Association between occupational lead exposure and immunotoxicity markers: A systematic review and meta-analysis

Abstract

Recent evidences suggest the role of chronic lead (Pb) exposure in altering immunological parameters. Present study aimed to systematically review existing literature and synthesize quantitative evidence on the association between chronic Pb exposure and changes in immunological markers. Observational studies reporting immunological markers such as leukocyte derivative counts (CD3⁺, CD4⁺, CD8⁺, CD45⁺, CD56⁺, lymphocyte, and total leukocyte), cytokine, Immunoglobulin (Igs), C-reactive protein (CRP) among Pb-exposed and unexposed controls were systematically searched from PubMed, Scopus and Embase digital databases from inception to January 2021. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were adhered during systematic review. Mean differences in the immunological markers between Pb-exposed and control groups were pooled using random-effects model. The heterogeneity was assessed using Cochran-Q test and I² statistic.
The review included forty studies reporting immunological markers in Pb-exposed and unexposed control groups. The occupational Pb-exposed group exhibited significantly higher BLL, impaired immunological markers, characterized by a marginal lowering in lymphocyte count, lymphocyte subsets (CD3⁺, CD4⁺, CD4⁺/CD8⁺ ratio), INF-γ and IgG levels, while CD8⁺, IgM, IgA, IgE, and cytokines (IL-4, IL-6, IL-10, and TNF-α) exhibited a trend of higher values in comparison to the control group. Further, inflammatory marker viz., total leukocyte count was significantly higher among Pb-exposed. The included studies exhibited high levels of heterogeneity. In conclusion, Occupational Pb exposure alters the immunological markers such as the circulating cytokines and leukocyte counts. However, high-quality, multicentered studies are required to strengthen present observations and further understand the Pb's role on the immune system.
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Association between lead exposure and DNA damage (genotoxicity):
systematic review and meta-analysis
Preprint · July 2022
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Archives of Toxicology
https://doi.org/10.1007/s00204-022-03352-9
REVIEW ARTICLE
Association betweenlead exposure andDNA damage (genotoxicity):
systematic review andmeta‑analysis
RajuNagaraju1· RavibabuKalahasthi1· RakeshBalachandar2· BhavaniShankaraBagepally3
Received: 20 July 2022 / Accepted: 27 July 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022
Abstract
Studies suggest that chronic lead (Pb) exposure may induce deoxyribonucleic acid (DNA) damage. However, there is no
synthesised evidence in this regard. We systematically reviewed existing literature and synthesised evidence on the associa-
tion between chronic Pb exposure and markers of genotoxicity. Observational studies reporting biomarkers of DNA damage
among occupationally Pb-exposed and unexposed controls were systematically searched from PubMed, Scopus and Embase
databases from inception to January 2022. The markers included were micronucleus frequency (MN), chromosomal aberra-
tions, comet assay, and 8-hydroxy-deoxyguanosine. During the execution of thisreview, we followed the Preferred Reporting
Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Mean differences in the biological markers of DNA
damage between Pb-exposed and control groups were pooled using the random-effects model. The heterogeneity was assessed
using the Cochran-Q test and I2 statistic. The review included forty-five studies comparing markers of DNA damage between
Pb-exposed and unexposed. The primary studies utilised buccal and/or peripheral leukocytes for evaluating the DNA dam-
age. The pooled quantitative results revealed significantly higher DNA damage characterised by increased levels of MN and
SCE frequency, chromosomal aberrations, and oxidative DNA damage (comet assay and 8-OHdG) among Pb-exposed than
the unexposed. However, studies included in the review exhibited high levels of heterogeneity among the studies. Chronic
Pb exposure is associated with DNA damage. However, high-quality, multicentred studies are required to strengthen present
observations and further understand the Pb’s role in inducing DNA damage. CRD42022286810.
* Bhavani Shankara Bagepally
bshankara@gmail.com; bagepally.bs@gov.in
1 Biochemistry, Regional Occupational Health Centre
(Southern), ICMR-National Institute ofOccupational Health,
Bengaluru, Karnataka, India
2 Division ofHealth Sciences, ICMR-National Institute
ofOccupational Health, Ahmedabad, Gujarat, India
3 Non-Communicable Diseases, ICMR-National Institute
ofEpidemiology, Tamil Nadu, Chennai, India
Highlights
The synthesized evidence indicates that chronic Pb exposure is associated with DNA damage.
The DNA damage markers with Pb exposure include higher levels of micro-nuclei (with nuclear buds and nucleoplasmic
bridges), sister chromatid exchange frequency, chromosomal aberrations and oxidative DNA damage.
Keywords DNA damage· Chromosomal aberrations· 8-Hydroxy deoxyguanosine· Lead exposure· Comet length·
Micronuclei
Introduction
Lead (Pb),a heavy metal with multiple desirable proper-
ties, viz. easy moulding, relatively inert and many others,
has been extensively used in various industries, including
automobiles, paint, ceramics, batteries, etc. (Flora etal.
2012). Therefore, workers in these industries are potentially
exposed to high levels of Pb. Any levels of Pb in the biologi-
cal samples (including blood) may be deemed potentially
harmful. The Center for Disease Control (CDC) released
reference blood Pb levels acceptable for community adults

Archives of Toxicology
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(i.e. without obvious occupational Pb exposure) is 3.5µg/
dL(Centers for Disease Control and Prevention (CDC) 2012;
Kalahasthi etal. 2022).
