Intan
Nur’azizah Rahman1, Katharina Oginawati2,
Yuyun Ismawati3, Sonia Buftheim4,
Dwi Aris Agung Nugrahaningsih5
Faculty of Civil and
Engineering, Institut Teknologi Bandung, Indonesia1,2
Nexus3Foundation, Indonesia3,4
Faculty of Medicine, Public
Health, and Nusing, Universitas
Gadjah Mada, Indonesia5
Email: [email protected], [email protected], [email protected], [email protected], [email protected]
KEYWORDS DNA
damage; occupational lead exposure; paint industry workers; PbB |
ABSTRACT Lead-based paint is a main source of lead exposure to paint industry
workers and causes an imbalance of Reactive Oxygen Species (ROS) and
antioxidants, causing a genotoxic effect. Pb in the blood (PbB)
level and DNA damage are frequently used as exposure and effect biomarker of
lead. The purpose of this study to determine the
relationship between PbB level and DNA damage due to occupational
lead exposure in paint industry workers. The research design uses a
cross-sectional epidemiological study involving 52 workers from three paint
manufacturers in Indonesia. Blood samples were taken for PbB
analysis using ICP-MS, while DNA damage was analyzed using the Comet Assay
method. The PbB average obtained was 4.36±1.60 µg.dL-1,
where 17 workers (32.69%) exceeded the safe limit value of PbB (5 µg.dL-1). Meanwhile, the
influential factors of PbB are the working period
and alcohol consumption (p=0.029). The level of DNA damage was represented as
Tail DNA (%), and the average was 9.62±0.19
%. All respondents in this study were categorized as under low damage (Class
2). There was no significant relationship between PbB
and Tail DNA (%) and has a negative correlation (p=0.878; r=-0.022). The
study concludes that there was no difference in Tail DNA (%) between PbB ≥ 5 µg.dL-1 and PbB < 5 µg.dL-1 (p=0.876). It means that
lead exposure in this finding has not reached a level that can significantly
cause DNA damage. However, it is necessary to monitor PbB
levels in workers to minimize genotoxic or other effects. |
INTRODUCTION
Lead (Pb)
is a xenobiotic that is widely distributed and highly persistent (Singh et
al., 2018). Lead
(Pb) characteristics are low melting point, ductility, high malleability,
corrosion resistance, and low cost. These functions made it widely used in
various industrial sectors, which can accumulate in the environment and cause
serious health problems for humans (Sangeetha
and Umamaheswari, 2020). IARC classifies
lead as a class 2A carcinogenic substance, in which the agent is possibly
carcinogenic to humans (O’Connor
et al., 2018). It is
also unnecessary for human health, and harmful to the human body (Wani et
al., 2015).
Lead-based
paints are the primary source of lead poisoning in the industrial environment (Abdollahi
et al., 1996).
Occupational environments lead to higher lead levels in the occupational area,
so there can be more opportunities for lead exposure (Singh et
al., 2018). Paint
products may contain lead when the paint's raw materials are contaminated with
lead during the mixing process or cross-contamination from other products in
the same industry. Paint manufacturers add Pb to paint to be a pigment, dryer,
or anti-corrosion, however, the lead compound most often added to paint is
pigment (Brosché
et al., 2014) for
improve colors such as lead chromate (PbCrO4) for yellow pigments
and lead carbonate (PbCO3) for white pigments. Also, it is
protective, which makes the paint last longer, increases paint adhesion to the
surface, and can make the paint layer remains strong but remains flexible and
resists cracking longer (O’Connor
et al., 2018).
The issue
of lead-based paint has attracted the attention of international organizations
and non-governmental organizations (NGOs). The International Community, led by
UNEP and WHO, is actively supporting the global elimination of lead-based
paints by 2020 due to its severe health risks. However, lead-based paints are
still widely produced and used in developing countries, including Indonesia (O’Connor
et al., 2018). Based on
Ismawati et al. (2021), lead-based paints in Indonesia are still widely
manufactured and sold. Of 120 samples tested, 39% have lead concentrations
above 10,000 ppm. The threshold of Pb in paints (dry) is 600 ppm (Badan
Standarisasi Nasional, 2014). IPEN recommends 90 ppm as a safe and achievable
concentration of lead in paints worldwide (Brosché
et al., 2014).
