This study evaluated genotoxic effects in workers exposed to low concentrations of benzene. Fuel tanker drivers had higher airborne benzene exposure (mean 306.7 μg/m3) than filling station attendants (mean 23.3 μg/m3) and controls (mean 4.6 μg/m3). The frequency of micronuclei was higher in older fuel tanker drivers (>40.5 years) than controls, suggesting age promotes genotoxic effects of benzene at exposures over an order of magnitude below regulatory limits. No differences were found between attendants, controls, or with duration of employment, providing no evidence of genotoxicity at very low exposures. Further studies are needed to clarify effects at

1. 9th International Symposium on Biological Monitoring in
Occupational and Environmental Health (ISBM 2013)
GENOTOXIC EFFECTS OF OCCUPATIONAL
AND ENVIRONMENTAL EXPOSURE TO LOW
CONCENTRATIONS OF BENZENE
Lovreglio P. 1, Maffei F.2, Carrieri M.3, D’Errico M.N.1, Drago I.1, Hrelia P.2,
Bartolucci G.B.3, Soleo L.1
1Interdisciplinary
Department of Medicine, Section of Occupational Medicine “E.C. Vigliani”,
University of Bari, Italy
2Department of Pharmacology, University of Bologna, Italy
3Department of Molecular Medicine, Section of Occupational Medicine, University of Padova, Italy
8-11 September 2013, Manchester, UK

2. INTRODUCTION
BENZENE
Occupational toxicant
Environmental toxicant
Main sources:
- automobile exhaust
- cigarette smoke
Exposure to benzene urban pollution
(always
at
low
or
very
low
concentrations) can affect:
- … not only the general population …,
- … but also workers in urban traffic,
such as traffic wardens, bus drivers,
taxi drivers, etc.
Because of its effects on human health (also
carcinogenic), the use of benzene in industrial
applications has been strictly limited by
specific regulations in western nations.
Nevertheless, occupational exposure to low or
very low concentrations is still possible in
workers in:
- upstream/downstream petrochemical industry;
- chemical synthesis industry;
- coke ovens;
- fuel distribution.

3. INTRODUCTION
Benzene has a well known clastogenic effect in humans
CHROMOSOME DAMAGE
BIOMARKERS
Frequency of chromosome
aberrations (CA) in
peripheral blood
lymphocytes
Frequency of micronuclei
(MN) in peripheral blood
lymphocytes
Workers exposed to concentrations
of benzene exceeding or near to
the TLV-TWA of the ACGIH, i.e.
1600 μg/m3
Workers
exposed
to
lower
concentrations, up to 2 orders of
magnitude below the TLV-TWA
Both workers exposed to benzene
concentrations exceeding or near
to the TLV-TWA and workers
exposed to lower concentrations
than the TLV-TWA ….
Evident increase in the
frequency of CA, reported
in numerous studies.
Few studies, with limited
information.
….. studies have shown
conflicting results and
poor agreement.
For the genotoxic effects of low and very low concentrations of benzene, like those
currently present in the workplace and living environment, the exposure-response
relationship is controversial yet.

4. OBJECTIVE OF THE RESEARCH
To study possible genotoxic effects of benzene, by
determining the frequency of CA and MN in the
peripheral blood lymphocytes of workers occupationally
exposed to low and very low concentrations of the
toxicant.

5. METHODS
19 male fuel tanker drivers
EXPOSED
24 male filling station attendants
CONTROLS
31 male workers with no occupational exposure to benzene,
resident in the same geographic area
All subjects were administered a questionnaire probing:
- general characteristics
- current and previous work experience
- smoking history and alcohol consumption
- clinical history
- use of medicinal drugs and exposure to ionizing radiation in the last 12 months
- possible exposure to non occupational sources of benzene.
Exclusion criteria for subjects enrolled in the study:
- previous treatment with ionizing radiation or chemotherapeutic drugs;
- radiography in the 12 months before the sampling;
- a previous diagnosis of cancer;
- for exposed workers, less than 1 year's work in the current job.

6. METHODS
EXTERNAL DOSE BIOMARKER: Airborne benzene
- passive personal sampling (Radiello®) for 8 hours;
- blinded analyses performed by GC-FID; limit of detection (LOD)=3.0 µg/m3.
INTERNAL DOSE BIOMARKERS: blinded analyses performed on urine samples collected at the
end of the environmental sampling.
- t,t-muconic acid (HPLC–UV method at 264 nm, after solid phase extraction (SPE); LOD=10 µg/L)
- SPMA (HPLC–ESI–MS/MS; LOD=0.20 µg/L)
- Urinary benzene (GC/MS analysis; LOD=0.02 µg/L)
- Urinary creatinine (colorimetric method; LOD=0.01 mg/dL).
EFFECT BIOMARKERS:
• peripheral blood collection before the start of the environmental sampling;
• heparinized blood samples were code labeled, transported at room temperature and cultured for
cytogenetic tests within 24 hours of collection;
• all the phases following blood collection were performed blinded.
- CA by standard technique; at least 100 metaphases were counted and scored for each subject;
assessment was made of total CA and separately, of chromosome and chromatid breaks; gaps were
excluded from the analyses.
- MN by standard cytokinesis block technique; for each subject, 2000 binucleated cells (BN) were
examined; the frequency was expressed as total MN per 1000 cells analyzed.
- Nuclear Division Index (NDI): calculated by scoring at least 1000 cells per series of cultures,
adopting the formula NDI = (M1+2M2+3M3+4M4)/n,
[M1–M4 =number of cells with 1-4 nuclei; n = total number of cells scored].

