See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/50269088

Cytogenetic Biomonitoring on a Group of

Petroleum Refinery Workers

Article in Environmental and Molecular Mutagenesis · July 2011

DOI: 10.1002/em.20641 · Source: PubMed

CITATIONS READS

9 81

6 authors, including:

Emiliano Basso Maddalena Papacchini

Università Degli Studi Roma Tre INAIL Istituto Nazionale per l'Assicurazione con…
20 PUBLICATIONS 98 CITATIONS 47 PUBLICATIONS 566 CITATIONS

SEE PROFILE SEE PROFILE

Giovanna Tranfo Antonella Mansi

INAIL Istituto Nazionale per l'Assicurazione con… INAIL Istituto Nazionale per l'Assicurazione con…
72 PUBLICATIONS 519 CITATIONS 35 PUBLICATIONS 224 CITATIONS

SEE PROFILE SEE PROFILE

All content following this page was uploaded by Giovanna Tranfo on 19 March 2015.

The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document
and are linked to publications on ResearchGate, letting you access and read them immediately.
 Environmental and Molecular Mutagenesis 52:440^447 (2011)

Research Article

Cytogenetic Biomonitoring on a Group of Petroleum

Refinery Workers
Emiliano Basso,1 Chiara Cevoli,1 Maddalena Papacchini,2
Giovanna Tranfo,2 Antonella Mansi,2 and Antonella Testa1*
1
Section of Toxicology and Biomedical Sciences, Research Centre ENEA
Casaccia, Rome, Italy
2
Italian National Institute for Occupational Safety and Prevention (ISPESL),
Rome, Italy

Workers employed in petroleum refineries are
exposed to a wide range of toxic compounds
(benzene, polycyclic aromatic hydrocarbons,
heavy metals, etc.) with known mutagenic and
carcinogenic potential. In this study, we investi-
gated by using the cytokinesis block micronucleus
(CBMN) assay on human peripheral blood lym-
phocytes (PBL) whether general occupational ex-
posure in petroleum refineries resulted in early bi-
ological effects, which would be indicative of
adverse health effects in the long term. In this
study, out of more 500 workers enrolled in the
study, 79 male subjects (46 nonsmokers and 33
smokers), employed in two different Italian petro-
leum refineries, and a total of 50 male control
subjects (34 nonsmokers and 16 smokers) were
selected by using very strict selection criteria. The
comparison of chromosome damage in PBL
between exposed and control populations pointed
out a significant increase of micronuclei in the
exposed group, correlated with the length of
employment. Results confirm that smoking is the
principal confounding factor for the responses. In
conclusion, our results are indicative of a potential
genotoxic risk related to the complex occupational
exposure in petroleum refineries, despite the
measures adopted in the plants, and corroborate
the need to increase safety measures to avoid expo-
sure to chemical agents. Environ. Mol. Mutagen.
52:440–447, 2011. V C 2011 Wiley-Liss, Inc.

Key words: petroleum refinery; micronucleus; cytogenetic monitoring; smoke

INTRODUCTION reported to be less than 3 mg/m3, with higher levels in

In the past decades, the occupational exposure has rep-
resented a steady problem for the industrial activity
because workers are continuously exposed to a wide
range of potentially toxic agents, and the biological moni-
toring has acquired an increasing importance in the
evaluation of risks to human health as an integral part of
strategies to improve conditions of occupational safety
and health [Roma-Torres et al., 2006].
Among the many industrial fields, petrochemical indus-
try represents a big source of job, and it hires a lot of
people. As reported by IARC, ‘‘world-wide, the petroleum
refining industry employs about 500,000 persons in more
than 700 plants. Process operators and maintenance work-
ers may be exposed to a large number of substances
which occur in crude oil, process streams, intermediates,
catalysts, additives and final products. Aliphatic and aro-
matic hydrocarbons and hydrogen sulfide have commonly
been measured in the air of working environments. Less
commonly, polycyclic aromatic compounds have been
detected at specific process units. In general, the concen-
trations of benzene in modern refineries have been
some operations. Exposure via the skin to high-boiling
materials may also occur’’ [IARC, 1989]. Moreover, the
IARC reported that occupational exposures in petroleum
refining are probably carcinogenic to humans (Group 2A)
related to skin cancer and leukemia [IARC, 1989]. For
this reason, a large number of epidemiological studies in
the petroleum industry have been performed to address
the issue of carcinogenicity of petroleum chemicals.
Grant sponsor: Ministry of Public Health Project—Occupational benzene
exposure: Development of advanced biosensors for environmental moni-
toring; Grant Number: PMS/024/2003.
*Correspondence to: Antonella Testa, Section of Toxicology and Bio-
medical Sciences, Research Centre ENEA Casaccia, Via Anguillarese
301, Rome 00123, Italy. E-mail: antonella.testa@enea.it
Received 23 June 2010; provisionally accepted 2 December 2010; and in
final form 8 December 2010
DOI 10.1002/em.20641
Published online 2 March 2011 in Wiley Online Library (wileyonlinelibrary.
com).

C 2011 Wiley-Liss, Inc.