The
pathway for lead to enter the body can be through air, food, or drink (Wani et
al., 2015).
Meanwhile, the routes of absorption of lead are through ingestion, inhalation,
or the skin. Exposure to lead occurs mainly through gastrointestinal (GI)
tracts and the respiratory system (Sangeetha
and Umamaheswari, 2020). However, the
dermal route is not significant in the general population, but dermal exposure
most likely occurs in persons working with lead-containing materials. Although
inorganic Pb exposure to the dermal route is rarely studied because it
contributes little to absorption (Wani et
al., 2015), several
absorption kinetics parameters, such as Kp and diffusion rate, are produced to
be used as a risk assessment for the work environment. The study produced Kp of
10-7 – 10-5 cm.hour-1 and diffusion rate
ranges of 10-7 - 10-4 mg.cm-2.hour-1
obtained from animal and human skins (Niemeier
et al., 2022). After
absorption, lead accumulates in the blood, soft tissues, and bone, and the
lead's half-life in these parts is 35 days, 40 days, and 20-30 years
respectively (Sangeetha
and Umamaheswari, 2020).
Blood is
commonly used as a biomarker because it is relatively easy to collect and is
one of the pathways through which most chemicals and their metabolites travel
within the body (Paustenbach
and Galbraith, 2006). The
concentration of lead in the blood (PbB) is an indication of Pb absorption. It
is the best parameter to be used as a biological marker to evaluate lead exposure
(La-Llave-León
et al., 2017). While
any measurable effect or response biomarkers, such as biomonitoring of enzymes
in the blood, indicate organ damage, microscopic and subcellular levels
(Timbrell, J. 2002). Lead toxicity can affect organ systems, induce various
biochemical, physiological, and genetic dysfunctions as well as cause damaging
effects, such as DNA damage (genotoxicity) (Singh et
al., 2018).
Individuals exposed to the paint experienced increased
levels of DNA damage (Cassini
et al., 2011). It occurs due to changes in the basic structure of DNA which affect
the physical-chemical properties of DNA which then affect the interpretation
and transmission of genetic information (Juan et
al., 2021). Occupational
exposure to lead is associated with DNA damage that can measure using Comet
Assay, a rapid, sensitive method suitable for biomonitoring studies (Olewińska
et al., 2010). This method is widely used
because it can detect DNA damage sensitively by measuring and analyzing DNA
damage in lymphocyte cells. The damage can be in the form of a single strand
break (SSB) or a double strand break (DSB) in opposite positions. The degree of
DNA damage was determined by the proportion of cells with comets (Danadevi
et al., 2003).
The major mechanism of Pb toxicity is lead can induced
oxidative stress (Collin et
al., 2022) which
describe as an imbalance between the generation of Reactive Oxygen Species
(ROS) and the ability of antioxidants. Pb is capable of inhibiting the
activities of antioxidant enzymes by interacting with a functional sulfhydryl
(SH) group in antioxidant enzymes, such as δ-aminolaevulinic acid dehydrase
(δ-ALAD), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase
(GPx), and glucose-6-phosphate dehydrogenase (G6PD), resulting increase
Reactive Oxygen Species (ROS) production and decreased antioxidants (Hemmaphan
and Bordeerat, 2022) which can cause
damage to cellular biomolecules (include lipids, proteins, and DNA) (Juan et
al., 2021).
Reactive
Oxygen Species (ROS) are the typical by-products of the electron transport
chain (ETC) during cellular respiration in aerobic organisms and are
additionally derived from catabolic oxidases, anabolic processes, and
peroxisomal metabolism. However, in excess, ROS species can cause a total of
approximately 100 different oxidative base lesions and 2-deoxyribose
modifications. The most reactive of ROS is OH radical (-OH) produced as a
by-product of Fenton's reactions of H2O2 with Fe2+ can
damage DNA. Another biologically significant and major oxidative base lesion
formed from hydroxylation of the C-8 residue of guanine is the saturated
imidazole ring 7,8 dihydro-8-oxo guanine (8-oxo-G). 8-oxo-guanine pairs
incorrectly with adenine instead of cytosine, thereby adding to the overall
mutational load, and is further oxidized to other deleterious secondary DNA
lesions because of its low oxidation potential. Besides attacking DNA bases,
ROS radicals can also compromise the DNA backbone causing an estimated 2300
single-strand breaks per cell per hour (Chatterjee
and Walker, 2017).