Chronic Pb exposure is associated with abnormalities in
neurological (Virgolini and Aschner 2021), nephrological
(Kuraeiad and Kotepui 2021), cardiovascular (Chen etal.
2021; Upadhyay etal. 2021), haematological (Upadhyay
etal. 2021), immunological (Kalahasthi etal. 2022) and
reproductive (Balachandar etal. 2020) functions. The mech-
anistic studies have shown that Pb exposure is associated
with impairment in antioxidant status of cells, i.e. depletion
of reduced glutathione pools and increase in reactive oxygen
species formation leading to organ toxicity (Qu etal. 2019).
Literature on mechanistic as well as epidemiological stud-
ies suggests the carcinogenetic risk of Pb (Lundström etal.
1997; Liao etal. 2016). International Agency for Research
on Cancer (IARC) classified inorganic Pb in group 2A, and
organic Pb in group 3 as Pb is related to excess risk of stom-
ach, lungs, kidney, balder and brain cancers (2010). Data
from Steenland etal. 2019 (Steenland etal. 2019) and others
conclude a robust positive correlation between blood lead
levels (BLL) and the incidence of cancers in the lungs, brain
and other organs (Kauppinen etal. 2000, 2001) The geno-
toxicity induced by Pb may cause a carcinogenic mechanism
and lead to cancer incidence (Carere etal. 1995), Centers for
Disease Control and Prevention 2013)
Studies used comet assay (tail length, tail moment, tail
DNA), chromosomal aberrations and micronuclei frequency
(MN) to screen workers' genotoxicity. The outcomes from
such studies are contradictory, as many studies exhibited no
or mild change [16–20], while others showed higher levels
of DNA damage and chromosomal aberrations in exposed
individuals (Wu etal. 2002; Palus etal. 2003; Das and De
2013; Yedjou etal. 2015; de Souza etal. 2018; Balasubra-
manian etal. 2020; Batra etal. 2020; Meng etal. 2021;
Duydu 2022). By considering the impact of Pb on genetic
material, the pooled results would provide the best estimates
of the effects of Pb exposure on induction of DNA damage
and genotoxicity mediated various disorders. This would
provide evidence for policy implications. Therefore, the
present systematic review and meta-analysis synthesised
the association between BLL with different biomarkers of
genotoxicity among workers. The present study observations
would provide insights and comprehension of the pieces of
evidence on Pb-induced genotoxicity.
Methods
The systematic review was registered at Prospero (Reg
No PROSPERO 2022CRD42022286810) and executed as
per Preferred Reporting Items of Systematic reviews and
Meta-Analysis (PRISMA) (Page etal. 2021). Observational
studies reporting the comparison of genotoxicity and /
or DNA damage between occupationally Pb-exposed and
healthy controls were systematically searched. The search
was performed in PubMed- Medline, Scopus, and Embase
online repositories. The last search was performed on 10th
January 2022. The search terms and strategies adopted in
the current review are tabulated in Supplementary Table1.
The search parameters were constructed by using the con-
ventional PICO approach. The “Participant” (individuals
occupationally exposed to Pb), “Intervention” (i.e., expo-
sure to Pb), “Comparator” (individuals without occupational
exposure to Pb) “Outcome” (DNA damage and genotoxicity
parameters) were used. The search on “Outcome” measures
included parameters on DNA damage (comet assay), i.e.
the percentage of tail DNA, tail intensity, tail length, tail
moment and Olive tail moment (OTM), sister chromatid
exchange (SCE) frequency, micronuclei frequency (MN)
(micro-nucleated, Bi-nucleated) and other nuclear abnor-
malities like pyknosis, condensed chromatin, karyorrhexis,
nuclear buds, nucleoplasmatic bridges and mitotic index and
oxidative DNA damage markers (8-hydroxy-2-deoxyguano-
sine), respectively. The studies which included parameters
on chromosomal analysis, i.e. Gaps, chromatid aberrations
(chromatid breaks, chromatid deletions, chromatid rings,
dicentrics, acentric fragments, gaps) and chromosomal aber-
rations (chromosomal breaks, chromosomal deletions, chro-
mosomal rings, dicentrics, acentric fragments, gaps) were
also considered. A sensitivity and precision maximizing
strategy was adopted to identify relevant studies, and addi-
tional keywords identified during the search were included
in the systematic search.
Screening andreviewing ofstudies
The authors independently screened the titles and abstracts
of all citations, resulting from the systemic search of vari-
ous electronic databases for their potential inclusion (NR
and KRB). Authors (NR and KRB) independently reviewed
the full text of articles scrutinized during screening. The
final list of studies meeting the inclusion and exclusion cri-
teria was prepared after removing duplicates based on the
authors’ mutual consensus (NR, KRB, and BSB) (Fig.1).
The studies meeting the following inclusion criteria were
selected: (1) Published in English; (2) Occupational inves-
tigation; (3) article must contain an exposure group and
control group; (4) the two groups were comparable in terms
of age and health status; and (5) availability of outcome vari-
ables in mean ± standard deviation (SD) or can be convert-
ible into mean ± S.D form. Studies were excluded according
to the following criteria; (1) Studies involving participants
with only < 18years of age; (2) Case reports, reviews, let-
ters to editors, editorials, and methodological papers; (3)
duplicated data, and incomplete information studies; and (4)

Archives of Toxicology
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animal experiments and basic research. Lateral search for
potential studies using the studies identified during the full-
text review was additionally attempted. The Rayyan online
platform was used for screening and selection of studies
(Ouzzani etal. 2016).