V

A review of cohort studies on more than 350,000 petro-
leum workers in the United States, the United Kingdom,
Canada, Australia, Finland, Sweden, and Italy reported no
increased mortality from digestive (stomach, large intes-
tine, liver, or pancreas), lung, bladder, kidney, or brain
cancer [Wong and Raabe, 2000]. For all petroleum work-
ers, a small increase in skin cancer mortality was found.
Significant increases in melanoma mortality were found
in some small groups of refinery workers in the United
Kingdom and upstream operation workers in Canada
[Wong and Raabe, 2000].
A major concern relative to exposure of these workers
is constituted by benzene, classified by IARC as carcino-
genic to humans (Group 1) [IARC, 1982]. For this reason,
the threshold limit value (TLV) for benzene has been
reduced from 100 ppm (1946) to the current value of
0.5 ppm (1.6 mg/m3) by the American Conference of
Governmental Industrial Hygienist (ACGIH). Benzene is
a natural component of crude oil (less than 1% by
weight); therefore, it is found in some refinery products
like petrol [Schnatter, 2000], and it is also the main com-
ponent of the organic solvents that are emitted by
petroleum refinery [Rao, 2007]. Several studies on occu-
pational exposure to benzene reporting induction of chro-
mosomal aberrations [Zhang et al., 2005], in particular
monosomy and trisomy of specific chromosomes [Zhang
et al., 2005, 2007], are available in the literature. More-
over, translocations between chromosomes 8 and 21
[t(8;21)], often associated with acute myeloid leukemia
[Su et al., 2004], and between chromosomes 14 and 21
[t(14;21)], associated with non-Hodgkin’s lymphoma, are
reported. Other studies reported association between
micronuclei (MN) and benzene exposure [Liu et al.,
1996; Roma-Torres et al., 2006], whereas some other
investigations did not find the same results [Carere et al.,
1995; Surrallés et al., 1997].
Despite the additives containing lead were reduced or
eliminated, a greater amount of aromatic compounds is
mixed in petrol for the antiknock purpose. Despite many
epidemiological data are present in literature, there are only
very few cytogenetic monitoring studies on petroleum
workers. In the last years, biological monitoring has been
used in cooperation with traditional epidemiological studies
to have a ‘‘molecular’’ vision of effects from exposure to
toxic compounds. In this context, cytogenetic biomarkers
are widely utilized in the field of molecular epidemiology
to investigate potential genotoxic effects induced by xeno-
biotic agents on human health [Bonassi and Au, 2002].
A study on a group of petroleum refinery workers
reported a significant increase in chromosome aberrations
related to high-level benzene exposure [Kim et al., 2004].
Similarly, a follow-up from 1990 to 2003 on a group of
benzene distillers employed in an oil refinery reported an
increase in chromosomal aberrations and sister chromatid
exchanges (SCE) respect to control subjects [Tompa
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Biomonitoring in a Petroleum Refinery 441
et al., 2005]. Another study on a group of workers from a
petroleum refinery aromatic plant reported significant
increases in MN and chromosomal aberrations [Roma-
Torres et al., 2006]. Recently, another investigation shows
that the frequencies of MN and chromosomal aberrations
were significantly higher in workers of a petroleum refin-
ing industry than in unexposed controls [Kim et al.,
2008]. A study carried out in Spain during clean-up oper-
ations after an oil spill demonstrated that, in hired work-
ers, there was an increase in MN, especially in manual
cleaners. Moreover, the genetic damage measured by
comet assay revealed a positive correlation with exposure
to heavy metals [Pérez-Cadahı́a et al., 2008a,b].
In this study, we evaluated the genotoxic effects owing
to occupational exposure in a group of petroleum refinery
workers by using the cytokinesis block MN (CBMN)
assay on peripheral blood lymphocytes (PBL). The
CBMN assay is widely used in cytogenetic monitoring
studies as biomarker of chromosome damage. This tech-
nique, proposed as a ‘‘cytome’’, can provide a lot of
information on chromosome instability and altered cell
viability caused by exogenous genotoxins [Fenech, 2006].
Recently, the predictive power of CBMN toward cancer
risk has been highlighted [Bonassi et al., 2007; Murgia
et al., 2008].
MATERIALS AND METHODS
Selection of Subjects
Out of more than 500 workers enrolled in the study, 79 male subjects
(46 nonsmokers and 33 smokers), employed in two different Italian pe-
troleum refineries, were selected by using two different questionnaires:
(a) the ‘‘personal health questionnaire’’ proposed by Carrano and Natara-
jan [1988] for the evaluation of ‘‘lifestyle confounding factors’’ and (b) a
specific questionnaire on individual professional exposure (e.g., individ-
ual benzene exposure, length of employment, etc.). Individuals having
potential confounding factors (other than smoking), like drug and alcohol
consumption, recent radiodiagnostic exposure, subjects with previous
treatment by chemotherapy and radiotherapy and with recent viral or
bacterial infections, and individuals with known genetic defects and
major illnesses were excluded. All individuals provided informed consent
in accord with current law; all subjects received a letter outlining the
aims of the research study, in which they were informed that the results
would be published and that they would not receive any individual
results. A total of 50 male control subjects (34 nonsmokers and 16
smokers) employed in administrative offices were selected from usual
blood donors by using to the same selection criteria. All smoker subjects
(occupationally exposed and controls) reported that they smoked more
than 10 cigarettes/day (mean, 13 cigarettes/day).
Exposure Assessment
Workers employed in petroleum refineries are exposed to a wide range
of toxic compounds, but because most operations are closed processes,
exposures are expected to be minimal under normal operating conditions.
However, there is a potential for exposure to chemicals such as phenol,
furfural, glycols, methyl ethyl ketone, amines, hydrogen sulfide, carbon
monoxide, trace polycyclic aromatic hydrocarbons (PAHs), mercaptans,
ammonia, dry chemical demulsifiers, acids, caustic (sodium hydroxide),
Environmental and Molecular Mutagenesis. DOI 10.1002/em
442 Basso et al.

