 100 nM of each
probe for 20 min at 37°C in the dark. After incubation,
the leukocytes were washed and resuspended in
300 μL PBS. Twenty-thousand events were acquired
and analyzed using a BD AccuriÔ C6 Plus flow cy-
tometer (BD Biosciences, USA). Data were expressed
as median intensity of green (MTG) and red (MTR)
fluorescence (MFI) in the total leukocyte population.
Additionally, the MTR/MTG ratio was calculated to
estimate the mitochondrial membrane potential in
relation to mitochondrial mass.
Plasma lipid peroxidation
Lipid peroxidation levels in plasma samples were
assessed by thiobarbituric acid reactive substances
(TBARS), according to the method described by
Lapenna et al. (2001). The samples were mixed with
distilated water, 1% phosphoric acid, 0.6% thio-
barbituric acid (TBA), and incubated for 60 min at
95oC. The reaction product was determined at 532 nm
using spectrophotometry. The results were calculated
Bonfanti-Azzolin et al.
using a standard curve with different concentrations of
malondialdehyde (MDA) and expressed as nmol
MDA/mL plasma (Lapenna et al., 2001).
Plasma sulfhydryl groups Results
Thiol groups were assessed in plasma by the method of Demographic data and leukocyte count
Boyne and Ellman (1972), which consists of the re-
duction of 5.5’-dithio(bis-nithrobenzoic) acid (DTNB)
in pH 7.0. The absorbance was determined at 412 nm
using spectrophotometry. The results were calculated
using a standard curve with different concentrations of
glutathione (GSH) and expressed as μM GSH/mL
plasma.
Plasma total antioxidant capacity
The total antioxidant capacity in plasma samples was
assessed by the iron-reducing capacity (FRAP), ac-
cording to Benzie and Strain (1996). The samples were
mixed with FRAP reagent (containing acetate buffer,
pH = 3.6; tripyridyl-s-triazine; FeCl3.6H2O, and
water) and incubated for 30 min at 37oC. The ab-
sorbance was determined at 593 nm using spectro-
photometry. The results were calculated using a
standard curve with different concentrations of FeSO4
and expressed as mM Fe II/mL of plasma.
Plasma butyrylcholinesterase activity
Butyrylcholinesterase (BChE) activity in plasma sam-
ples was assessed by measuring butyrylcholine hy-
drolysis in reaction with 50,50-dithio (bis-nitrobenzoic
acid), according to Ellman et al. (1961). The reaction of
BChE was initiated by adding 0.8 mM butyrylth-
iocholine iodide (BuSCh), and the absorbance was
determined at 405 nm. The results are expressed as
μmol BuSCh/h/g of protein. Total protein was deter-
mined using a Total Protein Kit (Labtest, Brazil), ac-
cording to the manufacturer’s instructions.
Statistics
The non-categorical data were presented as mean ±
standard deviation or median and interquartile range,
and the categorical data were presented as absolute (n)
and relative frequency (%). The Shapiro–Wilk test was
performed to assess the data distribution. Parametric
data were compared using a Student’s t-test, and
nonparametric data were compared using Mann–
Whitney’s U test. The correlation between variables
755
was determined using Pearson’s correlation test. A
significance level of 5% was adopted (*p < 0.05, **p <
0.01, ***p < 0.001).
Demographic data and leukocyte counts are shown in
Table 1. No statistically significant differences in age,
sex, and body mass index were observed between the
controls (n = 20) and metalworkers (n = 40). The mean
working time in the metalworking industry was 3.22 ±
2.10 years, and all metalworkers reported using per-
sonal protective equipment (PPE). Segmented neu-
trophil count was significantly reduced, and band
neutrophil, monocyte, and lymphocyte counts were
significantly higher in metalworkers than in controls.
Increased apoptosis in leukocytes
from metalworkers
The percentage of cells in early and late apoptosis were
significantly higher in leukocytes from metalworkers
than in controls (p < 0.01 and p < 0.001, respectively),
leading to a significant reduction in cell viability (p <
0.001) (Figure 1). No differences were observed in the
percentage of necrotic cells (data not shown). Addi-
tionally, a moderate positive correlation was detected
between the percentage of apoptotic cells and working
time in the metalworking industry (r = 0.5236, p <
0.05), and a moderate inverse correlation was detected
between the percentage of viable cells and working
time in the metalworking industry (r =
0.5040, p <
0.05).
Increased mitochondrial mass and membrane
potential in blood from metalworkers
Mitochondrial mass (MTG) and membrane potential
(MTR) were significantly higher in leukocytes from
metalworkers than in controls (p < 0.05 and p < 0.001;
respectively; Figure 2). The MTR/MTG ratio showed
that the increase in mitochondrial membrane potential
occurred despite the increase in mitochondrial mass in
leukocytes from metalworkers (p < 0.001; Figure 2).
Oxidative stress in blood from metalworkers
Intracellular production of reactive species was sig-
nificantly higher (p < 0.01); Figure 3(a) and (b)) in
756 Toxicology and Industrial Health 37(12)

Table 1. Demographic data and leukocyte count.