Data extraction, analysis, andmanagement
The Microsoft excel ver. 2016 was used to extract relevant
details from studies to achieve planned objectives. The
details of publications (author(s), title, journal, and year of
publication), participant details (study location, age, gender,
and clinical details), Pb exposure details (source(s), duration
and Pb levels in biological samples) and outcome measures
(DNA damage and genotoxicity markers) were extracted
from the primary studies and recorded. The corresponding
authors of the primary studies were contacted by email when
relevant data was unavailable. The authors were contacted
and reminded on at least two occasions with an interval
of two weeks between the reminders before excluding the
study/declaring “non-availability of data”. However, these
studies were part of the critical evaluation for generating
necessary evidence.
Data on central tendency (mean/median) and dispersion [
(SD) / Standard error (SE) / Interquartile range / 95% confi-
dence interval (CI)] for available parameters were indepen-
dently extracted from the included studies (NR and KRB)
and verified for consistency before further analysis (KRB).
The outcome variables reported in units other than the con-
ventional units/standard units were converted to standard
/ conventional units [Blood Pb as μg/dL, SCE frequency
and chromosomal aberrations as per cell, MN frequency,
Tail DNA, tail intensity as a percentage, tail length in µM
and 8-hydroxy-2-deoxyguanosine (8-OHdG) in ng/ml etc.]
using standard conversion factors. The measures of central
tendency & data dispersion, when provided (Carere etal.
1995; Iarmarcovai etal. 2005; Chen etal. 2006; Kašuba
etal. 2012; Das and De 2013; Jannuzzi and Alpertunga
2016; Alabi etal. 2020) alternate to mean (e.g., median and
mode) and SD (e.g., 95% CI, Interquartile range, standard
error of the mean) appropriate conversions were adopted to
pool the results (Hozo etal. 2005; Wan etal. 2014). Mean
differences of the outcome variables (where available) were
pooled between Pb- exposure and unexposed/control groups
when ≥ 3 studies were available for quantitative assessment.
For studies reporting more than one group in the control and
exposed group (Wu etal. 2002; García-Lestón etal. 2012;
Dobrakowski etal. 2017), the grand mean and SD were cal-
culated (Altman etal. 2000). Key details recorded in the data
extraction sheet are summarized in Supplementary Table2.
Heterogeneity, sensitivity, subgroup, andrisk
ofbias assessment
The heterogeneity among included studies was assessed
using visual inspection of forest plots, the Cochran-Q test,
and I-squared (I2) statistics. Either I2 > 25% or Cochrane-
Q < 0.1 was regarded as evidence for the presence of het-
erogeneity among the included studies. The random-effects
model of Der Simonian and Laird was used after confirming
Fig. 1 PRISMA flow chart. The flow chart illustrates the number of articles included and excluded at various steps

Archives of Toxicology
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heterogeneity (DerSimonian and Laird 2015). Further, the
sources of heterogeneity were explored by fitting the co-
variables such as age and duration of exposure in the meta-
regression model depending on the availability of data mini-
mum of ten studies are essential for meta-regression analysis
(Bagepally etal. 2021; Kalahasthi etal. 2022). The variable
responsible for reducing I2 by 50% in the meta-regression
model was regarded as the potential source of heterogeneity;
subsequently, a bubble plot was used to explore the covari-
ate’s influence. Asymmetrical funnel plot or significant
Egger’s test (p < 0.05) of the effect measures were regarded
as evidence for potential publication bias (Hayashino etal.
2005; Peters etal. 2010). However, the funnel plot and Egg-
er’s test of effect measures were valuable when adequate
(i.e. > 10) primary studies were available for pooling the par-
ticular outcome variable. Contour-enhanced funnel plot was
additionally explored to investigate the sources of biases.
Lastly, the influence of the type of sample used (i.e. buccal
cells vs peripheral leukocytes) was explored by subgroup
analysis. However, in view of fewer studies, the proposed
sensitivity & subgroup analyses (i.e. < 3 studies under each
subgroup) were not executed for the genotoxicity param-
eters. Data was recorded using a Microsoft Excel sheet and
analyzed using Stata version 16 (2019) (StataCorp 2019).
Two-sided p < 0.05 was considered statistically significant.
Assessment oftherisk ofbias
New Castle Ottawa scale (NOS) was used to evaluate the risk
of bias for each of the included studies (Wells etal. 2013).
The details of NOS followed in this study are described in
our previous publication (Kalahasthi etal. 2022).
Results
The search in databases retrieved 4050 studies. Subse-
quently, with duplicate removal and screening, Forty-five
studies were selected for the review and data synthesis.
The study selection details at various screening steps are
shown in the PRISMA selection flow chart (Fig.1). The
particulars of extracted data related to assessments, primary
study participants, and their occupation-related details from
the selected studies are listed in Supplementary Table2.
Included studies’ participants were occupationally exposed
to Pb as smelters, welders, Pb battery manufacturing & recy-
cling workers, automobile workers, petrol station attendants,
E-waste workers, and workers involved in the foundry, paint-
ing work and chemical plants with occupational Pb usage.