spent caustic, spent catalyst (Merox), catalyst dust and sweetening agents
(sodium carbonate and sodium bicarbonate), monoethanolamine (MEA),
diethanolamine (DEA), methyldiethanolamine (MDEA), and hydrocar-
bons. If diatomaceous earth is used in filtration, it can contain silica in
very fine particle size, making this a potential respiratory hazard. The
potential for exposure to hydrogen sulfide and sulfur dioxide exists in the
production of asphalt. During blending, sampling, and compounding, per-
sonal protection from steam, dusts, mists, vapors, metallic salts, and other
additives is appropriate. Skin contact with any formulated grease or lubri-
cant should be avoided. Working around transformers and switches that
may contain a dielectric fluid requires special handling precautions.
The volatile organic compound (VOC) emissions from the refinery are
mainly alkanes, but also include about 5% alkenes and 15% aromatic
hydrocarbons, among which the major concern regards benzene because
of its ascertained carcinogenic properties. According to the Italian law,
the extent of exposure of workers to carcinogens must be assessed by
means of periodical measurements, and workers are subjected to the
compulsory medical surveillance.
The ACGIH states a TLV for occupational airborne benzene exposure
of 1.6 mg/m3 (time weighted average on 8 hr [TWA]), whereas the
Italian law foresaw a maximum benzene concentration in urban air of
10 lg/m3 at the time of the study (since January 1, 2010, the value has
been lowered to 5) as an annual average.
Benzene exposure during the entire work-shift (approximately 8 hr)
was measured at the breathing zone level in all subjects (n 5 79), using
personal diffusive samplers containing an active carbon cartridge (Radi-
ello1). The Radiello Passive Air Sampling System does not require any
electrical power, what makes it ideal for an industrial site where flamma-
ble vapors could be present. Its characteristics are light weight, small
size, and high and constant sampling rate. Analysis was performed by
gas chromatography-flame ionization detector (GC/FID) after desorption
of benzene from the active carbon with carbon disulfide, with a detection
limit of 0.2 lg/m3.
Even control subjects are exposed to benzene, which is a ubiquitous
pollutant of the environment mainly because it is a component of auto-
motive exhaust emission, but very likely at lower concentrations. Smok-
TABLE I. Baseline Characteristics of the Population Under
Study (Exposed and Controls)
Exposed Controls
All 79 50
Smokers 33 16
Non-smokers 46 34
Mean (SD) age (years) 38.6 (10.7) 37.1 (7.0)
Range 23–64 19–50
Mean (SD) length of employment (years) 13.4 (11.7) —
Range 0.5–44 — In Table I, the baseline characteristics of the examined
Mean (SD) exposure (benzene) (mg/m3) 0.093 (0.11) — population are reported. In this study, we analyzed a group
Range 0.00022–0.81 —
SD, standard deviation.
ers, both workers and controls, absorb significant doses of benzene,
which is produced by cigarette smoke.
Lymphocyte Culture
Peripheral blood samples were collected locally at the medical presid-
ium of the two refineries by venipuncture into heparinized tubes. Blood
samples were transported by night at room temperature to the cytoge-
netic laboratories of the ENEA Research center in Rome and put in cul-
ture within 20 hr from the blood collection. For lymphocyte culture, 0.5
ml of whole blood was added to 4.5 ml of RPMI Dutch Modification
1640 medium (Euroclone, Milan, Italy) supplemented with 10% heat-
inactivated fetal calf serum (Euroclone), 2% phytohemagglutinin (Gibco-
Invitrogen, Carlsbad, CA), 1.5% penicillin–streptomycin (5,000 U/ml
and 5,000 mg/ml) (Euroclone), and 1% L-glutamine (Euroclone). Cul-
tures were grown at 378C. For the MN study, cultures were incubated at
378C for 72 hr; 44 hr after incubation, cytochalasin B (Sigma, St. Louis,
MO) was added at a final concentration of 6 lg/ml to arrest cytokinesis.
Air-dried preparations were stained by the conventional Giemsa
method. The presence of MN was evaluated by scoring a total of 1,000
binucleated (BN) cells with well-preserved cytoplasm for each donor. In
addition, another 1,000 lymphocytes were scored to evaluate the percent-
age of cells with one, two, three, and four or more nuclei. The nuclear
division index (NDI) was used for measuring cell proliferation kinetics.
Statistical Analysis
The distribution of all parameters was evaluated by the Kolmogorov–
Smirnov test and showed significant departures from the normal distribu-
tion. Therefore, the nonparametric Mann–Whitney U-test (significance
taken as P < 0.05) was applied to compare exposed and controls to eval-
uate genetic damage. To evaluate the relationship between the genetic
damage and confounding factors such as age, length of employment,
smoke, and benzene exposure, we performed a multiple regression analy-
sis, considering time by time as dependent variable MN% or BNMN%
and as independent variables age, length of employment, smoke, and
benzene exposure. We reported the R and R2 values to show the good-
ness of fit and the P-value for each independent variable to test whether
the regression model predicts the independent variables in a statistically
significant manner. Moreover, to better evaluate the relationship between
BNMN frequencies and age both in exposed and control subjects, the
Spearman’s rank correlation test was used. Statistical analysis of data
was performed using Graph Pad software Instat version 3.02 (Graph Pad
Software, San Diego, CA).
RESULTS
of 79 male occupationally exposed workers (mean age,
38.6 6 10.7; mean working age (years), 13.4 6 11.7)

