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Biomarkers of Susceptibility Following Benzene Exposure: Influence of

Genetic Polymorphisms on Benzene Metabolism and Health Effects

Article  in  Biomarkers in Medicine · January 2016

DOI: 10.2217/bmm.15.106

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Review

Biomarkers of susceptibility following

benzene exposure: influence of genetic
polymorphisms on benzene metabolism
and health effects

Benzene is a ubiquitous occupational and environmental pollutant. Improved Damiano Carbonari1,

industrial hygiene allowed airborne concentrations close to the environmental Pieranna Chiarella1, Antonella
context (1–1000 μg/m3). Conversely, new limits for benzene levels in urban air were Mansi1, Daniela Pigini1, Sergio
set (5 μg/m3). The biomonitoring of exposure to such low benzene concentrations are Iavicoli1 & Giovanna Tranfo*,1
1
INAIL Reaserch, Department of
performed measuring specific and sensitive biomarkers such as S-phenylmercapturic Occupational & Environmental Medicine,
acid, trans,trans-muconic acid and urinary benzene: many studies referred high Epidemiology & Hygiene, Via Fontana
variability in the levels of these biomarkers, suggesting the involvement of polymorphic Candida 1 - 00040 Monte Porzio Catone
metabolic genes in the individual susceptibility to benzene toxicity. We reviewed the (RM), Italy
*Author for correspondence:
influence of metabolic polymorphisms on the biomarkers levels of benzene exposure
Tel.: +39 069 418 1436
and effect, in order to understand the real impact of benzene exposure on subjects g.tranfo@ inail.it
with increased susceptibility.

First draft submitted: 1 July 2015; Accepted for publication: 5 October 2015; Published
online: 14 January 2016

Keywords: benzene • biomarker of exposure • biomarker of susceptibility • environment

• genetic polymorphism • leukemia • occupational setting

Benzene is a ubiquitous pollutant widespread trans,trans-muconic acid (t,t-MA) and uri-

in both occupational and environmental set-
tings  [1] . Many years have passed since ben-
zene was classified, for the first time, as Car-
cinogen (Group I) by International Agency
for Research on Cancer (IARC) [2] . Since
then, due to improved industrial hygiene,
occupational benzene exposure has steadily
declined, so that no significant difference
could be found between environmental and
occupational benzene exposure, with concen-
trations ranging between 1 and 1000 μg/m3
(<0.001–0.312 ppm) [3,4] . On the other side,
more stringent environmental regulations
have set new limits to control benzene con-
centration in urban air [5] . The achievement of
these results has pointed out that biomonitor-
ing of exposure to such low benzene concen-
trations requires the use of one or more spe-
cific and sensitive biological marker that can
be used for risk assessment of exposed popu-
lations: S-phenylmercapturic acid (S-PMA),
nary benzene (U-B) [4] , whose determination
is allowed by the recent availability of highly
sensitive and selective analytical techniques.
Many studies over the years referred a high
variability in levels of biomarkers following
benzene exposure, suggesting that polymor-
phisms of genes involved in benzene metabo-
lism may significantly modify the individual
susceptibility to benzene toxicity [6] . These
findings may be useful to understand the real
impact of benzene exposure on subgroup of
subjects with increased susceptibility. In this
review, we analyzed the literature published
since year 2001 regarding the relationship
between metabolic polymorphisms and the
benzene urinary biomarkers.
Bibliographic search strategy
Articles were retrieved from Scopus and
PubMed databases using optimized search
part of
strategies developed in the Occupational

10.2217/bmm.15.106 © 2016 Future Medicine Ltd Biomark. Med. (2016) 10(2), 145–163 ISSN 1752-0363 145
Review  Carbonari, Chiarella, Mansi, Pigini, Iavicoli & Tranfo
Medicine field. We compiled a list of search terms
(either Medical Subjects Heading [MeSH] or non-
MeSH) seeming pertinent to biological monitoring for
benzene exposure. Limits were set for articles added
to Scopus and PubMed databases from 2001 to 2015.
We carried out different researches using the follow-
ing key words or search strings in both databases:
• Benzene AND (biological monitoring OR bio-
marker) AND genetic polymorphism;
• Genetic polymorphisms AND benzene metabolism;
• Benzene exposure AND leukemia; benzene suscep-
tibility AND leukemia.
Exclusion criteria were: language other than Eng-
lish, book chapters.
All articles have been carefully read and selected in
relation to the relevance for the objectives of the study.
In total 110 papers have been selected. Some papers
were retrieved as cross-references.
Noncancerous & cancerous effects of
benzene on the human health
The toxic and carcinogenic effects of benzene on
humans have been widely characterized. In experi-
mental and epidemiologic studies, it has been reported
that chronic exposure to benzene is associated with
decrease in the production of both erythrocytes, leu-
kocytes and platelets from bone marrow in humans,
resulting in aplastic anemia, thrombocytopenia and
pancytopenia. B-cell and T-cell proliferation is also
reduced by benzene, altering the immune system
response [7] . Furthermore, after exposure to very high
benzene concentrations, a hematotoxic syndrome
called chronic benzene poisoning (CBP) has been
described, the effects of which can continue years
after the workers were removed from the source of
exposure [8] .
Nowadays it is widely accepted that benzene exerts
its carcinogenic effect on the bone marrow and
lympho-hematopoietic system [2] . The key of ben-
zene carcinogenicity resides in its metabolism, where
electrophilic metabolites are produced together with
highly reactive oxygen species (ROS) [9,10] . These
compounds induce a condition of oxidative stress
onto lipids, proteins and nucleic acids. The oxida-
tion of such biological cell components results in cell
damage, DNA lesions and strand breaks, favoring
mutagenesis and chromosomal aberrations as well as
alteration of RNA transcription and protein transla-
tion process [11] . As a result, the benzene uptake by
bone marrow may affect the stem cell differentia-
tion and proliferation program leading to malignant
146 Biomark. Med. (2016) 10(2)
transformation of hematopoietic cells [12] . Nowadays,
although many studies on the cancerous effect of
benzene have been carried out, the precise molecu-
lar mechanism of benzene-induced ­leukemogenesis is
still unclear.
Benzene exposure in the environmental &
occupational settings
Environmental exposure to benzene is quite variable
because it is influenced by several factors like individ-
ual lifestyles, weather conditions, living environments
and geographical location. The main environmental
sources of benzene exposure are related to fuel emission
exhaust from motor vehicles and evaporation losses
from handling, distribution and storage of fuels [13] .
Another major source of environmental benzene
exposure is tobacco smoking for both mainstream and
second-hand smokers [14] . It has been found that smok-
ers receive 90% of their total benzene exposure from
smoking and that benzene concentration in the breath-
ing air of smokers can be 10–20-times higher than in
nonsmokers, with a peak level of 0.16 ppb [15,16] . Other
sources may also contribute to the cumulative benzene
indoor concentration. Household items like paints,
adhesives, marking pens, rubber products and tapes
were estimated to contribute overall 0.1–140 ppm [17] .
Considerably lower exposure to benzene (<1% of
the total body burden) can occur from other ways like
consumption of cooked food (1–190 ppb of benzene
concentration) water and beverage [18] .
Occupational exposure to benzene mainly occurs in
the petrochemical industry, coke oven and steel plants
where benzene emissions are a consequence of the pro-
ductive processes. In this regard, many studies report
that workers handling petroleum derivatives like
aviation workers, service station workers, bus drivers,
police, cargo tanker workers and urban workers may be
exposed to benzene at concentrations ranging between
0.003 and 30.8 ppm in about 8-h shifts [4,19–22] . Occu-
pational exposure to benzene may be also due to its
deliberate use when it is utilized as a reagent (synthe-
sis of other chemicals starting from benzene) or final
product (benzene synthesis). Finally, benzene is also
an important raw material utilized in the manufac-
turing of synthetic rubber, gums, lubricants and other
­pharmaceuticals and agricultural chemicals [1] .
In order to control benzene exposure in occupa-
tional and nonoccupational settings, many regulations
were adopted in several countries. To reduce the con-
tribution of exhaust emission to the airborne benzene
concentration, the Directive 98/70/EC [23] established
since 2000 a content of benzene in gasoline less than
1% v/v instead of the previous 5%. The same author-
ity, with the Directive 2000/69/EC [5] , established at
future science group