Continuous
oxidative stress can cause severe molecular and cellular changes that can lead
to cell death (Singh et
al., 2018).
Moreover, any resulting damage, if not repaired, will lead to mutations and
possibly disease (Lodish, 2004). Pb replaces
calcium and zinc in enzymes involved in DNA processing and repair, resulting in
increased genotoxicity when combined with other DNA-damaging agents such as
tobacco smoke or UV A. Interestingly, abnormal DNA repair capacity was reported
in lead-exposed workers (Hemmaphan and Bordeerat, 2022)
Based on this mechanism, it is necessary to evaluate
the genotoxic effect in the form of DNA damage using comet assay on workers in
the paint industry in Indonesia, because the genotoxic effects of paint industry
workers in Indonesia have never been reported. The purpose of this study was to
investigate the relationship between blood Pb (PbB) levels and DNA damage in workers of occupational
exposure to lead in the paint industry in Indonesia.
RESEARCH METHOD
Research Design
The
research design used an observational epidemiological study type
cross-sectional to determine exposure and effects at one time quantitativelly.
Sampling was taken from 52 paint industry workers who are in three different
locations in Indonesia, namely Industry A, Industry B, and Industry C. The kind
of sampling technique used is non-probability sampling, namely purposive
sampling. The selection of respondents is not random, but by setting inclusion
criteria, including, males, aged 25-50 years, and willing to sign informed
consent, while the exclusion criteria were not living near landfills and or
industrial areas.
The
respondents were divided into two groups based on exposure, the exposed groups
and the controls groups. The groups in contact with lead-containing materials
were included as the exposed group, while the group that had indirect contact
with lead-containing materials was included as the unexposed group or as a
control. The sample size in this research refers to NIOSH Occupational Exposure
Sampling Strategy Manual (Leidel et al., 1997). This research has been reviewed
and approved by the Padjadjaran University Research Ethics Commission with
document number 1066/UN6.KEP/EC/2022.
Data Collection
Primary
data collection is in the form of blood sampling and interviews with
respondents. Blood collection was carried out by a phlebotomy-certified health
professional from Prodia Clinic Laboratory and stored in a trace element
Na-Heparin tube (Royal Blue-Top). As much as 6 mL samples of blood were
preserved in an ice box for PbB analysis later. Meanwhile, 2 mL of blood was
collected in a vacutainer tube then the sample was preserved in an ice box for
DNA damage analysis later. Meanwhile, interviews were conducted to determine
the characteristics of respondents.
Measurement of Pb in
Blood and DNA Damage
Pb Level
in Blood (PbB) was analyzed using the ICP-MS methods in Prodia Clinic
Laboratory, while DNA damage was measured using the Alkaline Comet Assay
according to the method of Singh et al., (1988) with some modifications in the
Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada. It
uses horizontal submarine electrophoresis 300 mA; 50V. DNA
damage was analyzed using ImageJ software for evaluating 50 cells per slide.
Statistical Analysis
Statistical
analysis in this research used SPSS 22 software. The confidence interval used
was 95%, with an error of 5%. Descriptive statistics are used to describe the
characteristics of the respondents, PbB values, and DNA damage (%Tail DNA). The
non-parametric comparison test used was Kruskal Wallis and Mann Whitney U to
determine the factors that influence PbB. Then the Rank Spearman test was used
to determine the correlation between PbB and DNA damage.
RESULTS AND
DISCUSSIONS
Characteristics of
Research Subjects
This
research was conducted on 52 respondents from three paint industries in
different locations in Indonesia. The number of respondents involved in each of
the industries A, B, and C are 20, 12, and 20 respondents respectively. The
interval aged of workers was 25-50 years with an average of 34 years. The
working periods variable also varies among respondents, 2.5-29 years with an
average of 11 years. Each subject has worked in their respective industries for
2.5 – 29 years with an average of 11 years. Table 1 summarizes the characteristics of the subjects
participating in this study, while Table
2 summarizes the classifications of worker characteristics and habits,
which will be evaluated for their effect on either PbB levels or DNA damage.
Table 1.
Main characteristics of research subjects (n=52).