TABLE II. Frequencies of Micronuclei in Control Subjects

Smokers (n 5 16) Non-smokers (n 5 34) Total (n 5 50)
Mean (SD) Range Mean (SD) Range Mean (SD) Range

Age 39.76 (7.54) 16–50 35.56 (6.35)a 24–50 37.1 (7.0) 19–50
MN tot (%) 5.63 (3.88) 1–13 3.48 (2.22) 1–11 4.35 (3.12) 1–13
BNMN (%) 4.88 (3.03) 1–10 3.35 (2.01) 1–11 3.96 (2.53) 1–11
NDI 1.70 (0.14) 1.5–1.9 1.74 (0.13) 1.5–2 1.7 (0.1) 1.5–2

Statistical analysis performed by Mann–Whitney U-test: aP < 0.01.

BNMN, binucleated cells with micronuclei; MN, total micronuclei; NDI, nuclear division index; SD, standard deviation.
 Environmental and Molecular Mutagenesis. DOI 10.1002/em
Biomonitoring in a Petroleum Refinery 443

TABLE III. Frequencies of Micronuclei in Exposed Subjects

Length of Exposure to MN tot BNMN
Subjects Age employment (years) Smoke benzene (mg/m3) (%) (%) NDI

E1 56 34 No — 14 11 1.3
E2 34 3 No — 14 13 1.2
E3 52 — No — 9 9 1.5
E4 57 34 No — 12 10 1.3
E5 56 33 No — 13 13 1.2
E6 34 11 No — 12 12 1.2
E7 55 32 No 0.064 10 9 1.2
E8 33 13 No 0.065 5 5 1.1
E9 40 16 No 0.063 3 3 1.5
E10 23 2.5 No 0.197 5 5 1.2
E11 33 2 No 0.107 6 6 1.1
E12 38 13 No 0.02 23 20 1.4
E13 53 30 No 0.197 25 21 1.4
E14 39 13 No 0.075 7 7 1.2
E15 57 35 No 0.083 16 13 1.2
E16 31 3 No 0.115 9 8 1.4
E17 30 2.5 No 0.08 11 11 1.4
E18 30 2 No 0.188 9 9 1.3
E19 36 11 No 0.039 6 6 1.1
E20 25 0.6 No 0.075 1 1 1.3
E21 36 8 No 0.019 2 2 1.3
E22 34 7 No 0.075 7 7 1.4
E23 31 11 No 0.077 2 2 1.3
E24 35 11 No 0.107 7 7 1.2
E25 56 36 No 0.019 21 14 1.2
E26 64 33 No 0.107 2 2 1.3
E27 42 13 No 0.077 7 7 1.4
E28 40 18 No 0.083 21 20 1.5
E29 55 35 No 0.019 8 8 1.5
E30 33 2 No 0.107 8 5 1.4
E31 31 2 No 0.013 3 3 1.3
E32 39 18 No — 14 14 1.4
E33 55 35 No 0.024 5 5 1.3
E34 33 7 No 0.209 8 8 1.4
E35 58 31 No 0.00043 13 12 1.4
E36 36 14 No 0.0003 12 10 1.5
E37 56 31 No 0.00043 23 18 1.3
E38 55 26 No 0.022 10 10 1.1
E39 37 11 No 0.006 10 10 1.5
E40 32 — No 0.095 3 3 1.4
E41 26 1 No 0.024 6 6 1.3
E42 36 13 No — 5 5 1.5
E43 32 7 No — 4 2 1.2
E44 64 44 No 0.00022 12 11 1.3
E45 32 13 No — 8 6 1.4
E46 31 7.5 No 0.034 4 4 1.2
E47 36 14 Yes — 2 2 1.4
E48 40 13 Yes 0.063 5 4 1.4
E49 25 1 Yes 0.075 6 6 1.4
E50 30 2.5 Yes 0.065 1 1 1.5
E51 24 2 Yes 0.075 2 2 1.5
E52 30 2 Yes 0.197 4 4 1.4
E53 30 2.5 Yes 0.197 1 1 1.4
E54 31 0.5 Yes 0.209 4 4 1.7
E55 42 17.5 Yes 0.083 6 5 1.4
E56 40 17 Yes 0.051 3 3 1.3
E57 38 8 Yes 0.024 2 2 1.5
E58 33 5 Yes 0.065 7 7 1.4
E59 47 6 Yes 0.016 6 6 1.3
E60 30 1 Yes 0.075 2 2 1.3
E61 49 25 Yes 0.071 17 14 1.2
Environmental and Molecular Mutagenesis. DOI 10.1002/em
444 Basso et al.