Among 45 studies included in the meta-analysis, seven
biomarkers of DNA damage (MN, 8-OHdG, chromosomal
aberrations, the comets assay, cell apoptosis, telomere
length, and necrosis rate) were reported in biological
materials like buckle cells and peripheral leukocytes/ lym-
phocytes. The MN frequency (n = 20) is the most commonly
monitored biomarker. In contrast, telomere length (Wu
etal. 2012), necrosis (Kašuba etal. 2012) and apoptosis
rate (Kašuba etal. 2012) were investigated independently by
single individual studies and hence synthesis of quantitative
information using meta-analysis was not executed for these
parameters. The risk of bias assessment results using the
NOS is reported in Supplementary Table3.
BLL
Forty studies reported BLL comparison between Pb-exposed
and controls. Majority ofthese studies observed significantly
higher BLL among the exposed group than the control
group. The included studies were classified based on BLL
as per the guidelines of New York state for health work-
ers (New York state) few studies observed BLL > 10μg/
dL (elevated BLL) among the controls (Duydu etal. 2001;
Hamurcu etal. 2001; Vaglenov etal. 2001; Wu etal. 2012;
Balasubramanian etal. 2020; Leelapongwattana and Bor-
deerat 2020). The Pb-exposed in the included studies exhib-
ited wide range of BLL, majority of the studies observed
elevated Pb levels in blood (i.e. BLL between 25 and 40μg/
dL) (Bilban 1998; Duydu etal. 2001; Hamurcu etal. 2001;
Fracasso etal. 2002; Minozzo etal. 2004; Grover etal. 2010;
García-Lestón etal. 2012; Jannuzzi and Alpertunga 2016;
Pawlas etal. 2017; Balasubramanian etal. 2020; Batra etal.
2020; Kašuba etal. 2020; Duydu 2022), while few observed
seriously elevated levels (i.e. 40–80μg/dL (Vaglenov etal.
2001; Palus etal. 2003; Karakaya etal. 2005; Kašuba etal.
2010; Olewińska etal. 2010; Wu etal. 2012; Singh etal
2013) and one study observed very high levels of BLLs
(> 80μg/dL) (Das and De 2013). The effect measure of
mean difference in BLL from studies demonstrates that the
study participants with occupational Pb exposure (n = 2570)
had significantly higher BLL than age-matched controls
(n = 1981). The pooled mean difference in BLL is 23.55
(95%CI 19.96–27.15) μg/dL, and there was high heterogene-
ity between the studies (I2 = 99.72) (Supplementary Fig.1).
The subgroup, sensitivity, and meta-regression analyses did
not aid in exploring and identifying the factors attributing
to heterogeneity. There is publication bias, as suggested by
the asymmetry in the funnel plot (p = 0.271); the contour-
enhanced funnel plot indicates the possible existence of
other biases. (Supplementary Fig.2).
Micronuclei frequency
MN frequency is an extensively studied biomarker to inves-
tigate chromosomal damage induced by cytotoxic agents (/
genotoxicity) (Fenech 1998). Twenty studiesincluded in
current review reported MN frequency in mononucleated

Archives of Toxicology
1 3
cells (MNC). Few studies (Pinto etal. 2000; Khan etal.
2010; Singh etal 2013; Aksu etal. 2019; Alabi etal. 2020)
utilized buccal cells as the source of sample, while others
used peripheral leukocytes (Bilban 1998; Hamurcu etal.
2001; Vaglenov etal. 2001; Palus etal. 2003; Minozzo
etal. 2004; Chen etal. 2006; Grover etal. 2010; Kašuba
etal. 2010, 2012, 2020; García-Lestón etal. 2012; Yu etal.
2018; Balasubramanian etal. 2020; Leelapongwattana and
Bordeerat 2020; Meng etal. 2021).
Nineteen out of twenty studies observed considerably
higher MN frequency among the Pb-exposed than the con-
trols, with most studies reporting statistical significance.
Consistent with the observations from primary studies,
the pooled results revealed significantly higher MN fre-
quency in the Pb-exposed with a mean difference of 1.50
(95%CI 1.17–1.84)% than the controls, with high hetero-
geneityamong studies (I2 = 99.74%) (Fig.2A). The asym-
metric funnel plot is suggestive of potential publication
bias (p = 0.011) and plausible other biases as suggested by
the contour-enhanced funnel plot (Supplementary Fig.3).
The subgroup analysis with the type of sample (i.e. buccal
cells vs. peripheral leukocytes) used for assessing the MN
frequency did not indicate the source of heterogeneity or
alterthe direction of results. The subgroup pooled mean
difference in MN involving buccal cells and peripheral
leukocytes was respectively 1.78 (95%CI 0.00–3.57)%
and 1.37 (95%CI 1.05–1.69) %with I2 99.93% and 98.98%
(Fig.2A).
In addition, ten studies reported MN frequency in binu-
cleatedcells (BNC) harvested frombuccal cells (Grover
etal. 2010; Khan etal. 2010; Singh etal 2013; Aksu etal.
2019), and peripheral leukocytes (Carere etal. 1995;
Vaglenov etal. 2001; Palus etal. 2003; Meng etal. 2021).