5 μg/m3 (calendar year or annual mean) the limit value
for benzene concentration in urban air, equivalent to
1.54 ppb.
In many occupational settings, except for those of
developing countries where higher levels of exposure
are still reported, occupational exposure to benzene is
quite lower than the limit value (3.25 mg/m3, 1 ppm)
set by European Directive 97/42/EC [24] while the
American Conference of Governmental Industrial
Hygiene (ACGIH) set the Threshold Limit Value
(TLV®) to 1.6 mg/m3 (0.5 ppm) [25] .
Recently, many studies have observed that the same
toxic and carcinogenic effects caused by exposure to
benzene levels of tens of ppm have been found in work-
ers exposed to less than 1 ppm [12,26,27] . Other stud-
ies focused on low-dose benzene metabolism show a
superlinear behavior of both benzene induced hema-
totoxic effects [12] and dose-specific metabolism [28,29]
at exposure levels lower than 1 ppm. This last param-
eter is calculated by dividing the background-adjusted
concentration of each metabolite, and their sum (total
metabolites) by the corresponding airborne benzene
concentration  [30] . Kim et al. found a ninefold reduc-
tion in dose-specific metabolism ranging from 0.03 to
88.9 ppm [28,29] . These results are in accordance with
others reporting that four volunteers, inhaling 40 ppb
of 13C-benzene for 2 h time period, metabolized the
benzene more rapidly than workers exposed to ppm
levels  [31] . A superlinear relationship between low-dose
(under 1 ppm) of benzene exposure and specific ben-
zene-related protein adducts levels are also reported [32] .
Rappaport et al. (2009 and 2010) explained this behav-
ior hypothesizing a benzene metabolism with two path-
ways, catalyzed by two different enzymes. This model
predicted that a high affinity (low dose) pathway was
responsible for about 73% of all benzene metabolism at
nonsaturating (ppb) air concentrations [33,34] .
Benzene metabolism & biomarkers of
exposure
Since it is widely accepted that the cause of the benzene
toxicity can be attributed to its metabolism, the focus
of the most recently works is about the balance of its
related intermediates and the fractional distribution of
metabolites formed.
Benzene uptake occurs mainly by inhalation in
both occupational and nonoccupational exposure.
This compound undergoes biotransformation within
the liver but can be metabolized also in the lung and
bone marrow. Inhaled benzene is readily absorbed into
the blood at 40 to 70% of airborne dose by passive
­diffusion through alveolar capillary membranes.
The metabolism of benzene is rather complex and is
shown in Figure 1. First benzene is oxidized to benzene
future science group
Biomarkers of susceptibility following benzene exposure  Review
oxide (BO) a molecule in equilibrium with oxepin.
Recent findings hypothesized, for exposures in the
ppm range, that this oxidation would be catalyzed,
mainly in the liver, by two isoforms of cytochrome
P450 (CYP) oxidase: 2E1 and 2B1, with high and
low affinity, respectively [33,34] . BO, following oxida-
tion and opening of the ring, is converted into t,t-MA
which is excreted in the urine. A small fraction of BO
(about 1% of total benzene uptake at 0.1–10 ppm of
exposure) is detoxified through conjugation with glu-
tathione (GSH), by glutathione-S-transferases (GSTs),
and excreted in urine as S-PMA [28,29] . Unmetabolized
benzene may also be found in urine (U-B) after pas-
sive diffusion from blood to urine (<0.1% of internal
dose)  [35] . The majority of BO spontaneously rear-
ranges to phenol (PH) which is oxidized by CYP2E1
to hydroquinone (HQ), catechol (CAT) and then to
1,2,4 benzenetriol (BT). CAT, a further major toxic
metabolite, can also derive by oxidation from benzene
dihydrodiol (BDHD), formed by the hydrolysis of
BO by microsomal epoxide hydrolase (EPHX1). The
intermediates HQ, CAT and BT can undergo further
oxidation to the respective and much more toxics semi-
quinones (1,4-SQ; 1,2-SQ and 1,2,4-SQ) and benzo-
quinones (1,4-BQ and 1,2-BQ). These reactions are
catalyzed by myeloperoxidase (MPO) in the target
organ (bone marrow). In this tissue, the MPO activ-
ity is balanced by the two- or four-electron reduction
catalyzed by the NAD(P)H:quinone oxidoreductase 1
(NQO1), which thus has a protective effects compared
with MPO.
Considerable evidence suggests that detoxification
of the high reactive benzene intermediates, like HQ
and BQs, may induce ROS production, DNA damage
and mutations [36] . Since benzene exposure is associ-
ated to the production on these reactive intermediates
able to induce various forms of genetic damage, accu-
rate estimation of human exposure to even low levels
of benzene is therefore needed for the protection of
both occupational and environmental health effects.
Numerous studies have examined the suitability of
potential biomarkers which could be highly specific
and sensitive, on the basis of their relationships with
external exposure [4,19,26,28,29] .
t,t-MA and S-PMA are considered the most sensi-
tive urinary biomarkers for benzene exposure in the
range 0.1–1.0 ppm [29,37] , but at concentration below
0.1–0.5 ppm their validity has been questioned [4,38] .
In humans exposed to benzene air concentrations
between 0.1 and 10 ppm, Rappaport and co-workers
observed that PH represents the most abundant (70–
85%) urinary benzene metabolite whereas HQ, t,t-MA
and CAT represent the 5–10%, and S-PMA represents
less than 1% of metabolites [33] . In 2013, ACGIH pro-
www.futuremedicine.com 147
Review  Carbonari, Chiarella, Mansi, Pigini, Iavicoli & Tranfo

OH
O OH
O
S OH
N O
H Benzene Benzene diol epoxide

S-Phenylmercapturic acid, CYP2E1

GS CYP
SPMA
T
OH
O
Epoxide hydrolase
Benzene oxide OH
Benzene dihydrodiol
Nonenzymatic
O Rearrangement
Oxepin
CYP HO
CYP2E1
HO HO

Phenol Catechol
O
O CYP2E1
T,T-Muconaldehyde MPO NQO1

OH O
O HO OH
HO O
OH HO OH
Hydroquinone
O
T,T-muconic acid, 1,2,4-benzenetriol 1,2-Benzoquinone
TTMA MPO NQO1

O O

1,4-Benzoquinone

Figure 1. Benzene metabolic detoxification pattern.

posed t,t-MA and S-PMA as biomarkers of low levels for
the occupational exposure to benzene. The two metab-
olites replaced PH as marker for biological monitoring,
being the latter unsuitable for measuring exposure at
concentration below 16.25 mg/m3 (5 ppm) being also
a metabolite of other aromatic compounds [39] . t,t-MA,
having a median half-life of 5.1 ± 2.3 h [40] , has been
the biomarker mostly used in the routine biological
monitoring because of the widespread availability of
ultraviolet high-performance liquid chromatography
(HPLC-UV) technology. However, t,t-MA is not a
specific biomarker of benzene exposure since its excre-
tion is influenced by the metabolism of sorbic acid,
an anti-fungal preservative used in food and cosmet-
ics  [41,42] . On the other hand, S-PMA is considered a
more specific and sensitive biomarker of exposure for
very low doses of airborne benzene (up to two orders
below the threshold value of 0.5 ppm) so that, in some
cases, it is the first choice for monitoring occupational
exposures  [39] . The mean half-life of S-PMA ranges
from 9 to 13 h with a second phase of slow elimination
that has a half-life of 45 h [43] . Many studies report a
good correlation between S-PMA with personal expo-
sure, U-B and other metabolites [1,43–47] . Neverthe-
less, the urinary concentration of S-PMA is affected
by the enzyme activity of the polymorphic GSTT1
and GSTM1 genes [3,44,48] . Cigarette smoke contains
benzene that affects both t,t-MA and S-PMA levels in
the urine, thus it should be taken into account dur-
ing the assessment of the exposure to benzene in the
workplaces. Unmetabolized benzene may also be used
to determinate the internal exposure.
Nowadays the available analytical techniques are
sensitive enough to detect benzene in three different
biological matrices: blood, urine and exhaled air [13] .
Although dependent on the dose, the biological half-
life of benzene is approximately 10 h or less. Hoet
et al. [1] found that blood benzene concentration (B-B)
was a specific marker of internal dose, which showed
a good correlation with urine benzene concentration
(U-B) and S-PMA; B-B was also able to differentiate
between smokers and nonsmokers. Many authors also
referred that U-B is a better discriminatory marker of
internal dose at low benzene exposure levels [4,6,47] .