TABLE III. Continued

Length of Exposure to MN tot BNMN
Subjects Age employment (years) Smoke benzene (mg/m3) (%) (%) NDI

E62 32 8 Yes 0.209 3 3 1.2

E63 61 37 Yes — 7 7 1.2
E64 40 16 Yes — 6 6 1.3
E65 30 5 Yes 0.016 6 6 1.4
E66 32 9 Yes 0.019 2 2 1.3
E67 38 14 Yes 0.05 6 6 1.2
E68 24 1 Yes 0.08 1 1 1.4
E69 34 13 Yes 0.107 5 5 1.3
E70 27 — Yes 0.016 5 5 1.5
E71 31 2 Yes 0.075 2 2 1.4
E72 31 6 Yes 0.02 2 2 1.3
E73 51 17 Yes 0.123 5 5 1.3
E74 33 9 Yes 0.313 3 3 1.4
E75 30 — Yes 0.019 3 3 1.5
E76 34 13 Yes 0.81 1 1 1.4
E77 32 1 Yes 0.201 6 5 1.4
E78 27 1.5 Yes 0.201 4 4 1.4
E79 46 — Yes 0.139 4 4 1.3
TOT 79
Mean (SD) 38.79 (10.82) 13.69 (11.86) 0.09 (0.11) 7.27 (5.57) 6.66 (4.70) 1.34 (0.11)

BNMN, binucleated cells with micronuclei; MN, total micronuclei; NDI, nuclear division index; SD, standard deviation.

compared with 50 healthy male subjects (mean age, 37.7

6 7.0).
Mean benzene exposure of the 79 workers was found
to be 0.093 mg/m3 (range, 0.0002–0.8100). This figure
is significantly lower than the ACGIH TLV-TWA of
1.6 mg/m3, but higher than the limit value for urban air
of 0.01 mg/m3 as annual average, to which controls are
expected to have been exposed.
In Tables II and III, the total frequencies of MN
(MN% tot), cells BN with MN (BNMN%), and the NDI
detected in controls and occupationally exposed subjects
are reported, respectively. Data on age, smoking habit,
length of employment and individual benzene exposure
(only for exposed group) are also reported in the tables. Fig. 1. Analysis of genetic damage in exposed and control subjects (all).
Figure 1 shows the results of the statistical comparison BNMN, binucleated cells with micronucleus; MN, total micronuclei.
(Mann–Whitney U-test) between occupationally exposed Statistical analysis performed by Mann–Whitney U-test: *P < 0.001.
and control groups. The occupationally exposed group
shows a significant increase (P < 0.001) in BNMN % between control subgroups (smokers and nonsmokers) no
(6.7 6 4.7) and in MN % (7.3 6 5.6) with respect to statistically significant differences were found (data not
controls (3.8 6 2.5 and 4.2 6 3.0), respectively. In non- shown). Figure 3 shows the relationship between BNMN
smoker subjects, this trend is confirmed for both BNMN frequency and age in occupationally exposed and control
% (8.5 6 5.0 vs. 3.4 6 2.0) and MN % (9.5 6 6.0 vs. groups by using linear regression analysis. The results
3.5 6 2.2) (P < 0.001) (Fig. 2a), whereas between smok- indicate an effect of age on the MN frequency in both
ers subgroups we found a lower chromosome damage in groups (exposed and controls). The multiple regression
the exposed smokers (BNMN % 4.0 6 2.6; MN % analysis between cytogenetic biomarkers (BNMN %, MN
4.2 6 3.0) respect to control smokers (BNMN % 4.5 6 %) and some confounding factors like length of employ-
3.0; MN % 5.6 6 3.9) (Fig. 2b). Finally, occupationally ment, smoking habit, age of subjects, and individual ex-
exposed smokers (BNMN % 4.0 6 2.6; MN % 4.2 6 posure to benzene showed a significant correlation
3.0) showed a significantly lower damage (P < 0.001) between genetic damage and age, length of employment,
with respect to occupationally exposed nonsmokers and smoking habit (Table IV). We tried to evaluate the
(BNMN % 8.5 6 5.0; MN 9.5 6 6.0) (Fig. 2c), whereas prevalence of BN cells containing one, two, three, or
 Environmental and Molecular Mutagenesis. DOI 10.1002/em
Biomonitoring in a Petroleum Refinery 445

Fig. 2. Analysis of genetic damage in (A) exposed nonsmokers and control nonsmokers; (B) exposed smok-
ers and control smokers; and (C) exposed smokers and exposed nonsmokers. BNMN, binucleated cells with
micronucleus; MN, total micronuclei. Statistical analysis performed by Mann–Whitney U-test: *P < 0.001.