Variables |
Min. |
Max. |
Mean ± SD |
Ages
(Years) |
26 |
50 |
33.67±5.39 |
Working
Periods (Years) |
2.5 |
29 |
10.61±4.79 |
Body
Weight (kg) |
50 |
105 |
75.81±14.05 |
Table
2. Classifications of characteristics and habits
of all respondents
No |
Factors |
Classifications |
N |
1 |
Ages |
26-35 (years) |
35 (67.0%) |
36-55 (years) |
17 (33.0%) |
||
2 |
Industry Locations |
A |
20
(38.5%) |
B |
12
(23.0%) |
||
C |
20
(38.5%) |
||
3 |
Group
of Exposures |
Exposed group |
35
(67.0%) |
Control group |
17
(33.0%) |
||
4 |
Working
Periods |
<10 years |
19
(36.5%) |
≥10 years |
33
(63.5%) |
||
5 |
Smoking
Habits |
Smoking |
25
(48.1%) |
Non-smoking |
27
(51.9%) |
||
6 |
Alcohol
consumptions |
Yes |
5
(10.0%) |
No |
47
(90.0%) |
Blood Pb (PbB) Levels
Exposure
biomarkers are one of the important parameters in toxicology to evaluate lead
exposure, which indicates a measure of the interaction between biological
systems and lead. Thus, exposure can be roughly determined by measuring the
dose, but it cannot be assumed that all doses are absorbed. Therefore, the
estimation of exposure is the concentration of chemicals in the blood lead
level (PbB) as an exposure biomarker. The result of PbB measurements was
expressed in µg.dL-1 units, showing the mass of lead in 100
mL of blood from ICP-MS measuring. The result of the PbB value is shown in Table 3.
Table
3. Descriptive
summary of PbB
Parameters |
PbB (µg.dL-1) |
Min |
1.40 |
Median |
4.00 |
Max |
8.10 |
Mean ± SD |
4.36 ± 1.60 |
Figure 1. Distributions of Pb in the blood (PbB)
level’s in all respondents
Based on Table 3., the PbB average of all
respondents in three industries was 4.36±1.60 µg.dL-1, where 17
respondents (32.69%) exceeded the safe limit value of PbB, 15 of 17 respondents
from industry C, one from industry A, and industry B, respectively, shown at Figure 1. According to the Centre
for Disease Control (CDC) guidelines, the acceptable range for adult blood
lead (PbB) levels is 10 µg.dL-1, whereas based on NIOSH, the maximum value of PbB was 5
µg.dL-1
(Singh, et al. 2020). The PbB average of
each Industry A, B, and C was 3.2±1.4 µg.dL-1, 3.68±0.87 µg.dL-1
and 5.57±1.57 µg.dL-1 respectively. However, respondents
of Industry C also have the largest PbB concentration range (1.80-8.10 µg.dL-1)
and have the largest average PbB value. The Kruskal-Wallis test was used to
find out the average difference between the three industries.
The PbB values in Industry C
have significantly larger than in Industry A and B (p<0.05), as shown
in Table 4., while the box plot
of these industries is shown in Figure
2. The high presence of PbB levels in Industry
C’s respondents is thought to be due to exposure to lead obtained from
lead-based paints that exceed the limit value. Based on a study conducted by
Ismawati et al. (2021), industry C uses Pb in paints of more than 600 ppm, and
for example, the lead content in the paint for the yellow color was 14,000 ppm,
which means it is far from the quality standards set by SNI 8011-2014. Based on
the California
Department of Public Health, if the PbB value was 5-9 µg.dL-1, it is
necessary to identify a history of potential sources of lead exposure in the
workplace and minimize contact with lead, as well as monitor these PbB levels,
by carrying out inspections PbB levels every three months until the PbB value
is less than 5 µg.dL-1 (Grandjean
et al., 1981).
Figure 2. Box plot of PbB value based on industry location
Besides
industry locations, several confounding factors will be analyzed for their
effect on biomarkers of lead exposure (PbB). The confounding factors that have
been evaluated for their significance on blood lead levels are exposure groups,
working periods, smoking habits, and alcohol consumption. These factors were
also evaluated using the Kruskal Wallis to find out the differences in the
three factors while using Mann-Whitney U for two factors as a post hoc test.