148 Biomark. Med. (2016) 10(2) future science group


Other studies, however, reported that U-B was able
to distinguish smokers from nonsmokers but a large
­inter-individual variability was also observed [1,22] .
The human detoxification systems work in order
to minimize the potential of damage from xenobiot-
ics, like benzene; several reports have shown that indi-
vidual susceptibility to the adverse effects of benzene
exposure is partly attributable to the genes involved in
the biosynthesis of metabolic enzymes [49,50] . Indeed,
metabolic polymorphisms may alter the balance
among benzene intermediates responsible for the dam-
aging effects, especially at low levels of exposure when
the metabolism could be intensified [32–34] .
Polymorphic genes of benzene metabolism
as biomarkers of susceptibility
Biomarkers of susceptibility are indicators of an
increased sensitivity to the effects of an environmental
agent that can be objectively measured in a biological
system or a sample [13] . Since the genetic susceptibil-
ity may affect the individual response to xenobiotics in
different environmental and occupational contexts, a
growing number of studies are exploring how genetics
and exposure interact in the etiology of diseases [50] .
The carcinogenic effect of benzene on the cell acts
through the production of toxic metabolites as well
as by induction of oxidative stress and DNA damage.
In particular, DNA base lesions such as 8-hydroxy-
2′-deoxyguanosine (8-OHdG) generation, are highly
mutagenic and in the absence of functional DNA
repair enzymes, they can lead to malignant transfor-
mation. Since the variety of polymorphic genes within
the human population affects the function and the
efficacy of any enzyme activity, we discuss here three
categories of polymorphisms influencing benzene
metabolism: polymorphic genes involved in benzene
biotransformation; polymorphic genes involved in the
oxidative stress; polymorphic genes involved in the
DNA repair mechanism.
Polymorphic genes involved in benzene
biotransformation
CYP2E1 is involved in the first step of benzene bio-
transformation pathway [33] . Oxidation of benzene by
CYP2E1 to reactive intermediates is a prerequisite of
cellular toxicity as well as a limiting step in metabolites
excretion. CYP2E1 polymorphisms have been reported
in the promoter gene, within the binding sequence
for the transcription factor HNF-1 (CYPE1*5B RsaI/
PstI sites) and in the exon 6 (CYP2E1*6 DraI site).
Actually it is not possible to identify an overall effect
of these polymorphisms on the enzymatic function
of CYP2E1 [51] . In this regard, recent studies focused
on the effects of CYP2E1 genotypes on the biomarker
future science group
Biomarkers of susceptibility following benzene exposure  Review
levels of benzene with conflicting results [3,4,51,52] .
Kim et al. [52] reported a reduction of the urinary con-
centration of the benzene metabolites t,t-MA, PH and
HQ in Chinese workers carrying the homozygous vari-
ant genotype CYP2E1*5B (C105T) than those with at
least one wild-type allele. This observation was in agree-
ment with a reduction of the enzyme activity caused by
the lower expression of the CYP2E1 variant allele com-
pared with the wild-type [53] . In the same study, the
difference of the metabolite concentrations of t,t-MA,
PH and HQ between the two genotypes increased at
airborne benzene concentration above 0.1 ppm. On
the contrary, Fustinoni et al.  [4] reported a signifi-
cantly increased excretion of t,t-MA in heterozygous
subjects compared with the wild-type homozygote for
CYP2E1*5B allele, under conditions of benzene expo-
sure between 2 and 147 ppb (time weighted averaged
on the working shifts); in the same study, similar find-
ings were reported for CYP2E1*6 genotype. Subjects
carrying the heterozygote and homozygote variants
excreted much higher concentrations of t,t-MA than
those having the homozygote wild-type ones. These
results were also confirmed after adjustment for several
covariates. Other authors also considered the effects
of CYP2E1*5/*6 genotypes on other benzene metabo-
lites like S-PMA, PH, B-B and U-B. Some of them
reported no effects on S-PMA excretion levels [4,51,52,54]
or blood benzene [55] , while others reported a reduc-
tion of U-B in subjects with CYP2E1*5B heterozygote
variant compared with the wild-type homozygote [4] .
Conflicting results were also reported about the effect
of CYP2E1*5B on PH urinary levels [51,52] . Finally,
many authors referred of no effects of CYP2E1*5/*6
genotypes on benzene metabolites [1,3,56] . The explana-
tion of these conflicting findings could be found on
the questionable real effect of the genotype on the pre-
dicted CYP2E1 enzyme activity and on the rare fre-
quency of allele variants that is very low among Cauca-
sian (1–5%) and much higher in Oriental populations
(19–28%) [50] . The rarity of the CYP2E1 allele variant
may affect the statistical power of the studies often per-
formed on little cohorts of subjects. Further investiga-
tions on gene expression and/or on post-transcriptional
regulation could help to elucidate the real impact of the
CYP2E1 genotypes on benzene metabolism in order to
improve the ­interpretation of the data obtained from
biomonitoring.
The GST super gene family consists of several
gene subfamilies including glutathione s-transferase
M1 (GSTM1), glutathione s-transferase T1 (GSTT1)
and glutathione s-transferase P1 (GSTP1)  [48] . Both
GSTT1 and GSTM1 are involved in the detoxification
of BO to S-PMA [28,29] . Genetic variants of GSTM1
and GSTT1 consist of the complete deletion of the
www.futuremedicine.com 149
150
Table 1. Description of the studies published since 2001 concerning biomarkers of internal dose and metabolic polymorphisms of exposed workers in
different countries.
Study (year) Country Exposed subjects Environmental Personal exposure Exposure level (ppm) Biomarkers of Metabolic Ref.
monitoring internal dose polymorphisms
Carbonari Italy 301 male Not performed Measurement by 0.021 mg/m3 (0.0065 T,t-MA, S-PMA CP1A1*2A (mspI_site), [3]
et al. (2014) petrolchemical Radiello® during ppm) at the end of the CYP2E1*5B (rsaI_site),
workers the entire work work shift NQO1*2 C609T
shift (∼8 h) (hinfI_site), MPO
G463A, GSTM1, GSTT1,
GSTA1*A/B, EPHX1
Tyr113His and EPHX1
His139Arg
Mansi et al. Italy 315 Not performed     T,t-MA, S-PMA GSTM1, GSTT1, GSTP1 [49]

Biomark. Med. (2016) 10(2)

(2012) petrolchemical at the end of the Ile105Val
workers work shift
Carrieri et al. Italy 28 male Not performed Measurement by 0.034 mg/m3 (0.01 T,t-MA, S-PMA GSTT1, GSTM1 [44]
(2012) petrolchemical Radiello® during ppm) [0.002–0.895 at the end of the
workers the entire work mg/m3] [0.0006–0.275 shift
shift (∼8 h) ppm]
Angelini et al. Italy 70 urban Municipal Measurement by 19.33 μg/m3 (0.006 S-PMA at the CYP2E1*5B (rsaI_site), [104]
Review  Carbonari, Chiarella, Mansi, Pigini, Iavicoli & Tranfo