Fig. 3. Linear regression analysis between BNMN and age in exposed

and control subjects. BNMN, binucleated cells with micronucleus; R2,
coefficient of determination. Fig. 4. Box plot showing the trend of genetic damage with the increase
in length of employment. The length of employment has been divided in
classes to emphasize the relationship between the two parameters.
BNMN, binucleated cells with micronucleus.
TABLE IV. Multiple Regression Analysis of Confounding
Factors on BNMN and MN Frequencies in Exposed Subjects
Biomarker Independent variables Partial R2 R2 P-value DISCUSSION
BNMN Age 0.0820 0.0023
Length employment (years) 0.1144 0.0107
This study investigated whether general occupational
Smoke 0.1455 0.0334 exposure in petroleum refineries resulted in early biologi-
Benzene exposure 0.0713 0.6356 cal effects, which would be indicative of adverse health
0.3769 effects in the long term. The comparison of chromosome
MN Age 0.0820 0.0010 damage in PBL between exposed and control populations
Length employment (years) 0.1144 0.0077
Smoke 0.1455 0.0489
pointed out a significant increase in MN in the exposed
Benzene exposure 0.0713 0.5040 group.
0.3944 We found out that the chromosome damage increase is
correlated with the length of employment, and this finding
is in agreement with other previous studies on occupa-
more MN, and we verified that the amount of genetic tional exposure in petroleum refineries [Roma-Torres
damage was independent of individual benzene exposure et al., 2006].
(data not shown). Moreover, our results show a positive correlation
Figure 4 shows a positive trend of BNMN with the between age of subjects and MN frequency in both con-
increase in working age. The linear regression analysis trol and occupationally exposed groups, confirming other
performed on NDI and individual benzene exposure literature data [Bonassi et al., 2001; Roma-Torres et al.,
did not show any relationship between the two parameters 2006]. One of the compounds that may be responsible for
(R2 5 0.006984) (data not shown). at least part of chromosome abnormalities observed in
Environmental and Molecular Mutagenesis. DOI 10.1002/em
446 Basso et al.
these workers could be benzene, even if the average expo-
sure levels measured (mean 5 0.093 mg/m3) were many
times lower than TLV (ACGIH, 2009), but our data did
not show any correlation between benzene individual
occupational exposure and genetic damage.
Smoking is an important confounding factor that should
be carefully considered in biological monitoring studies
because it is itself a well-known cancer risk factor. For
these reasons, we improved the statistical analysis com-
paring smokers and nonsmoker subgroups separately.
Occupationally exposed nonsmokers show a very signifi-
cant increase in MN frequency compared with nonsmoker
controls, while occupationally exposed smokers show a
lower but not significant MN frequency compared with
control smokers. Moreover, control smokers show a sig-
nificant increase in MN frequency compared with non-
smoker controls. This finding was unexpected, considering
the results of a huge biomonitoring study demonstrating
that significant effect of smoking is likely limited to sub-
jects smoking 30 and more cigarettes/day [Bonassi et al.,
2003], while our control smokers declared to smoke less
than a mean of 13 cigarettes/day. Surprisingly, exposed
smokers show a significant decrease in MN frequency
compared with occupationally exposed nonsmokers, sug-
gesting a possible protective role of smoke, in case of ex-
posure to mutagens sharing the same detoxifying pathway
based on the glutathione (GSH) as previously reported
[Ray et al., 2002; Testa et al., 2005]. The GSH is a ubiq-
uitous molecule that acts as scavenger for electrophilic
xenobiotics and is the substrate for the glutathione S-
transferase (GST), an enzyme involved in the detoxifying
pathway of many toxic and carcinogen substances. Oesch
et al. [1994] found an increase of GSH in the smokers,
and Ray et al. [2002] demonstrated that the synthesis of
GSH is inducible. Other evidences showed a higher level
of GSH in epithelial pulmonary cells in response to an
increase in oxidative stress caused by cigarette smoking