The statistical test results of these factors are presented in Table 4.
Table
4.
Statistical test results of several factors that influence PbB
No. |
Factors |
Categories |
N |
p-value |
1 |
Industry
Locations |
A |
20 (38.5%) |
0.000* |
B |
12 (23.0%) |
|||
C |
20 (38.5%) |
|||
2 |
Exposure
Groups |
Exposed Groups |
35 (67.0%) |
0.441 |
Controls |
17 (33.0%) |
|||
3 |
Working
Periods |
<10 years |
19 (36.5%) |
0.017* |
≥10 years |
33 (63.5%) |
|||
4 |
Smoking
Habits |
Smoking |
25 (48.1%) |
0.384
|
Non-smoking |
27 (51.9%) |
|||
5 |
Alcohol
consumption |
Yes |
5 (10.0%) |
0.029* |
No |
47 (90.0%) |
*p-value is
statistically significant (p<0.05)
Factors
that gave a significance p <0.05 besides industrial locations are working
periods and habits of consuming alcohol. So, working periods and alcohol
consumption can affect the Pb in blood (PbB) levels. This study is similar to
Batra et al. (2020) studies that the concentration of Pb in the blood increases
with years of exposure (p<0.05) and there was a positive correlation between working periods and PbB (p-value=0.016; r=0.333). Therefore, we conclude that
the longer the duration of Pb exposure, the higher the level of lead in the
blood (PbB).
For the alcohol consumption factors, this study is
similar to Grandjean
et al. (1981) that PbB
detected in workers who are consuming alcohol is higher than in workers
who don't consume alcohol, and also there was a
positive correlation between alcohol consumption and PbB
(p-value=0.031; r=0.300). The significance value of the study was
p<0.05, which means that it is statistically significant, that people who
consume alcohol have increased blood lead levels. Moreover, daily consumption
of 13.5 mL of pure ethanol per day can contribute 0.5-1.0 µg leads.100 mL-1
of blood. Meanwhile, this study's exposure groups and smoking habits were not
significant to PbB values (p> 0.05).
DNA Damage
DNA damage
as effect biomarkers of lead is a change in the basic structure of DNA that
does not replicate on its own when DNA is replicated. DNA damage can be in the
form of chemical additions or disruptions to DNA bases (creating abnormal
nucleotides or nucleotide fragments) or breaks in one or both strands of DNA (Bernstein & Nfonsam, 2013), which can be
detected using Alkaline Comet Assay methods. One of the most used parameters to
describe DNA damage is the Tail DNA (%) which represents the fluorescence
intensity relative to the head and tail of DNA. The average Tail DNA (%) of all
respondents was 9.62±0.19
%. Based on Figure 3, all
respondents in this research are categorized as low damage (Class 2) (Pereira
et al., 2010).
Figure
3. Distributions
of Tail DNA’s (%) worker
Table
5. Descriptive
summary of DNA Tail (%)
Parameters |
%Tail DNA |
|
Min |
9.11 |
|
Median |
9.67 |
|
Max |
10.25 |
|
Mean |
9.62 |
|
SD |
0.19 |
|
Mann Whitney U test was also used for evaluating the
significance between two category PbB values, divided
by two groups based on safety value according to NIOSH, PbB
< 5 µg.dL-1 and PbB
≥ 5 µg.dL-1 shown in Figure 4.
Moreover, they had no significant differences (p=0.876) between the two PbB levels
groups, it shown in Table 6. This
study contrasts with Kalaayathi et al. (2013), there was
a statistically significant relationship between the Comet parameters and lead
level groups in respondents (p <0.05). The differences were due to larger
PbB values, and the groups were classified between PbB ≥ 10 µg.dL-1 and PbB <10 µg.dL-1. Whereas in this
study, it had not yet reached the PbB value for causing significant effects in
DNA damage.
Table 6. Tail DNA (%) of
the two groups based on PbB values
Parameters |
PbB (µg/dL) |
n |
Min. |
Max. |
Mean |
SD |
p-value |
Tail
DNA(%) |
<5 |
35 |
8.96 |
10.25 |
9.60 |
0.22 |
0.876 |
≥5 |
17 |
8.96
|
10.25
|
9.61
|
0.22 |
Figure 4.