All studies consistently observed higher MN frequency in
BNC in the Pb-exposed than in the controls. We noticed
contrasting results among studies with low and high BNC
in the exposed population vs the control group, however,
two of these studies observed a trend, while the remain-
ing studies reported significant differences between the
duo (Carere etal. 1995; Grover etal. 2010). The pooled
difference was 1.97 (95% CI 1.19–2.74) with high hetero-
geneity (I2 = 99.42%) (Fig.2B). Asymmetric funnel plots
and contour-enhanced funnel plots suggest possible pub-
lication bias (p = 0.048) and other biases (Supplementary
Fig.4). The subgroup analysis exploring the role of tissue
(i.e buccal cells vs peripheral leukocytes) exhibitedmean-
MN frequencydifference between Pb-exposed and con-
trols, asrespectively 3.03 (95%CI 1.56–4.51, I2 = 99.50)
% and 1.23 (95%CI 0.60–1.85, I2 = 96.93%)% (Fig.2B).
Fig. 2 Forest plot for MN frequency among mononucleus and binucleus cells. Group differences in MN frequency among mononucleus (A) and
binucleus cells (B) between the occupationally Pb exposed and unexposed workers

Archives of Toxicology
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Other biomarkers ofcytogenic alteration
The other biomarkers of cytogenic alteration such as con-
densed chromatin (CC), lobed nucleus (LN), nuclear buds
(NB), mitotic index (NDI), nucleoplasmatic bridges (NPB),
pyknosis (PYC) and karyorrhexis (KARY) were available
in addition to MN frequency. The CC was quantified by
two studies (Aksu etal. 2019; Alabi etal. 2020) in buccal
cells, and both studies reported higher percentage of CC
among Pb exposed than controls. Alabi etal. (2020) was
the only study to report lobed nucleus (the nucleus is seg-
mented into two or more connected lobes), wherein the Pb
exposed group exhibited a significantly higher percentage
of the lobed nucleus than controls (Alabi etal. 2020). The
NB frequency was reported by five of the included studies;
four of them used peripheral leukocytes (Grover etal. 2010;
Kašuba etal. 2010, 2012, 2020), and one with buccal cells
(Aksu etal. 2019). Aksu etal. (2019) observed consider-
ably more frequent NB amongPb-exposed than controls,
while the other studies reportedno significant difference.
The pooled difference between Pb-exposed and controls was
0.04 (95%CI – 0.28 to 0.36) % with high between-study het-
erogeneity (I2 = 99.62%) (Supplementary Fig.5).
The NDI frequency was reported among eight of the
included studies. Palus etal. (Palus etal. 2003) was not part
of the quantitative analysis in view of the non-availability of
data dispersion details. All studies reported NDI frequency
using peripheral leukocytes (Carere etal. 1995; Minozzo
etal. 2004; Chen etal. 2006; Kašuba etal. 2010, 2012, 2020;
Leelapongwattana and Bordeerat 2020), where Kasuba
etal. (2020) and Minozzo etal. (2004) observed statistical
significance. The pooled mean difference between the duo
was – 0.0003 (95%CI – 0.00 to 0.00) % with low between-
study heterogeneity (I2 = 1.75%) (Supplementary Fig. 6).
The frequency of NPB was reported in three of the included
studies (Kašuba etal. 2010, 2012, 2020), and two of these
observed significant differences. The pooled mean difference
between the Pb-exposed and controls was 0.08 (95%CI 0.02
to 0.13) % with no obvious heterogeneity (I2 = 0.00%) (Sup-
plementary Fig.7).
The frequency of PYC (Aksu etal. 2019; Alabi etal.
2020) and KARY (Khan etal. 2010; Alabi etal. 2020) were
reported using the buccal cells of the participants. The PYC
and KARY frequency were considerably higher among Pb-
exposed workers compared to the controls. The pooled mean
KARY difference between the Pb-exposed and controls was
1.67(95%CI – 0.12 to 3.46)% (Supplementary Fig.8), with
high (99.95%) between-study heterogeneity. Funnel plots
and contour-enhanced funnel plots were not analyzed due
to fewer available studies (n < 10). The results from included
studies demonstrated a strong and consistent association
between Pb exposure and high rates of MN frequency in
buccal cells and lymphocytes of occupationally Pb exposed
than controls.
Sister chromatid exchange (SCE)
Ten of the included studies reported SCE frequency among
lymphocytes (Mäki‐Paakkanen etal. 1981; Carere etal.
1995; Bilban 1998; Dönmez etal. 1998; Pinto etal. 2000;
Duydu etal. 2001; Wu etal. 2002; Wu etal. 2004; Palus
etal. 2003; Duydu 2022). Majority of the studies observed
trend of(p > 0.05) higher SCE levels among exposed group
than the controls (Bilban 1998; Dönmez etal. 1998; Pinto
etal. 2000; Duydu etal. 2001; Palus etal. 2003; Duydu
2022). Consistent with the results from primary studies, the
pooled mean difference between the duo was 1.35 (95%CI
0.87to1.82) per cell, with high heterogeneity (I2 = 93.21%)
(Fig.3A). Theresults could be potentially biased bypublica-
tion and other biases as suggested by the asymmetric funnel
plot (p = 0.036) and contour-enhanced funnel plot (Supple-
mentary Fig.9).