(2011) policemen monitoring station Radiello® during ppm) [13.46–31.41 μg/ end of work CYP2E1*5 -6/8 repeats,
(unexposed: 40 in suburban area the entire work m3] [0.004–0.01 ppm] shift NQO1C609T (hinfI_
indoor workers) 10–15 μg/m3 shift (∼6 h) site), MPO G463A,
[0.003–0.005 ppm] EPHX1 Tyr113His, EPHX1
His139Arg, GSTT1,
GSTM1, GSTP1Ile105Val
and Ala114Val
Manini et al. Italy 100 traffic Not performed Measurement by Traffic policemen and S-PMA at the NQO1*2 C609T (hinfI_ [48]
(2010) policemen, 37 Radiello® during taxi drivers: 6 μg/m3 end of work site), GSTT1, GSTM1,
taxi drivers, 102 the entire work (0.002 ppm), gasoline shift GSTA1*A/B
gasoline pump shift (∼8 h) pump attendants:
attendants 15 μg/m3 (0.005 ppm)
Hoet et al. Europe 110 male Not performed Measurement [0.1–4.3 ppm] B-B, U-B, S-PMA, CYP2E1*6, CYP2E1*7B, [1]
(2009) petrolchemical at the breathing t,t-MA at the CYP2E 1*1D, GSTM1,
workers zone level during end of work GSTT1, GSTP1 Ile105Val
the entire work shift EPHX1 Tyr113His and
shift EPHX1 His139Arg
Lin et al. China 70 male workers Not performed Measurement by a TWA® : 7.2 ± 15 ppm; T,t-MA, S-PMA GSTM1, GSTT1, GSTP1 [47]
(2008) chemical diffusive sampling [0.0052–70 ppm] at the end of the Ile105Val
synthesis factory method during median: 0.62 ppm work shift
the entire work
shift

future science group

 Table 1. Description of the studies published since 2001 concerning biomarkers of internal dose and metabolic polymorphisms of exposed workers in
different countries (cont.).
Study (year) Country Exposed subjects Environmental Personal exposure Exposure level (ppm) Biomarkers of Metabolic Ref.
monitoring internal dose polymorphisms
Buthbumrung Thailand 109 school­ Measurement by Measurement by a Urban schoolchildren: T,t-MA, B-B, U-B CYP2E1*5B (rsaI_site), [56]

future science group

et al. (2008) children in urban passive sampler passive sampler set [5.50 ± 0.40 ppb]; NQO1*2 C609T (hinfI_
area (unexposed: urban area: 17.75 at the breathing rural schoolchildren: site), GSTM1, GSTT1,
62 boys ± 2.23 ppb; inside zone for 8 h [2.54 ± 0.23 ppb] GSTA1*A/B
schoolchildren in school: 8.25 ±
rural area) 0.78 ppb; rural
area: 4.49 ± 0.59
ppb; inside school:
2.71 ± 0.38 ppb
Kim et al. China 250 shoe-making Not performed Full-shift Median: 0.512 ppm Urinary- CYP2E1*5B (rsaI_site), [52]
(2007) and clothes- measurement by [0.002–6.40 ppm] benzene, NQO1*2 C609T (hinfI_
manufacturing passive samplers urinary-PH, site), NQO1*1 C645T,
workers (136 during the entire urinary-CAT, MPO G463A, GSTM1,
controls) work shift urinary-HQ, t,t- GSTT1, GSTP1 Ile105Val,
MA, S-PMA at EPHX1 Tyr113His, EPHX1
the end of the His139Arg
work shift
Chanvaivit Thailand 62 exposed: Laboratory: 26.56 ± Measurement by Laboratory workers: B-B, t,t-MA at CYP2E1*5B (rsaI_site), [55]
et al. (2007) 31 laboratory 5.44 ppb; gasoline diffusive badges 24.40 ± 5.82 ppb; the end of the NQO1*2 C609T (hinfI_
workers, 31 service stations: during the entire gasoline service work shift site), GSTT1
gasoline service 46.81 ± 5.10 ppb work shift (8 h) attendants: 112.41
attendants (34 ± 13.92 ppb; control
controls) workers: 1.39 ± 0.17
ppb
Manini et al. Italy 37 taxi drivers Measured by Measurement by 5.9±1.7 μg/m3 [0.002 U-B, t,t-MA, NQO1*2 C609T [22]
®
(2006) municipal air Radiello during ppm] S-PMA at the (hinfI_site), GSTT1,
monitoring station: the entire work beginning and GSTM1, GSTA1*A/B,
3.25 ± 0.50 μg/ shift (∼11.1 ± 1.7 h) at the end of the EPHX1 Tyr113His, EPHX1
m3 [1 ± 0.01 ppb] work shift His139Arg
measurement on
taxicab: 7.7 ± 2.0
μg/m3 [2.4 ± 0.62
ppb]

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Biomarkers of susceptibility following benzene exposure 

151
Review
 Table 1. Description of the studies published since 2001 concerning biomarkers of internal dose and metabolic polymorphisms of exposed workers in

152
different countries (cont.).
Study (year) Country Exposed subjects Environmental Personal exposure Exposure level (ppm) Biomarkers of Metabolic Ref.
monitoring internal dose polymorphisms
Fustinoni Italy Exposed: 78 Not performed Measurement by Genoa: bus drivers: U-B, t,t-MA, CYP2E1*5B (rsaI_site), [4]
et al. (2005) gas station Radiello® during 21 μg/m3 (6 ppb), S-PMA at the CYP2E1*6 (draI_site),
attendants the entire work 9 μg/m3 (2.8 ppb); beginning and NQO1*2 C609T (hinfI_
and 77 urban shift (∼6 h) Milan: traffic at the end of site) 
policemen in policemen: 22 μg/m3 monitored
Milan, 153 bus (6.8 ppb), gas station period
drivers in Genoa attendants: 61 μg/m3
(unexposed: 107 (18.8 ppb), 6 μg/m3
office workers) (1.8 ppb)

Biomark. Med. (2016) 10(2)

Avogbe et al. Benin 29 taxi drivers, Not performed Estimated by Rural area: 2 μg/m3 S-PMA GSTT1, GSTM1, GSTP1 [60]
(2005) 37 roadside evaluating the (0.6 ppb), suburban Ile105Val, NQO1*2 C609T
residents, S-PMA excretion area: 13 μg/m3 (4 ppb), (hinfI_site)
42 suburban levels cityroad: 40 μg/m3
residents (27 (12.3 ppb), taximoto
rural controls) drivers: 121 μg/m3 (37.2
ppb)
Review  Carbonari, Chiarella, Mansi, Pigini, Iavicoli & Tranfo

Qu et al. China 130 factory Not performed Measurement by a 0.06–122 ppm T,t,-MA, S-PMA CYP2E1*5B (rsaI_site), [51]
(2005) workers (51 diffusive sampling Median: 3.2 ppm at the end of CYP2E1*6 (draI_site),
controls) method during work shift NQO1*2 C609T (hinfI_
the entire work site), MPO G463A,
shift GSTT1
Sorensen Estonia 50 underground Measurement by Measurement by Underground T,t-MA, S-PMA GSTT1, GSTM1, GSTP1 [58]
et al. (2004) mine workers a passive sampler a passive sampler workers: 190 ± 50 μg/ at the beginning Ile105Val
(unexposed: underground: 290 during the entire m3 [58.5 ± 15.4 ppb]; and at the end
50 male surface ± 44 μg/m3 [89.2 ± work shift (∼8 h) surface workers: 114 ± of work shift
mine workers) 13.5 ppb]; Surface: 35 μg/m3 [35.1 ± 10.8
130 ± 27 μg/m3 ppb]
[40.0 ± 8.3 ppb]
Sorensen Denmark 40 subjects living Not performed Measurement by 2.53 μg/m3 (0.8 ppb) T,t-MA, S-PMA GSTT1, GSTM1, GSTP1 [59]
et al. (2003) and working Radiello® during [1.86–3.61 μg/m3] at the beginning Ile105Val, NQO1*2
in central a period of 5 days (0.6–1.1 ppb) and at the end C609T (hinfI_site)
Copenhagen (∼110 h) of work shift
Verdina et al. Italy 118 outdoor Not performed Measurement by TWA-7h outdoor B-B, t,t-MA, CYP2E1*5B (rsaI_site) [54]
(2001) police officers Radiello® during workers: 9.3 μg/m3 S-PMA at the GSTT1, GSTM1, NQO1*2
(unexposed: the entire work [2.9 ppb] TWA-7h end of work C609T (hinfI_site)
51 Indoor police shift (∼7 h) indoor workers: 3.7 shift
officers) μg/m3 [1.13 ppb]