[Rahman et al., 1996a,b, 1998].
In conclusion, our results are indicative of a potential
genotoxic risk related to the occupational exposure in petro-
leum refineries despite the measures adopted in the plants
and corroborate the need to increase safety measures to
avoid exposure. As the petroleum refinery environment is
characterized by a complex mixture of chemicals, it is diffi-
cult to associate the increased chromosome damage
observed to specific exposures [Barregard et al., 2009].
Since the biological effects of such a mixture are unpredict-
able until now, we cannot exclude that the genotoxic effect
observed is the end-result effect of more than one chemical
agent with different mechanisms of action. Unfortunately,
very little information is available on adverse health effects
of chemical mixtures [Carpenter, 1998].
Exposure to a sole substance in the occupational field
is infrequent, and assessment of exposure to complex
mixtures of chemicals is a major challenge. Interactions
can occur at low exposure doses, and, even if there are no
toxicokinetic interactions between the mixture compo-
nents, there could still be combined effects of two or
more substances on a common organ or system. Under
this situation, the limits set may not offer adequate pro-
tection of workers against possible toxic effects [Viau,
2005].
For the majority of cases where potential additive or
synergistic effects were identified, there is a lack of toxi-
cological data in the literature [Vyskocil et al., 2007].
Therefore, these kind of studies in occupational exposure
should be a priority objective for research in this field.
As it has been recently demonstrated that the frequency
of MN is itself predictive of increased cancer risk, rather
than just a reflection of exposure, our data suggest that
the population under study could be at increased risk for
cancer. This conclusion raises questions about the relative
efficacy of current prevention measures. Because oil refin-
ery is a closed process, exposures are expected to be min-
imal under normal operating conditions; however, there
are many refinery utilities and miscellaneous supporting
activities related to hydrocarbon processing in which safe
work practices and/or appropriate personal protective
equipment are needed for exposures to chemicals and
other hazards such as noise and heat. Therefore, more
attention should be paid to activities like process sam-
pling, inspection, maintenance, marine, tank car, and tank
truck loading and unloading, and turnaround activities.
REFERENCES
Barregard L, Holmberg E, Sallsten G. 2009. Leukaemia incidence in
people living close to an oil refinery. Environ Res 109:985–990.
Bonassi S, Au WW. 2002. Biomarkers in molecular epidemiology stud-
ies for health risk prediction. Mutat Res 511:73–86.
Bonassi S, Neri M, Puntoni R. 2001. Validation of biomarkers as early
predictors of disease. Mutat Res 480–481:349–358.
Bonassi S, Neri M, Lando C, Ceppi M, Lin YP, Chang WP, Holland N,
Kirsch-Volders M, Zeiger E, Fenech M;HUMN collaborative
group. 2003. Effect of smoking habit on the frequency of micro-
nuclei in human lymphocytes: Results from the Human MicroNu-
cleus project. Mutat Res 543:155–166.
Bonassi S, Znaor A, Ceppi M, Lando C, Chang WP, Holland N, Kirsch-
Volders M, Zeiger E, Ban S, Barale R, Bigatti MP, Bolognesi C,
Cebulska-Wasilewska A, Fabianova E, Fucic A, Hagmar L, Jok-
sic G, Martelli A, Migliore L, Mirkova E, Scarfi MR, Zijno A,
Norppa H, Fenech M. 2007. An increased micronucleus fre-
quency in peripheral blood lymphocytes predicts the risk of can-
cer in humans. Carcinogenesis 28:625–631.
Carere A, Antoccia A, Crebelli R, Degrassi F, Fiore M, Iavarone I, Isac-
chi G, Lagorio S, Leopardi P, Marcon F, Palitti F, Tanzarella C,
Zijno A. 1995. Genetic effects of petroleum fuels: Cytogenetic
monitoring of gasoline station attendants. Mutat Res 332:17–26.
Carpenter DO. 1998. Polychlorinated biphenyls and human health. Int J
Occup Med Environ Health 11:291–303.
Carrano AV, Natarajan AT. 1988. International Commission for Protec-
tion Against Environmental Mutagens and Carcinogens. ICPEMC
publication no. 14. Considerations for population monitoring
using cytogenetic techniques. Mutat Res 204:379–406.