Chromosomal aberrations
Six of the included studies reported chromosomal aberra-
tions (Bilban 1998; Pinto etal. 2000; Karakaya etal. 2005;
Grover etal. 2010; Das and De 2013; Balasubramanian
etal. 2020) in lymphocytes, with consistent evidences of
chromosomal aberrations among occupational Pb exposed
workers. The pooled mean difference between Pb-exposed
and controls was 2.25 (95%CI 0.30–4.20) per cell with high
between-study heterogeneity (I2 = 99.80%) (Fig.3B). Three
studies reported chromosomal breaks (Carere etal. 1995;
Bilban 1998; Grover etal. 2010) in lymphocytes, while in
view of non-availability of data dispersions, Carere etal.
1995 wasn’t part of the quantitative analysis. All three
studies observed higher chromosomal breaks among the
Pb exposed than the controls. The chromosomal acentrics,
dicentrics and rings were higher among Pb exposed group
(Bilban 1998). However, the pooled difference was not
available for these parameters due to a single study (Bilban
1998). Funnel plots and contour-enhanced funnel plots were
not analyzed due to fewer available studies (n < 10).
Chromatid aberrations
Two studies reporting chromatid aberrations observed higher
chromatid aberrations among Pb exposed group compared to
the control group (Pinto etal. 2000; Balasubramanian etal.
2020). In case of chromatid acentrics, studies (Bauchinger
etal. 1976; Grover etal. 2010) monitored aberrations in lym-
phocytes and found considerably higher levels among Pb-
exposed than in controls. The Pb-exposed group exhibited
higher chromatid breaks with pooled mean difference 0.91

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1 3
(95%CI – 0.07 to 1.89, I2 = 99.79%) per cell (Bauchinger
etal. 1976; Bilban 1998; Grover etal. 2010) (Supplemen-
tary Fig.10). The dicentrics (Bauchinger etal. 1976; Grover
etal. 2010) were higher among the Pb-exposed. The funnel
plots and contour-enhanced funnel plots were not analyzed
due to fewer available studies (n < 10).
Comet assay parameters
The studies included in the current review evaluated the
parameters of the comet’s tail, such as its intensity, DNA
content, moment and length, to assess the DNA damage.
Nine studies monitored the percentage of DNA content in
the lymphocyte comet tail (Fracasso etal. 2002; Olewińska
etal. 2010; García-Lestón etal. 2012; Jannuzzi and Alper-
tunga 2016; Dobrakowski etal. 2017; Akram etal. 2019;
Balasubramanian etal. 2020; Batra etal. 2020; Meng etal.
2021) interestingly, all studies reported a significantly higher
percentage of DNA in the Pb-exposed group than in the con-
trols. Consistently, the pooled mean difference in the per-
centage of tail length between the duo revealed the same
with a statistical significance, i.e. 7.92 (95%CI 4.77–11.07)
%, with high heterogeneity amongthe studies (I2 = 99.56%)
(Fig.4A). Five of the included studies reported tail inten-
sity using peripheral leukocytes (Kašuba etal. 2012, 2020;
Kayaalti etal. 2015; Pawlas etal. 2017; Aksu etal. 2019).
The pooled mean difference between Pb-exposed and con-
trols was 1.79 (95%CI 0.28–3.30% with high heterogeneity
(I2 = 86. 60%) (Fig.4B). The length of the comet’s tail was
reported in nine of the included studies using peripheral leu-
kocytes (Fracasso etal. 2002; Grover etal. 2010; Olewińska
etal. 2010; Kašuba etal. 2012, 2020; Dobrakowski etal.
Fig. 3 Forest plot for Chro-
mosomal aberrations. Group
differences in Sister chromatid
exchange (A) and chromosomal
aberration (B) between the
occupationally Pb exposed and
unexposed workers

Archives of Toxicology
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2017; Pawlas etal. 2017; Akram etal. 2019; Balasubrama-
nian etal. 2020). The majority of the studies observed sig-
nificantly longer comet’s tail among the Pb-exposed as com-
pared to controls (Fracasso etal. 2002; Grover etal. 2010;
Olewińska etal. 2010; Kašuba etal. 2012; Dobrakowski
etal. 2017; Balasubramanian etal. 2020), The pooled mean
difference in comet’s tail length between the duo was 6.31
(95%CI 2.23–10.38) µm with high between-study heteroge-
neity (I2 = 98. 60%) (Fig.4C). The tail moment was reported
by seven studies using peripheral leukocytes (Fracasso etal.
2002; Grover etal. 2010; Olewińska etal. 2010; Kašuba
etal. 2012; Kayaalti etal. 2015; Dobrakowski etal. 2017;
Pawlas etal. 2017; Akram etal. 2019). The tail moment of
Pb-exposed was higher as compared to the controls, with
pooled tail moment difference of 10.03 (95%CI 5.71–14.35)
and heterogeneity (I2 = 99.07%) (Fig.4D). The OTM was
analyzed in lymphocytes (Iarmarcovai etal. 2005; Akram
etal. 2019), and studies found higher levels of OTM among
samples of the Pb-exposed group than in the controls. The
comet length was reported by Akram etal. 2019 (Akram
etal. 2019) and Palus etal. 2003 (Palus etal. 2003) in iso-
lated lymphocytes of the participants; similar to the other
parameters of the comet assay, the comet length was con-
siderably higher among Pb-exposed than in controls. A sin-
gle study using lymphocytes reported the DNA in the head
region (Akram etal. 2019). The observation of DNA con-
tent of the comet’s head was lower among the Pb-exposed
than the controls was consistent with the other comet assay
parameters, suggestive of a relatively greater DNA damage
among the Pb-exposed.