future science group


genes and the loss of the corresponding enzyme activ-
ity [57] . In GSTP1 genotype, a single-nucleotide substi-
tution (A to G) at nucleotide 313 causing the change
of Isoleucine to Valine, decreases the GSTP1 enzyme
activity  [47] . GSTM1 and GSTT1 are the most studied
genetic variants of the benzene metabolic genes, since
they have given the most concordant results, especially
toward the S-PMA excretion. Due to the specificity of
the reaction catalyzed by these enzymes, the effect of
their variants is detectable also at low levels of expo-
sure. On the basis of the predicted enzyme activity
many authors reported a significant reduction of the
excretion of S-PMA in subjects carrying the GSTT1
null genotype [44,48,49,51,52,58–60] . Carbonari et al.  [3]
demonstrated that in GSTT1 null genotypes, the aver-
age S-PMA excretion was about 50% with respect
to the positive ones, for the same benzene exposure.
Lin et al. [47] showed that GSTT1 positive subjects had
2.2-times higher average urinary SPMA level than
the GSTT1-deficient subjects. These findings give us
very important information, because the influence of
GSTT1 genotype on S-PMA excretion is shown to be
independent from the benzene airborne concentration:
in these studies the exposure levels of the populations
on which the correlation between GSTT1 genotype
and S-PMA excretion was observed, ranged from
0.0006 to 122 ppm. Otherwise, other authors failed
to detect any effect of GSTT1 genotype on S-PMA
­excretion levels  [1,22,54,56,59] (Table 1) .
As it concerns the influence of GSTM1 genotype on
the urinary S-PMA levels, the results are very differ-
ent. Some authors showed a significant reduction in
S-PMA levels in subjects carrying the null genotype
with respect to the wild-type [22,48,52] . Recently, our
group reported a reduction of both S-PMA and t,t-
MA/S-PMA concentration ratio (R) associated with
the GSTM1 null genotype in smokers (high exposure
subjects). These results show that R is a valid tool to
evaluate the effect of genetic differences on the ben-
zene metabolite excretion, independently from the
personal benzene exposure [3,49] . Finally, other groups
failed to observe any influence of GSTM1 genotype on
S-PMA levels [1,44,47] .
Different results were obtained about the influ-
ence of GSTs polymorphisms on t,t-MA excretion
since this pathway is independent from their cor-
responding enzyme activities; many authors studied
this relationship failing to find any correlation [1,3,4,44,
47–49,52,54,56,58–60] .
More interesting findings were obtained with respect
to the influence of GSTA1–1 genetic polymorphisms
on the formation of benzene metabolites. Manini et al.
reported that subjects carrying the homozygous vari-
ant for GSTA1*A/B excreted significantly lower uri-
future science group
Biomarkers of susceptibility following benzene exposure  Review
nary S-PMA levels than those carrying at least one
wild-type allele [22,48] . Recently, our group confirmed
this result reporting higher urinary levels of S-PMA
and lower of R value in subjects carrying the wild-type
genotype than both the heterozygous and the mutant
genotypes  [3] . Moreover, Manini et al. concluded that
in subjects who are defective for the activity of one
GST enzyme, another isoform could effectively com-
pensate it [48] . Our results confirmed this speculation,
but highlighted that this is true only for GSTM1 and
GSTA1 defective subjects but not with GSTT1 [3] .
No effects of GSTP1 genotype on benzene metabo-
lism were reported by the authors who investigated this
hypothesis [1,3,47,52,58–60] .
Two EPHX1 genotypes, Tyr113His and His139Arg
were also studied in relation to their effect on the
benzene metabolism since it has been shown that
these polymorphisms influence the corresponding
enzyme activity [61] ; however, few groups studied these
polymorphisms as modifiers of markers of internal
dose [1,3,22,52] and the results were mostly inconsistent.
NQO1 catalyzes the two-electron reduction and
detoxification of quinones and their derivatives, avoid-
ing the formation of free radicals (semiquinones) and
ROS, hence protecting cells against the adverse effects
of quinones and their derivatives [62] .
NQO1*2 (C609T) is the more prominent polymor-
phism, in terms of both frequency and phenotypic
consequences. It consists of a proline to serine sub-
stitution at position 187 of the amino acid sequence
of the protein. The mutant NQO1*2 protein is very
unstable, and is rapidly ubiquitinated and degraded by
the proteasome. Thus, both NQO1 protein and activ-
ity are virtually undetectable in humans carrying the
NQO1*2/*2 genotype [63] .
The contribute of NQO1*2 polymorphism on
benzene metabolism is quite difficult to investigate
since it is not directly involved on urinary metabo-
lite excretion. Many studies tried to find possible
effects  [3,4,48,51,52,55,56,59] and one of these found that,
at very low level of exposure (0.0008 ppm), subjects
with mutant and heterozygote alleles had significantly
higher levels of t,t-MA than wild-types [59] . Others
failed to find any effect on t,t-MA excretion levels.
Again, some authors studied the influence of NQO1*2
on urinary S-PMA [3,4,22,48,51–53,59,60] . Kim et al.  [52]
found that heterozygous and mutant genotypes were
associate with significant decrease of S-PMA excretion
levels in comparison with the wild-type subjects, as a
consequence of a reduced NQO1 activity. Our group
showed a significant lower median R value in subjects
with wild-type NQO1 genotypes with respect to both
the heterozygous and mutant genotypes in nonsmokers
(low exposure conditions) [3] . Additional studies have
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Review  Carbonari, Chiarella, Mansi, Pigini, Iavicoli & Tranfo
also investigated the effect of NQO1 genotype on PH
production  [51,52] and on the level of U-B [4,22] . In the
first case only Kim et al. were able to observe, after
adjustment for sex, age, smoking and body mass index
(BMI), a reduction of PH excretion with hetero- and
mutant genotypes with respect to the wild-type [52] .
Conversely, no effect of NQO1 genotype was reported
on U-B excretion levels [4,37] .
Like NQO1, MPO is not directly involved in the
metabolic pathways leading to the synthesis of the ben-
zene metabolites [50] . It has previously been shown that
the polymorphism in the promoter region of the MPO
gene, –463 G>A affects MPO activity [64] and some
authors have also tried to show a possible influence of
this polymorphisms on benzene metabolite excretion
but with conflicting results [3,51,52] .
Polymorphic genes involved in the oxidative
stress
Some of the polymorphic genes discussed previously
are implicated in the response to the oxidative stress.
During benzene detoxification electrophilic metabo-
lites and ROS are generated causing protein, lipid
and DNA breaks eventually leading to chromosomal
aberrations. However the cell capability to counter-
act the stressful insults relies on the proper functional
activation of a battery of antioxidant enzymes. Heme
oxigenase (HO-1), NQO1, GST-P1, thioredoxin
(TRX), catalase (CAT), superoxide dismutase (SOD)
are examples of enzymes involved in such defense
­mechanism.
Different epidemiologic studies correlated the sus-
ceptibility to benzene toxicity with certain variants
of polymorphic genes involved in the oxidative stress
response. In a study conducted on Bangkok school-
children, Buthbumrung et al.  [56] demonstrated that
oxidative DNA damage was significantly higher in the
urban schoolchildren with respect to the rural ones.
They observed that the concentration of 8-hydroxy-
2′-deoxyguanosine (8-OHdG) in schoolchildren
leukocytes correlated with benzene exposure levels,
underlining the effect of benzene exposure is the
oxidative damage of DNA. Genetic polymorphism
of CYP2E1, NQO1 and GSTs did not show any sig-
nificant influence on the 8-OHdG level, although
a correlation was found in subjects carrying the
GSTM1*2/*2 genotype. These individuals excrete
lower concentrations of urinary MA in comparison
to those having the GSTM1*/1 genotype, concluding
that the GSTM1*2/*2 genotype may be a suscepti-
bility marker in schoolchildren regularly exposed to
environmental benzene. NQO1 is a cytoprotective
antioxidant responsive element (ARE)-induced gene
under the control of the Nrf2/Keap1 system [65] . Two
154 Biomark. Med. (2016) 10(2)
single nucleotide polymorphisms in the gene encod-
ing for NQO1 have been described in humans, the
NQO1*2 and the NQO1*3. As stated above, the most
prominent in terms of frequency and phenotypic
consequences is NQO1*2, a single nucleotide poly-
morphism, showing C to T change at position 609
of the NQO1 DNA sequence. The mutant NQO1*2
polypeptide is rapidly degraded by the ubiquitin pro-
teasomal system resulting in a lack of NQO1 protein
in individuals carrying the NQO1*2/*2 genotype.
The NQO1*2 polymorphism predisposes to ben-
zene toxicity and to various forms of leukemias [66] .
In his review reporting human and animal studies,
Ross describes three major pieces of evidence showing
the correlation between NQO1*2 polymorphism and
­benzene h­ ematoxicity and leukemia.
Another interesting work [60] investigated the level
of oxidative DNA damage (strand breaks and for-
mamidopyrimidine (FPG) DNA glycosylase sensitive
sites) induced on mononuclear blood cells of urban
residents from three locations in Cotonou, Benin
and rural residents exposed to ultrafine particles and
benzene. Here the S-PMA was used as biomarker
of benzene exposure. Polymorphisms in glutathi-
one peroxidase, NQO1 and GST genes were assessed
for modification of effect. Individuals with GSTT1
null genotype had lower urinary S-PMA excretion
in comparison to those carrying the plus genotype.
The excretion of urinary S-PMA correlated with the
presence of strand breaks and FPG sites in mono-
nuclear blood cells. The correlation between S-PMA
and strand breaks was strongest in subjects with
NQO1*1/*2 and NQO1*2/*2 genotypes and between
S-PMA and FPG sensitive sites in subjects with the
GSTP1*B/*B genotype. The study states that NQO1
and GST polymorphic genes may modulate the effect
of oxidative DNA damage in mononuclear blood cells
of resident people exposed to high air levels of urban
benzene and ultrafine particles.
Polymorphic genes involved in the DNA repair
DNA damage induced by benzene is critical for its
genotoxicity, making the cell prone to cell cycle arrest,
programmed death, mutagenesis, genomic instability
and cancer. Therefore, genetic variation in DNA-repair
genes may alter the individual’s susceptibility and
capacity to repair the benzene-induced DNA lesions.
Three are the major pathways involved in the removal
of benzene-induced DNA lesions: the base excision
repair pathway, the nucleotide excision repair pathway
and the double strand break repair pathway [67] . Many
researchers are trying to elucidate the influence of the
DNA repair polymorphic genes on the ability to main-
tain the genomic stability and to correlate the genetic
future science group