Controls Metalworkers P value
Age (years), mean ± SD 33.35 ± 11.46 30.53 ± 10.63 n.s.
Sex, n (%)
Male 14 (70) 31 (77.5) n.s.
Female 6 (30) 9 (22.5)
BMI, mean ± SD 25.05 ± 2.4 26.45 ± 3.8 n.s.
Time of work (years), mean ± SD - 3.22 ± 2.10 -
PPE use, n (%) - 40 (100) -
Sector of industry
Painting - 17 (42.5) -
Welding - 15 (37.5) -
Casting - 4 (10) -
Machining - 4 (10) -
Leukocyte count
Leukocyte, mean ± SD 6506 ± 1215 6176 ± 1498 < 0.001
Segmented neutrophil, mean ± SD 4074 ± 1066 2678 ± 808 < 0.001
Band neutrophil, mean ± SD 109 ± 48 199 ± 123 < 0.01
Lymphocyte, mean ± SD 1924 ± 425 2784 ± 789 < 0.001
Monocyte, mean ± SD 224 ± 119 327 ± 125 < 0.01
Eosinophil, median (IQ 25–75) 103 (5.2–190) 141 (10.25–208) n.s.
Basophil, median (IQ 25–75) 0 (0–0) 0 (0–0) n.s.
The table shows the comparison of demographic data and leukocyte counts between the control group (n=20) and the metalworkers
group (n=40). PPE: personal protective equipment; BMI: body mass index. Parametric data expressed as mean± standard deviation;
nonparametric data expressed as median and interquartile range. SD: standard deviation; n.s.: not significant.

leukocytes from metalworkers compared to controls.
Besides, non-proteic thiol groups (p < 0.001; Figure
3(d)) and iron-reducing capacity (p < 0.001; Figure
3(e)) were significantly reduced in plasma from
metalworkers compared to controls. However, no
significant differences were observed in plasma lipid
peroxidation levels (p>0.05; Figure 3(c)).
Peripheral cholinergic inflammation in blood
from metalworkers
Butiricholinesterase activity was significantly higher
in plasma from metalworkers (p < 0.01; Figure 4).
Discussion
To the best of our knowledge, this is the first study
describing the cytotoxicity associated with oxidative
stress and cholinergic inflammation in peripheral
blood from metalworkers. Initially, increased apo-
ptosis and reduced leukocyte viability was detected in
these individuals. The apoptotic process occurs
physiologically in cells of the immune system as part
of an inflammation control mechanism (Wong, 2011).
For example, neutrophils undergo apoptosis within
hours of being released into the bloodstream if there is
no tissue demand (Peng, 2006; McCracken and Allen,
2014). However, oxidative stress (Sato et al., 2003)
and inflammation (Maianski et al., 2003), patho-
physiological processes arising from the environment
work of metalworkers, can accelerate apoptosis in-
duction in leukocytes and affect immune homeostasis.
Apoptosis is positively related to the working time
of metalworkers; therefore, the increased cytotoxicity
in leukocytes may have been induced by exposure to
chemicals present in the MWF. Corroborating this
hypothesis, in vitro studies have reported that sub-
stances frequently present in MWF, such as nitrosa-
mines and formaldehyde, can induce apoptosis in
leukocytes and other cell models (Jablonski et al.,
2001; Zerin et al., 2015; Iwaniuk et al., 2019). Fur-
thermore, the increase in apoptosis observed in leu-
kocytes may be responsible, at least partially, for the
reduction in leukocyte and neutrophil count of met-
alworkers, affecting the number of cells available for
the immune responses.
Parallel to increased apoptosis, higher intracellular
production of reactive species by leukocytes was
detected in metalworkers, suggesting the connection
between such cellular processes. Immune cells
Bonfanti-Azzolin et al. 757

Figure 1. Increased apoptosis in leukocytes from metalworkers. (a) Graphs (left to right) showing increased early
apoptosis, late apoptosis, and reduced cell viability in leukocytes from metalworkers. Data are presented as median and
interquartile range. Significant differences were evaluated by T Student test. **P < 0.01, ***P < 0.001. (b) Representative
flow cytometry dot plot analysis of propidium iodide (PI) and Annexin V staining.