Oxidative DNA damage
The 8-OHdG is a product of oxidative DNA damage and is
considered a biomarker of genotoxic exposure while examin-
ing genome stability. Five of the included studies reported
8-OHdG in the study participants (Szymańska-Chabowska
etal. 2009; Leelapongwattana and Bordeerat 2020; Nsonwu-
Anyanwu 2021; Singh etal. 2021). The results of these stud-
ies marked a higher level of blood 8-OHdG content in the
exposed population than control. Similarly, the pooled mean
difference between the duo was high, i.e. 32.45 (95%CI
10.32–54.59, I2 = 96.35%)ng/ml with high heterogeneity
(Supplementary Fig.11).
Fig. 4 Forest plot for comet assay. Group differences in % of DNA in tail (A), tail intensity (B), tail length (C) and tail moment (D) between the
occupationally Pb exposed and unexposed workers

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Others
Telomere length
One study (Wu etal. 2012) examined telomere length in
peripheral blood lymphocytes of control and exposed
workers and demonstrated a significant decrease in tel-
omere length among the exposed population (1.91 ± 0.46
vs 1.66 ± 0.63).
Apoptosis andNecrosis rate
Kasuba etal. 2012(Kašuba etal. 2012)found a significant
increase in the apoptosis (5.5 ± 5.9% vs 19.6 ± 20.8%) and
necrosis rate (2.1 ± 2.9% vs 3.3 ± 5.7%) in whole blood of Pb
exposed workers as compared to the control group.
Discussion
Present systematically reviewed the current literature exam-
ining the impact of chronic Pb-exposure on DNA damage
and genomic instability using updated standard guidelines.
Studies describing the DNA damage or genetic instability
among occupationally Pb-exposed than the controls without
a history of noticeable occupational exposure were primarily
included. Occupational Pb-exposed workers were reported
with significantly higher BLL consistent with their exposure,
and exhibited relatively increased DNA damage, i.e. elevated
MN frequency, SCE frequency, total chromosomal aberra-
tions (chromosomal and chromatid aberrations), oxidative
DNA damage, apoptotic and necrosis rate. The comet assay
results were consistent with the above results, i.e. higher
DNA content, intensity, length and moment of the tail. In
contrast, telomere length was significantly decreased among
Pb-exposed workers compared to the respective controls.
The Pb-exposed group exhibited higher (23.55μg/dL)
BLL than the control group. This observation was consistent
across the included studies irrespective of the workplace's
nature and exposure duration. As there is no safe level for Pb
exposure, The CDC recommends investigating the potential
source among the adults with BLL ≥ 3.5μg/dL, which are
categorized as “elevated BLL” (2012) (https:// www. cdc. gov/
mmwr/ previ ew/ mmwrh tml/ mm625 4a4. htm). Similarly, few
studies reported BLL ≥ 10μg/dL among the controls (with
no obvious occupation Pb exposure) as well; however, sig-
nificantly lower BLL as compared to the corresponding Pb
exposed group.
The evidence from the current study noted a trend of
higher MN frequency among Pb exposed than unexposed
control groups. MN frequency is the common biomarker
used to monitor genotoxic exposure (Fenech etal. 2013), it is
a good indicator of clastogenesis (chromosomal breaks) and
aneugenic effects of xenobiotics on the genome (Augusto
etal. 1997; Fenech etal. 2013). It is a standardized and
validated method of predicting the risk of cancer (Fenech
etal. 2013; Nersesyan etal. 2021). In this review, 20 studies
reported higher MN frequency among Pb exposed group,
consistent with the pooled mean difference. The study from
Fench and Bonnasi demonstrated the influence of lifestyle
factors on DNA damage (i.e. MN frequency in the peripheral
leukocytes) (Fenech and Bonassi 2011), suggesting the care-
ful consideration of lifestyle factors while assessing the gen-
otoxic impact of the exposure (Fenech and Bonassi 2011).
Hamurcu etal. (2001) observed significantly higher MN fre-
quency among tobacco smokers without obvious Pb expo-
sure (controls) as compared to controls with non-smoking
(p = 0.048). However, the MN frequency was significantly
higher among Pb exposed (irrespective of the smoking sta-
tus) as compared to control groups (Hamurcu etal. 2001),
suggesting the association between Pb exposure and higher
MN frequency.
The MN frequency is typically scored in binucleated cells
after nuclear division in telophase by blocking cytokinesis
using cytochalasin-B; while recently, both mononucleosis
and binucelus were considered for MN frequency analysis
(Rosefort etal. 2004). The lymphocyte cytokinesis-block
micronucleus test (CBMN) is OECD recommended, robust
method of genotoxicity assessment (OECD 2010; Glei etal.
2016). Also, CBMN is a robust predictor biomarker for vari-
ous disorders (Bonassi etal. 2007; Furness etal. 2010).The
MN frequency from bi-nucleated cells and other parameters
viz. NB, NDI, pyknosis, karyorrhexis and NPB consistently
showed greater DNA damage among Pb-exposed than the
controls. Notably, NDI is the marker of cell proliferation.
In contrast, cells with greater chromosomal damage (lower
NDI) will either prematurely attain cellular death prior to
cell division or fail to complete the phase of cell division
(Ionescu etal. 2011). Therefore, the decreased NDI rate is
suggestive of genome instability.
The MN, NPB, and NB are biomarkers of genomic
instability (Podrimaj-Bytyqi etal. 2018; Ruiz-Ruiz etal.