variants with the increased susceptibility to certain
types of DNA lesions.
Using the micro nucleus frequency as marker
of genotoxic effect, Angelini et al.  [68] investigated
the influence of polymorphisms in the repair genes
APEX1, hOGG1, NBS1, XPD, XRCC1 and XRCC3 (see
referred paper for entire enzyme names) in peripheral
blood lymphocytes of 110 volunteers (70 urban traffic
wardens and 40 administrative staff members) exposed
to different levels of environmental benzene. A signifi-
cantly higher median micro nucleus frequency was
found in traffic wardens than in controls, despite none
of the analyzed polymorphisms was significantly asso-
ciated with the median micro nucleus frequency. The
APEX1 variant genotype was associated with signifi-
cantly lower median micro nucleus frequency in men,
not in women, evidencing a gene–gender i­nteraction
for the APEX1 genotype.
Xiao et al. studied the potential association between
the polymorphisms of ERCC1 (Excision repair cross-
complementation group 1) and ERCC2/XPD (excision
repair cross complementation group 2/xeroderma pig-
mentosum group D) with the risk of CBP. The case–
control study was carried out in the Northeast region
of China and involved 102 benzene poisoned patients
versus 204 healthy workers occupationally exposed
to benzene in the same factories with similar benzene
exposure environment [69] . The results highlight the
role of ERCC1 codon 118 TT polymorphisms as bio-
marker to CBP in the Chinese occupational population
since individuals carrying the ERCC1 codon 118 TT
genotype had an increased risk of CBP comparing with
the CC genotype.
The influence of polymorphic gene involved in the
DNA repair on the susceptibility to benzene was ana-
lyzed also in another study. Here the exposed subjects
were 50 bus drivers and 20 garagemen, working in the
center of Prague and 50 healthy volunteers. The first
two groups were supposed to be exposed to high levels
of air pollution [70] . The authors found that the carriers
of at least one mutant allele, hOGG1 (Ser/Cys or Cys/
Cys), tended to have higher levels of oxidative DNA
damage than the subjects with the wild-type (Ser/Ser)
genotype. The nonsmoking individuals with a variant
XPD 23 (Gln/Gln) genotype appeared to be more sus-
ceptible to the induction of DNA-strand breaks than
the heterozygotes (Lys/Gln) and homozygotes carrying
the wild-type allele (Lys/Lys). As stated by the authors,
DNA-strand breaks may be a prerequisite to damage
at the chromosomal level [71] . The findings imply that
individuals exposed to inhalable particulate matter
and carrying the XPD 23 (Gln/Gln) variant could be
at higher risk of cancer development than subjects with
the wild-type allele.
future science group
Biomarkers of susceptibility following benzene exposure  Review
Genetic polymorphisms & susceptibility to
leukemia
Although benzene may cause other noncancerous dis-
eases in humans including aplastic anemia, thrombo-
cytopenia and leukopenia, it is mainly known as a leu-
kemia-promoting carcinogen. The initial evaluation of
benzene carcinogenicity found sufficient evidence for
correlation with acute myeloid leukemia (AML) but
inadequate evidence for other hematological malignan-
cies  [2] . In 2012, IARC recognized adequate evidence
also for acute nonlymphocytic leukemia and limited
evidence for acute lymphocytic leukemia, chronic lym-
phocytic leukemia, multiple myeloma and non-Hodg-
kin’s lymphoma, highlighting the association between
benzene carcinogenicity and a variety of blood can-
cer [72–75] . Recent epidemiologic studies indicated also
correlation between maternal exposure to benzene and
occurrence of childhood leukemia, underlining the
role of environmental benzene concentration on the
human health during pregnancy and infancy [76,77] .
The main toxic effects of benzene comprise blood
cell number reduction and suppression of leukopoiesis,
resulting in increased susceptibility to infections. In
workers exposed to chronic dose, benzene can induce
chromosomal aberrations eventually leading to leu-
kemia  [77] . At cellular level, the biological effects of
benzene and its metabolites include DNA, protein and
lipid damage, via ROS induction. In particular, the
electrophilic intermediates 1,4-BQ and BO are able
to interfere with the cell functions forming covalent
adducts with the nucleophilic loci of Topoisomerase
II (a key enzyme for DNA replication) and affecting
other DNA-associated protein activity [78,79] . Instead,
HQ and CAT exert their toxic effects into the bone
marrow, causing suppression of hematopoiesis [78] .
In humans, a certain variable susceptibility to spe-
cific types of leukemia following benzene exposure
has been supposed to depend on the different activ-
ity of polymorphic genes involved in benzene metab-
olism  [11] . Several papers report associations between
genes having a direct or indirect role in benzene detoxi-
fication and leukemia onset. Here we discuss a small
number of studies conducted on diseased subjects and
selected polymorphic genes participating to benzene
metabolism (NQO1, CYP2E1 and GST ).
The role of the polymorphic gene NQO1 on ben-
zene metabolism and its protective activity achieved
through the detoxification of benzene-derived qui-
nones are well known [62] . However not all polymor-
phic NQO1 variants appear to be protective. A large
epidemiologic study of a benzene-exposed population
confirmed an association between an increased inci-
dence of benzene-induced hematotoxicity and subjects
having the mutant NQO1*2 (C609T, Pro187Ser), allele
www.futuremedicine.com 155
Review  Carbonari, Chiarella, Mansi, Pigini, Iavicoli & Tranfo
in comparison to those with the normal allele. Such
individuals exhibited a sevenfold greater risk of bone
marrow toxicity, with higher susceptibility to leukemia
and aplastic anemia [80] . This result is in agreement
with another study demonstrating transmission of
the variant NQO1*2 C609T allele is higher in fami-
lies whose children are affected by acute lymphocytic
­leukemia (ALL)  [81] .
Although there is no experimental evidence about
any effect of GSTP1 genotype on benzene metabolism,
a correlation of GSTP1 (A313G Ile105Val) gene poly-
morphism with the susceptibility to chronic myeloid
leukemia was analyzed, using a polymerase chain reac-
tion – restriction fragment length polymorphism (PCR-
RFLP) assay on 40 affected Egyptian patients, children
and adults, versus 40 healthy controls. The variant
allele IIe105Val was found to be associated with more
advanced phases of disease and with worse hemato-
logical and cytogenetic responses when compared with
the wild-type. Authors concluded GSTP1 (Ile105Val)
gene polymorphism conferred a significant association
with increased risk of chronic myeloid leukemia and
is associated with bad prognosis [82] . Another study
confirmed the influence of GSTP1 variants (alone or
combined with other GSTs) on children susceptibil-
ity to ALL. Results were obtained from 278 childhood
ALL patients compared with 378 healthy controls of
French–Canadian nationality. Subjects carrying the
GSTP1 Ile105Val variant showed association with an
increased risk of ALL. Furthermore authors found that
the combination of GSTP1 Ile105Val (GSTP1*B) and
GSTM1 null genotypes, further increased the risk of
ALL  [83] . The relationship between specific GST poly-
morphisms and leukemia occurrence was evaluated also
in a meta-analysis study of PubMed literature. Here
GSTM1-null genotype was found to be associated with
increased risk of AML in East Asians and GSTT1-null
genotype in Caucasians, with preference toward the
female gender [84] . However there are also studies where
a correlation between GSTP1 Ile105Val ­polymorphism
and leukemia did not emerge clearly [85] .
In another paper the possible association of three
CYP2E1 variants, alone or in combination, with the
risk of incidence of childhood ALL was investigated.
Polymorphic genes were analyzed by RFLP-PCR in
168 patients and 207 healthy controls of a Turkish pop-
ulation. The authors concluded that individuals carry-
ing combinations of CYP2E1*5B, *6 and *7B variants
together were more susceptible to the risk of develop-
ing childhood ALL [86] . Similar findings were obtained
in a meta-analysis study of papers reporting cases of
infant leukemia and polymorphic genes CYP2E1*5B
and NQO1 C609T, which were found to be highly
associated to the risk of childhood leukemia [87] .
156 Biomark. Med. (2016) 10(2)
Despite some data from the literature are contra-
dictory due to ethnic difference in the populations
or in the methodological approach, there is evidence
that polymorphic genes involved in benzene metabo-
lism influence the susceptibility to leukemia. From
this brief overview, more than one type of GST and
CYP2E1 polymorphism seems to be associated to a
higher susceptibility of developing leukemia whereas,
among NQO1 polymorphisms, the C609T variant
shows strong correlation with the disease risk. How-
ever, although polymorphisms influence the individual
metabolism of benzene, the genetic background is
not sufficient to explain leukemia occurrence follow-
ing exposure. The role of the environment, exposure
to other chemical and physical agents, diet, drug,
endogenous microbiome and stress should be taken
into account to evaluate the impact of complex dis-
eases such as the hematological malignancies. In other
words, genome wide association studies should be inte-
grated by exposome wide association studies in order
to identify more profound correlation between human
subjects and specific diseases [88] .
Conclusion & future perspective
Current studies suggest that chronic exposure to low-
levels of benzene, in both environmental and occupa-
tional settings, can determine long-term health con-
sequences, including cancer development. Therefore,
much interest has been focused on the identification of
biomarkers useful in monitoring exposed populations,
in order to improve the risk assessment.
Genetic polymorphisms as susceptibility biomark-
ers may be potentially useful to detect individuals
more susceptible to benzene hematotoxic effects, and
to allow a better interpretation of the results obtained
by biological monitoring. Actually, the only genes that
showed some consistent associations with both bio-
markers of exposure and effect, in line with the pre-
dicted enzyme activity, are GSTM1, GSTT1 and more
recently GSTA1. In fact, subjects carrying the genetic
variants associated to a reduced GSTs function, showed
a lower resistance to benzene toxicity. More recent
results have also shown a hierarchy among the GSTs,
hypothesizing a main and irreplaceable role of GSTT1
in the maintenance of GSTs functions.
These findings about the influence of metabolic
polymorphisms on the benzene detoxification path-
way may have a practical application in the improve-
ment of the occupational risk assessment of benzene.
First of all, the biological limit of S-PMA established
by ACGIH (BEI®) should be reconsidered on the basis
of the precautionary principle, taking as reference the
average levels of S-PMA measured in subjects with
lower GSTT1, GSTM1 and GSTA1 enzymatic activ-
future science group
 Biomarkers of susceptibility following benzene exposure  Review