naturally produce reactive species as part of the im-
mune response mechanism; however, increased pro-
duction of reactive species can induce oxidative stress
and cell damage (Pizzino et al., 2017) and can activate
several pro-death signaling pathways that induce cell
apoptosis (Lang et al., 2018; Li et al., 2018). Aug-
mented reactive oxygen species and oxidative stress
have been previously described in rats exposed to
MWF (Shvedova et al., 2002). Therefore, we suggest
that occupational exposure to MWF leads to increased
intracellular production of reactive species, which may
be responsible for the accelerated induction of
apoptosis.
Mitochondria are primarily responsible for the in-
tracellular production of reactive species
(Kudryavtseva et al., 2016; Bhatti et al., 2017). Thus,
our hypothesis is that the increase in reactive species in
leukocytes from metalworkers is responsible for in-
crease in mitochondrial mass and membrane potential
and that hyperpolarized mitochondria augment reac-
tive species production. In this way, the mitochondrial
membrane potential generated by proton pumps is an
essential component of energy generation during ox-
idative phosphorylation. However, hyperpolarized
mitochondrial membranes may represent a serious
burden, due to the excessive production of reactive
species (Zorova et al., 2018). Increased production of
reactive species damages mitochondria, especially
mitochondrial DNA. In response, the cells synthesize a
greater number of mitochondrial DNA copies to in-
duce mitochondrial biogenesis to meet the growing
respiratory demand. The increased number of mito-
chondria, in turn, generates more reactive species,
perpetuating a vicious cycle (Malik and Czajka, 2013).
Furthermore, previous studies have shown that ben-
zene, a hydrocarbon present in the metalworking in-
dustry as a contaminant of toluene, increases
mitochondrial DNA copies, indicating an increase in
the mitochondrial number to compensate for mito-
chondria damaged by oxidative stress in occupation-
ally exposed workers (Liu et al., 2003; Shen et al.,
2008).
In general, we suggest that the increase in apoptosis,
reactive species, mitochondrial membrane potential,
758 Toxicology and Industrial Health 37(12)

Figure 2. Mitochondrial analysis in leukocytes from metalworkers. (a) MTG and MTR analysis showing significantly
increased mitochondrial mass and membrane potential in leukocytes from metalworkers. Data are presented as median
and interquartile range. Significant differences were evaluated by U-Mann Whitney test. *P < 0.05, ***P < 0.001. (b)
Representative flow cytometry histogram analysis of MTG and MTR staining. MTG: MitoTrackerTM Green FM, MTR:
MitoTrackerTM Red FM, MFI: median of fluorescence intensity.

and mitochondrial mass detected in this study are
interrelated. Associated with these findings, this study
demonstrated a reduction in total plasma antioxidant
capacity and plasma thiol groups in workers. In line
with this, Kshirsagar et al. (2020) and Moro et al.
(2010) demonstrated a reduction in antioxidant de-
fenses in workers exposed to heavy metals and sol-
vents, respectively. In this context, antioxidant
defenses may be consumed to neutralize excessive
reactive species (Irazabal and Torres, 2020).
Thiol groups are present in peripheral blood as
cysteine side chain, antioxidant enzymes with thiol-
based reaction mechanism and low-molecular-weight
thiols. They act to maintain redox homeostasis in
various cellular compartments, protecting the organ-
ism from oxidative and xenobiotic stressors, and
partake actively in redox-regulatory and signaling
processes (Ulrich and Jakob, 2019). In this sense,
considering that the thiol modifications are fully re-
versible by thiol-based redox systems (Holmgren
et al., 2005), the lower level of thiol groups de-
tected in metalworkers suggests a consumption of
them greater than recycling capacity, indicating a
prooxidative status. This can be confirmed by the
reduced plasma total antioxidant capacity found in
metalworkers. Plasma antioxidant status is the result of
many different compounds and systemic metabolic
interactions (Ghiselli et al., 2000). This assay measures
the antioxidant capacity of all antioxidants in a bio-
logical sample, evaluating the combination effect of
them, and might give more biologically relevant in-
formation than that obtained from measuring con-
centrations of individual antioxidants (Ghiselli et al.,
2000; Kusano and Ferrari, 2008).
However, it was not possible to observe differences
in plasma lipoperoxidation levels between the groups
studied. Although this biomarker is commonly used to
assess damage caused by oxidative stress in workers
exposed to toxic compounds (Moro et al., 2010), this
study demonstrates that reduced plasma antioxidants,
reduced cell viability, and mitochondrial changes can
be more sensitive biomarkers of oxidative damage in
metalworkers and that apoptosis can occur indepen-
dently of lipid damage.
The higher activity level of BChE founded in
metalworkers could be related to oxidative and in-
flammatory status. Oxidative stress is a potent inducer
of inflammation (Miller et al., 2018). Recent data relate
Bonfanti-Azzolin et al. 759