2020), as they predict clastogens-induced aberrations in
cell division and structural chromosomal rearrangements
(Cheong etal. 2013; Ruiz-Ruiz etal. 2020). Current obser-
vations of increased NB and NPB congruent with MN fre-
quency among the Pb-exposed group suggest an elevated
risk of genotoxicity among the Pb-exposed group. Further,
increased cellular degeneration markers viz. pyknosis and
karyorrhexis among the Pb-exposed workers are consistent
with higher DNA damage associated with Pb exposure (Fer-
raz etal. 2016). Lastly, apoptosis and necrosis, the markers
of planned/regulated cellular death either due to ageing or
prematurely due to irreversible cellular injury (i.e. beyond
cellular repair) (Ruiz-Ruiz etal. 2020) were higher among
the Pb-exposed.

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The frequency of the peripheral lymphocyte chromo-
somal aberrations is considered a biomarker of genotoxic-
ity due to carcinogens of occupational and environmental
sources (Albertini etal. 2000; Norppa etal. 2006). Chromo-
somal aberrations may include the entire chromosomal or
only the chromatid, wherein the former is a better indicator
of cancer risk (Liou etal. 1999). The contrast between these
indicators is that S-independent clastogens cause those chro-
mosomal aberrations, while chromatid aberration is induced
by S-dependent mutagens (Norppa etal. 2006). Both chro-
mosome and chromatid aberrations are sub-divided into
exchanges and breaks, where breaks represent major aber-
ration in chromatid type, while breaks and chromosome rear-
rangements (dicentrics) are the foremost part of chromo-
somal aberration (Norppa etal. 2006). The current review
observed both chromatid and chromosomal type aberrations
were considerably higher among the Pb-exposed population,
suggesting the potential geno- and cytotoxicity associated
with chronic Pb-exposure.
The SCEs are biomarkers of genetic instability con-
ventionally used for hazard identification and risk assess-
ment among those occupationally exposed (Zeljezic and
Garaj-Vrhovac 2002; Duydu 2022; Zendehdel and Vahabi
2022). The SCEs are usually used to examine the cyto-
genic responses to carcinogen (chemical) exposure. SCEs
are considered a more sensitive biomarker of genotoxicity
than structural aberrations; however, they are less reliable
for assessing cancer risk (Tucker and Preston 1996). Cur-
rent observations of elevated SCE frequency among the Pb-
exposed suggest increased cytotoxicity with Pb exposure.
Comet assay test detects cellular level DNA damage,
which is simple and sensitive. The assay detects single-
strand breaks, alkali labile and cross-linking sites; hence it
is extensively employed in genotoxic regulatory studies. The
shape, size and amount of DNA within a comet are assessed
either by manual visualization or automated software appli-
cations to assess DNA damage (Kumaravel etal. 2009) par-
ticularly of occupational genotoxicity (Martino-Roth etal.
2003) such as those included in this review. Interestingly, all
parameters were consistently higher among the Pb-exposed
workers than in the controls, suggesting a potential associa-
tion between DNA damage and Pb exposure.
Enzymatic cleavage of the guanine base, 8-OHDG, is the
hallmark of general oxidative DNA damage (Martino-Roth
etal. 2003) and carcinogenesis (Shen etal. 1999; Lodovici
etal. 2000; Valavanidis etal. 2009) of occupational and
environmental exposures (Pilger and Rüdiger 2006). Cur-
rent results of high 8-OHDG among the Pb exposed group
suggest an association between oxidative stress (& possibly
carcinogenesis) and chronic Pb exposure.
This evidence appraisal is possibly the earliest to sys-
tematically review and document the association between
chronic Pb exposure, DNA damage, and cytotoxicity. The
primary studies included in the review exhibited high lev-
els of heterogeneity, risk of bias, fewer numbers limiting
the subgroup and meta-regression analyses, cross-sectional
design and low powered / quality. The current review sug-
gests the need for longitudinal studies with larger samples
and better quality to investigate the association between
chronic Pb exposure and investigate the pathways for effi-
cient DNA repair. Given the potential genotoxicity and
cytotoxicity of Pb, present observations suggest the need
for periodic screening of individuals (workers) employed in
Pb (or its associated) industries.
Conclusion
Current evidence synthesis infers that occupational Pb
exposure is associated with a higher frequency of MN and
SCE, chromosomal aberrations (cytotoxicity) and DNA
damage (comet assay and 8-OHDG) as compared to the
control group. Chronic Pb exposure is potentially genotoxic
(because of higher NB and NPB rates) and induces oxida-
tive DNA damage (elevated 8-OHDG). Current results could
provide a scientific basis while considering the revision of
strategies used to prevent occupational disease, particularly
occupational cancers among the Pb exposed workers. Fur-
ther, longitudinal & high-quality primary studies are crucial
for recommending regular screening for genotoxicity among
the Pb-exposed.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s00204- 022- 03352-9.
Acknowledgements None.
Author contributions NR: conceptualisation, data curation, formal
analysis, original draft.: KR: conceptualisation, data curation, meth-
odology, review and editing, inputs on the original draft. BR: inputs on
the original draft, review and editing. BBS: conceptualisation, formal
analysis, inputs on original draft, investigation, methodology, software,
review and editing.
Funding No funding support for this work.
Data availability statement Not applicable.
Declarations
Conflict of interest None.
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