Table 2. Frequency of genetic polymorphisms in different populations and their influence on urinary excretion levels
of trans,trans-muconic acid (t,t-MA) and S-phenylmercapturic (SPMA).
Enzyme Genotypes Population (%) Effect on biomarker
  Caucasian Asian African Influence on excretion
          S-PMA t,t-MA
CYP2E1  CYP2E1*5B       Uncertain Uncertain
  CC 92.4 [89] 59.5 [89] 97.0 [105]    
  CT 7.5 35.9 3.0    
  TT 0.1 4.6 0.0    
  CYP2E1*6       No No
  TT 85.4 [89] 48.3 [89] 64.0 [105]    
  TA 13.8 42.3 35.0    
  AA 0.8 9.4 1.0    
EPHX1 EPHX1 -28 T>C Tyr113His       No No
  TT 47.5 [89] 25.1 [107] 45.2 [106]    
  TC 34.7 44.2 42.8    
  CC 17.8 30.6 12.0    
  EPHX1+52 A>G His139Arg       No No
  AA 60.0 [89] 66.9 [107] 62.7 [106]    
  AG 35.0 28.3 34.9    
  GG 4.0 4.8 2.4    
NQO1 NQO1*2 C609T       Uncertain No
  CC 57.0 [45] 35.7 [108] 62.6 [110]    
  CT 36.0 44.4 30.8    
  TT 7.0 19.9 6.6    
MPO MPO G463A       No No
  GG 62.0 [89] 56.9 [109] NA    
  GA 35.0 37.1      
  AA 4.0 6.0      
GST-T1 GST-T1       Yes No
  Pos 79.0 [89] 47.1 [89] 58.0 [106]    
  Null 21.0 52.9 42.0    
GST-M1 GST-M1       Yes No
  Pos 51.0 [89] 35.3 [89] 64.2 [106]    
  Null 49.0 64.7 35.8    
GST-A1 GSTA1*A/B       Yes No
  AA▪ 33.0 [3] 81.0 [107] NA     
  AB▪ 55.0 17.0      
  BB▪ 12.0 2.0      
GST-P1 GSTP1 Ile105Val       No No
  AA 45.6 [3] 72.9 [107] 39.7 [106]    
  AG 43.9 23.7 44.0    
  GG 10.5 5.4 16.3    
Black small squares ▪ flanking the GSTA1*A/B gene polymorphisms indicate the two different alleles.
NA: Not available.