Figure 3. Oxidative stress in blood from metalworkers. (a) DCFH-DA analysis showing significantly increased
intracellular production of reactive species in leukocytes from metalworkers. (b) Representative flow cytometry
histogram analysis of DCFH-DA staining. (c) Thiobarbituric acid reactive substances analysis showing similar lipid
peroxidation in plasma from metalworkers and controls. (d) Thiol groups are significantly reduced in plasma from
metalworkers. (e) Iron-reducing capacity showing significantly reduced total antioxidant capacity in plasma from
metalworkers. Data are presented as median and interquartile range. Significant differences were evaluated by Student t-
test or Mann–Whitney U test, as appropriate. **P < 0.01, ***P < 0.001. DCFH-DA: dichlorodihydrofluorescein 20,70-
diacetate, MFI: median of fluorescence intensity, MDA: malondialdehyde, GSH: glutathione.

(Leon-Pedroza et al., 2015; Hussain et al., 2016). This

Figure 4. Increased butyrylcholinesterase in plasma from
metalworkers. Butyrylcholinesterase was significantly
augmented in plasma from metalworkers. Significant
differences were evaluated by Student t-test. **P < 0.01.
to oxidative stress and chronic low-grade inflamma-
tion, which is characterized by moderately elevated
circulating levels of inflammatory cytokines and in-
creased macrophage infiltration in peripheral tissues
is related to the development and progression of
chronic diseases, such as diabetes, and respiratory and
cardiovascular diseases (Custodero et al., 2018). The
plasma increase in butyrylcholinesterase (BChE) ac-
tivity has been described as a marker of chronic low-
grade inflammation because increased activity of
BChE in plasma indirectly reflects reduced butyr-
ylcholine concentrations, which act by inhibiting
Nuclear Factor Kappa B (NFKB) signaling and cy-
tokine production (Ben Anes et al., 2018; Ben Assayag
et al., 2010). Corroborating this hypothesis, re-
searchers reported that a plasma increase of BChE is
correlated with IL-6 and C-reactive protein in patients
with a stroke (Kliper et al., 2013). Increases in BChE
activity have been identified in pathologies, such as
Alzheimer’s disease (Geula and Darvesh, 2004), di-
abetes mellitus (Rao et al., 2007), obesity (Dantas
et al., 2011) and hypertension (Bose et al., 2019). In
this context, plasma cholinergic signaling may con-
tribute to the induction or maintenance of a low-grade
760
inflammatory state in metalworkers, making them
susceptible to the development of several chronic
pathologies (Hopf et al., 2019).
This study has some limitations. First, this study has a
reduced sample size, with a larger group of exposed in-
dividuals than in the control group, which might have
limited the statistical power of the analyses. Additionally,
it was not possible to determine the range of workers’
exposure to heavy metals and MWFs in the different
sectors of the metalworking industry, considering that
there may be multiple exposures among the recruited
individuals. Thus, it is not possible to establish the real
correlation between exposure type and the cellular and
molecular alterations described here. Despite this, we
obtained important findings demonstrating cytotoxic,
oxidative and inflammatory changes in metalworkers.
Conclusion
This study demonstrated that oxidative stress and
inflammation may be involved in the multiplicity of
health outcomes related to the working environment of
the metalworking industry. The presumed physio-
logical mechanism is that oxidative stress induces
inflammation, which ultimately increases oxidative
stress via a feedback mechanism. Thus, chronic in-
flammation can lead to adverse health effects. Since
occupational markers aim to prevent acute or chronic
intoxication, but are not intended to prevent chronic
pathologies to workers’ health, this study highlighted
the need to identify exposure biomarkers that are early
indicators of inflammatory and oxidative status.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
The author(s) disclosed receipt of the following financial
support for the research, authorship, and/or publication of
this article: This study was supported by grants from the
Fundação de Amparo a Pesquisa do Rio Grande do Sul
(FAPERGS); Conselho Nacional de Desenvolvimento
Cientı́fico e Tecnológico (CNPQ); and Programa de Bolsas
de Iniciação Cientı́fica da Universidade de Cruz Alta (PI-
BIC-UNICRUZ).
Authors’s Note
This protocol was approved by the Institutional Review
Board of the University of Cruz Alta (No. 2.442.325).
Toxicology and Industrial Health 37(12)
ORCID iD
Mariana M Parisi  https://orcid.org/0000-0002-2298-
7809
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