Fenech M. 2006. Cytokinesis-block micronucleus assay evolves into a
‘‘cytome’’ assay of chromosomal instability, mitotic dysfunction
and cell death. Mutat Res 600:58–66.
IARC. 1982. Some industrial chemicals and dyestuffs. IARC Monogr
Eval Carcinog Risk Chem Hum 29:1–398.
IARC. 1989. Occupational exposures in petroleum refining; crude oil
and major petroleum fuels. IARC Working Group on the Evalua-
tion of Carcinogenic Risks to Humans. IARC Monogr Eval Car-
cinog Risks Hum 45:1–322.
Kim YJ, Cho YH, Paek D, Chung HW. 2004. Determination of chromo-
some aberrations in workers in a petroleum refining factory.
J Toxicol Environ Health A 67:1915–1922.
Kim YJ, Choi JY, Paek D, Chung HW. 2008. Association of the NQO1,
MPO, and XRCC1 polymorphisms and chromosome damage
among workers at a petroleum refinery. J Toxicol Environ Health
A 71:333–341.
Liu L, Zhang Q, Feng J, Deng L, Zeng N, Yang A, Zhang W. 1996. The
study of DNA oxidative damage in benzene-exposed workers.
Mutat Res 370:145–150.
Murgia E, Ballardin M, Bonassi S, Rossi AM, Barale R. 2008. Valida-
tion of micronuclei frequency in peripheral blood lymphocytes as
early cancer risk biomarker in a nested case-control study. Mutat
Res 639:27–34.
Oesch F, Hengstler JG, Fuchs J. 1994. Cigarette smoking protects mono-
nuclear blood cells of carcinogen exposed workers from addi-
tional work exposure-induced DNA single strand breaks. Mutat
Res 321:175–185.
Pérez-Cadahı́a B, Laffon B, Porta M, Lafuente A, Cabaleiro T, López T,
Caride A, Pumarega J, Romero A, Pásaro E, Méndez J. 2008a.
Relationship between blood concentrations of heavy metals and
cytogenetic and endocrine parameters among subjects involved in
cleaning coastal areas affected by the ‘Prestige’ tanker oil spill.
Chemosphere 71:447–455.
Pérez-Cadahı́a B, Laffon B, Valdiglesias V, Pásaro E, Méndez J. 2008b.
Cytogenetic effects induced by Prestige oil on human popula-
tions: The role of polymorphisms in genes involved in metabo-
lism and DNA repair. Mutat Res 31:117–123.
Rahman I, Bel A, Mulier B, Lawson MF, Harrison DJ, Macnee W,
Smith CA. 1996a. Transcriptional regulation of gamma-
glutamylcysteine synthetase-heavy subunit by oxidants in human
alveolar epithelial cells. Biochem Biophys Res Commun 229:
832–837.
Rahman I, Smith CA, Lawson MF, Harrison DJ, MacNee W. 1996b.
Induction of gamma-glutamylcysteine synthetase by cigarette
smoke is associated with AP-1 in human alveolar epithelial cells.
FEBS Lett 396:21–25.
Rahman I, Bel A, Mulier B, Donaldson K, MacNee W. 1998. Differen-
tial regulation of glutathione by oxidants and dexamethasone in
alveolar epithelial cells. Am J Physiol 275:80–86.
View publication stats
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Biomonitoring in a Petroleum Refinery 447
Rao PS, Ansari MF, Gavane AG, Pandit VI, Nema P, Devotta S. 2007.
Seasonal variation of toxic benzene emissions in petroleum refin-
ery. Environ Monit Assess 128:323–328.
Ray S, Watkins DN, Misso NL, Thompson PJ. 2002. Oxidant stress
induces gamma-glutamylcysteine synthetase and glutathione syn-
thesis in human bronchial epithelial NCI-H292 cells. Clin Exp
Allergy 32:571–577.
Roma-Torres J, Teixeira JP, Silva S, Laffon B, Cunha LM, Méndez J,
Mayan O. 2006. Evaluation of genotoxicity in a group of workers
from a petroleum refinery aromatics plant. Mutat Res 604:19–27.
Schnatter R. 2000. Petroleum worker studies and benzene risk assess-
ment. J Toxicol Environ Health A 61:433–437.
Su XY, Busson M, Della Valle V, Ballerini P, Dastugue N, Talmant P,
Ferrando AA, Baudry-Bluteau D, Romana S, Berger R, Bernard
OA. 2004. Various types of rearrangements target TLX3 locus in
T-cell acute lymphoblastic leukemia. Genes Chromosomes Can-
cer 41:243–249.
Surrallés J, Autio K, Nylund L, Järventaus H, Norppa H, Veidebaum T,
Sorsa M, Peltonen K. 1997. Molecular cytogenetic analysis of
buccal cells and lymphocytes from benzene-exposed workers.
Carcinogenesis 18:817–823.
Testa A, Festa F, Ranaldi R, Giachelia M, Tirindelli D, De Marco A,
Owczarek M, Guidotti M, Cozzi R. 2005. A multi-biomarker
analysis of DNA damage in automobile painters. Environ Mol
Mutagen 46:182–188.
Tompa A, Jakab MG, Major J. 2005. Risk management among benzene-
exposed oil refinery workers. Int J Hyg Environ Health 208:509–516.
Viau C. 2005. Biomonitoring in occupational health: Scientific, socio-ethi-
cal, and regulatory issues. Toxicol Appl Pharmacol 207:347–353.
Vyskocil A, Drolet D, Viau C, Lemay F, Lapointe G, Tardif R, Truchon
G, Baril M, Gagnon N, Gagnon F, Bégin D, Gérin M. 2007. A
web tool for the identification of potential interactive effects of
chemical mixtures. J Occup Environ Hyg 4:281–287.
Wong O, Raabe GK. 2000. A critical review of cancer epidemiology in
the petroleum industry, with a meta-analysis of a combined data-
base of more than 350,000 workers. Regul Toxicol Pharmacol
32:78–98.
Zhang L, Yang W, Hubbard AE, Smith MT. 2005. Nonrandom aneu-
ploidy of chromosomes 1, 5, 6, 7, 8, 9, 11, 12, and 21 induced
by the benzene metabolites hydroquinone and benzenetriol. Envi-
ron Mol Mutagen 45:388–396.
Zhang L, Rothman N, Li G, Guo W, Yang W, Hubbard AE, Hayes RB,
Yin S, Lu W, Smith MT. 2007. Aberrations in chromosomes
associated with lymphoma and therapy-related leukemia in
benzene-exposed workers. Environ Mol Mutagen 48:467–474.
Accepted by—
U. Vogel