future science group www.futuremedicine.com 157

Review  Carbonari, Chiarella, Mansi, Pigini, Iavicoli & Tranfo
ity. In addition, also the exposure limit of airborne
benzene in occupational settings requires a revision.
The difference in occupational and nonoccupational
settings has no reason to exist yet. In light of the most
recent results, the health risk associated with low and
very low benzene exposures can be considerably greater
than those predicted by epidemiological studies on
workers exposed to airborne concentration from ten to
hundreds of ppm [28,29] .
The redefinition of benzene limit value should take
into account the strong variability in polymorphic gene
frequencies among Caucasian, Asian and African pop-
ulations  [89] . As shown in Table 2, the frequencies of
GSTT1, GSTM1 and GSTA1 are very different between
the three ethnic groups. Asians and Africans show an
increased frequency of null genotypes compared with
Caucasians, especially for the GSTT1 genotype, whose
enzymatic activity appears to be more important than
others in determining the overall ability to detoxify
benzene. Hence, within the occupational setting, the
benzene exposure limit should be set to a value such
to avoid underestimation of the risk caused by the
variability of the polymorphic gene frequencies in dif-
ferent populations, in order to protect even the most
­susceptible individuals.
Owing to limitations of a single biomarker in terms
of sensitivity and specificity, much effort has been
devoted to the use of a biomarker combination in order
to discriminate the high-risk subjects.
In our works we used the t,t-MA/S-PMA concen-
tration ratio R, first introduced by Lin et al.  [47] . This
parameter allows to evaluate the effect of the genetic
differences on the SPMA excretion levels indepen-
dently from the individual benzene exposure, using the
t,t-MA value as an estimate of the personal exposure,
in absence of airborne benzene measurements.
The advent of innovative technologies is allowing to
select novel biomarkers which might be used in com-
bination with the already known biomarkers to detect
and monitor the human response to toxic agents. Recent
studies are pointing to the epigenetic regulation on cell
gene expression as a new potential biomarker in ben-
zene exposed subjects [90–95] . Two ‘chromatin players’ of
the epigenome, in other words, DNA-methylation and
microRNA expression, are now being considered bio-
markers of benzene exposure, because they are involved
in the definition of a specific transcriptomic profile. In
particular, DNA methylation has been used to reflect
environmentally-induced epigenomic r­eprogramming
and to analyze the risk of disease [90,94,96,97] .
In vitro studies on TK6 lymphoblastoid cells exposed
to benzene have reported a global hypomethylation
changes through its active metabolites, including HQ
in a dose dependent manner [98] . Similar results have
158 Biomark. Med. (2016) 10(2)
been obtained also in L02 Hepatic cells, following HQ
and BQ exposure [94] . An exposure-related decrease of
DNA methylation was also observed in gasoline station
attendants and traffic policemen exposed to 18.8 and
6.8 ppm of airborne benzene, respectively [95] . These
findings have been partially confirmed by Seow et al.
who failed to find significant differences in DNA
methylation between exposed and controls at 0.5 ppm
of benzene exposure, although a reduced methylation
of LINE-1 DNA sequences and increased SPMA lev-
els were reported from the authors [93] . Aberrant DNA
methylation, including global DNA hypomethylation
and gene specific hypomethylation or hypermethylation
are frequently observed in AML and other malignant
cells  [99] . Hypermethylation of the tumor suppressor
gene p15 and hypomethylation of Melanoma Antigen
Family A-1 (MAGE-1) gene are frequently observed in
AML and other hematological cancers [100,101] : accord-
ingly, a reduced methylation of the leukemia’s antigen
MAGE-1 gene and an increased methylation of the
tumor suppressor gene p15 were observed in gasoline
station attendants and traffic policemen [95] . Hyper-
methylation of p15 and p16 has been also reported in
subjects with CBP [92] . On the contrary, a significant
reduction of p15 methylation was found among Bulgar-
ian petrochemical workers at increased SPMA excretion
levels  [93] ; the same study did not find any association
between both benzene exposure and SPMA levels with
MAGE-1 gene methylation levels. The discrepancies
among studies may be attributable to the d ­ ifferences in
the levels and source of exposure.
Moreover, a strong hypermethylation of ERCC3
gene was found to be associated with benzene exposure
in two groups of industrial workers exposed to high
levels (324 ppm/year and 100 ppm/year) with a long
history of exposure (20 ± 9 years) [92] .
A recent study focused on the research of new poten-
tial biomarkers and on action mechanisms of CBP
has also reported a strong hypomethylation of STAT-
3 gene in patients with CBP compared with healthy
controls [90] . In the JAK/STAT pathway, the transcrip-
tion factor STAT3 is one of most prominent factor for
­cancer and leukemia [102] .
Also microRNA expression profiling may be applied
to identify new biomarkers of benzene exposure due
to its ability to regulate gene expression at post-tran-
scriptional level and its important role during normal
hematopoiesis and leukemogenesis [103] .
To date, only one study has investigated the expres-
sion patterns of microRNA in the peripheral blood
lymphocytes of occupationally exposed subjects
affected by CBP versus a healthy control group: the
author observed that the key target genes regulated by
different microRNAs were implicated in adherent and
future science group
 Biomarkers of susceptibility following benzene exposure  Review

tight junctions, TGF-beta and Wnt signaling pathway
and other pathways involved in cancer [91] .
To conclude, the big changes in the exposure sci-
ence are revolutionizing the way to prevent the onset of
diseases, moving from the concept of a single causative
agent toward a holistic approach. Considering that the
vast majority of diseases results from the gene-environ-
ment interactions, the novel concept of measuring the
person’s exposome, including known and new biomark-
ers, will be the basis understanding the etiology of dis-
eases  [88] . Besides the use of biological samples such
as urine, the most consolidated matrix for the mea-
sure of dose biomarkers, saliva, buccal and mucosal
cells instead of blood will help in obtaining exposure
­assessment markers with less invasive methods.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial in-
volvement with any organization or entity with a financial
interest in or financial conflict with the subject matter or
materials discussed in the manuscript. This includes employ-
ment, consultancies, honoraria, stock ownership or options,
expert testimony, grants or patents received or pending, or
­royalties.
No writing assistance was utilized in the production of this
manuscript.

Executive summary
Noncancerous & cancerous effects of benzene on the human health
• Chronic exposure to benzene is associated with decrease in the production of erythrocytes, leukocytes and
platelets, resulting in aplastic anemia, thrombocytopenia and pancytopenia; B-cell and T-cell proliferation
is also reduced. After exposure to very high benzene concentrations, a hematotoxic syndrome called chronic
benzene poisoning has been described.
• The key of benzene carcinogenicity resides in its metabolism, where electrophilic metabolites are
produced together with highly reactive oxygen species, resulting in cell damage, DNA lesions and strand
breaks, favoring mutagenesis and chromosomal aberrations and leading to malignant transformation of
hematopoietic cells.
Benzene exposure in the environmental & occupational settings
• Benzene is an environmental and occupational pollutant and a well known carcinogen. To protect human
health from benzene-induced toxicity the Directive 2000/69/EC decreased the benzene limit value in the urban
air at 5 μg/m3 (1.54 ppb). In the occupational context, the limit of benzene exposure was set at 3.25 mg/m3
(1 ppm) by the Directive 97/42/EC. The main toxic effects of benzene exposure are bone marrow suppression
causing anemia, leukopenia, thrombocytopenia, aplastic anemia, pancytopenia. At very high concentration,
benzene may cause a hematotoxic syndrome called chronic benzene poisoning. The cancerous effect of
benzene is leukemia incidence.
Benzene metabolism & biomarkers of exposure
• Benzene toxicity should be sought in the metabolites derived from the biotransformation of this compound.
The biomonitoring of human exposure to low benzene concentrations requires the use of one or more specific
and sensitive biomarkers employed for risk assessment of exposed populations. Among these S-PMA, t,t-MA
and urinary benzene are recognized as the most suitable due to the recent development of highly sensitive
and selective analytical techniques for their detection.
Polymorphic genes of benzene metabolism as biomarkers of susceptibility
• Polymorphic genes can be considered biomarkers of susceptibility to the toxicity of benzene. Three categories
of polymorphisms influence benzene metabolism: polymorphic genes involved in benzene biotransformation,
responsible for the different capacity of human subjects to produce benzene metabolites; polymorphic genes
involved in the oxidative stress and polymorphic genes involved in the DNA repair mechanism.
Genetic polymorphisms & susceptibility to leukemia
• There is evidence from the literature that polymorphic genes involved in benzene metabolism influence
the susceptibility to leukemia. More than one type of GST and CYP2E1 polymorphism seem to be associated
with a higher susceptibility of developing leukemia whereas the C609T NQO1 polymorphism seems to show
strong correlation with the disease risk. However, although gene polymorphisms may influence the individual
metabolism of benzene, the genetic background is not sufficient to explain complex diseases such as
leukemias.

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