Box plot of Tail DNAs respondents based on PbB value
We used
the Rank Spearman method to find out the correlation between PbB and Tail DNA
(%). Based on Figure 5, there
was no significant relationship between PbB and Tail DNA (%), and it has a
negative correlation (p>0.05; r=-0.022). In contrast to research conducted by (Batra et
al., 2020), there was a significant relationship and a positive correlation
between PbB levels and Tail DNA (%) (p<0.05). The
contrasts could be due to different values in this research and differences in subject research and occupational exposure. Batra et al.
study involved building construction workers, painters, motor garage workers,
tinting and painting workers, and battery
workers involved in removing Pb electrodes, smelting, recycling Pb batteries,
and manufacturing and assembling Pb acid storage batteries. Furthermore, the
range of lead levels in the exposed group was much larger (38.03 ± 12.92) µg.dL-1 than in this study, and Batra et al. were
able to identify the cause of DNA damage in the form of Tail DNA (%) of (14.80
± 1.31)%.
Figure 5. Correlation between PbB and % Tail DNA
According to a study by Dobrakowski
et al. (2017), lead exposure to workers results in blood lead levels (PbB) of more than 20 µg/dL can significantly increase DNA
damage. It means that the lead exposure in this finding has not reached a level
that can significantly cause DNA damage. In addition, the human body contains DNA repair machinery that plays an
important role in protecting cells from DNA damage produced by exposure to
carcinogens and cytotoxic agents, as well as heavy metals (Hemmaphan
and Bordeerat, 2022).
Table 7. Statistical test results of several factors
that influence DNA Tail (%)
No |
Factors |
Categories |
N |
p-value |
1 |
Ages |
26-35 (years) |
35 (67.0%) |
0.464 |
36-55 (years) |
17 (33.0%) |
|||
2 |
Groups of Exposure |
Exposure |
20 (38.5%) |
0.114 |
Control |
12 (23.0%) |
0.114 |
||
3 |
Working Periods |
<10 years |
20 (38.5%) |
0.985 |
≥10 years |
35 (67.0%) |
|||
4 |
Smoking Habits |
Smoking |
17 (33.0%) |
0.627 |
Non-smoking |
19 (36.5%) |
|||
5 |
Alcohol consumption |
Yes |
33 (63.5%) |
0.449 |
No |
25 (48.1%) |
*p-value is statistically significant
(p<0.05)
Several factors affecting DNA Tail (%) have been
evaluated statistically. They have been evaluated for their
significance to DNA Tail (%). These factors
include age,
groups of exposure, working periods, smoking habits, and alcohol consumption.
These factors were also evaluated using the Mann-Whitney U test. The
statistical test results of these factors are presented in Table 7. Factors of age, groups of
exposure, working periods, smoking habits, and alcohol consumption in this
research do not influence the Tail DNA (%) (p>0.05). ROS accumulation can cause DNA damage, not only caused by Pb and
these factors, but ROS is also produced by ionizing radiation and UV radiation,
and also metabolic processes as well as various drugs and xenobiotics (Juan et
al., 2021). So further research must be monitored for ROS accumulation, for
example using 8-oxoG as a biomarker for oxidative DNA damage.
CONCLUSIONS
From the results of the study, it
can be concluded that: (1) The concentration of Pb in the blood (PbB)
of respondents from the paint industry was 4.36±1.60 µg.dL-1. As many as 17 workers
(32.69%) have exceeded the safe limit value of PbB, therefore is necessary to monitor blood lead levels in workers
periodically, (2) factors affecting PbB value are
industry locations, working periods, and alcohol consumption, (3) the result of
DNA damage in respondents of this research is categorized as low damage, and (4)
there is no relationship between blood Pb levels (PbB) and DNA damage (% Tail DNA) in respondents because the
PbB levels obtained have not caused any observed
effects. However, it is
necessary to monitor blood lead levels in workers due to lead exposure.
ACKNOWLEDGEMENT
The researchers express
their gratitude to IPEN and the GiveWell Foundation
for the funding support and also the paint industry who participated in this
study.
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Copyright holders:
Intan Nur’azizah
Rahman, Katharina Oginawati, Yuyun
Ismawati, Sonia Buftheim, Dwi Aris Agung Nugrahaningsih (2023)
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Devotion - Journal of Research and Community
Service
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