7. RESULTS
Table 1: General characteristics and lifestyle of the fuel tanker drivers, filling station
attendants and controls included in the study.
FUEL TANKER DRIVERS
N.
Mean±SD/% Median Range
Age (years)
19
42.1±7.5
42.0
Body mass
index (Kg/m2)
19
27.8±4.0
26.8
Duration of
employment
(years)
19
16.6±9.3
17.0
2
4
13
11
8
Alcohol
consumption
- teetotal
- <10 g/day
- >10 g/day
29-57
FILLING STATION
ATTENDANTS
N. Mean±SD/% Median Range
N. Mean±SD/% Median
24
31
41.7±9.1
31
26.7±4.8 25.8 20.9-44.4
20.8-35.9 24
5-37
40.7±9.6 40.0
19-60
26.1±3.9 26.5 20.3-37.2
13.5
CONTROLS
24
14.2±8.7
3-36
10.5%
21.1%
68.4%
7
7
10
29.2%
29.2%
41.6%
10
9
12
12
12
50.0%
50.0%
16
15
51.6%
48.4%
16
16.6±10.6
22-62
32.3%
29.0%
38.7%
57.9%
42.1%
43.0
Range
-
Smoking habit
Smoker
Non smoker
Cigarettes/day 11
(smokers)
21.5±10.6
20.0
6-50
12
17.7±11.1
20.0
Packs/year
(smokers)
20.4±11.2
21.5
3.3-40
12
27.0±17.6
25.0
11
2-40
1-62
16
23.4±19.4
11.0
5-40
12.7
7-61

8. Table 2: Airborne benzene concentrations in the fuel tanker drivers, filling station attendants
and controls.
FUEL TANKER DRIVERS
(N. 19)
Mean±SD Median Range
Airborne benzene
306.7±266.7 246.6 7.4-1017.1
(μg/m3)a
FILLING STATION
ATTENDANTS (N. 24)
Mean±SD Median Range
23.3±17.0
19.9
4.5-66.3
CONTROLS
(N. 31)
Mean±SD Median Range
4.6±2.6
4.3
<3.0-11.5
ap<0.001;
The biomarkers of internal dose, t,t-MA, SPMA and urinary benzene, resulted signficantly
higher in the fuel tanker drivers than in the other two groups (p<0.01).

10. Table 3: Biomarkers of effect in smokers and non smokers.
SMOKERS
NON SMOKERS
N.
Mean±SD
Median
Range
N. Mean±SD
Median
Range
CA (%)
39
3.7±1.9
4.0
1-8
35
3.6±1.7
3.0
1-8
Chromosome breaks (%)
39
1.0±1.0
1.0
0-3
35
0.7±0.7
1.0
0-2
Chromatid breaks (%)
39
2.7±1.1
3.0
1-6
35
2.9±1.3
3.0
1-6
MN / 1000 BN
37
8.0±2.7
8.0
2-13
35
7.8±3.0
8.0
3-16
NDI
37
1.8±0.2
1.8
1.3-2.3
35
1.7±0.3
1.7
1.4-2.2

11. Spearman correlations between effect biomarkers and general characteristics that
resulted significant in all the subjects analyzed as a single group.
MN – age
r=0.387, p<0.01
NDI – BMI
r=-0.255, p<0.05
- A strong correlation between age and duration of employment in fuel tanker
drivers and filling station attendants analyzed together (r=0.679, p<0.001);
- No significant correlation between the frequency of CA, and the frequency of MN
and NDI.

12. Figure 1: Frequency of micronuclei in the fuel tanker drivers, filling station
attendants and controls, each subdivided into two subgroups by age, with a cutoff value of 40.5 years (median value).
Similar results were obtained when analyzing the frequency of micronuclei in the
fuel tanker drivers and filling station attendants groups, each subdivided by
duration of employment (as a measure of time of benzene exposure in the job),
with a cut-off value of 16 years (median value).

13. Table 4: Stepwise multiple regression models for the genotoxicity biomarkers found to be
significantly dependent on at least one of the independent variables studied, considering fuel
tanker drivers, filling station attendants and controls both separately and together as a
single group.
Micronuclei
Independent variables
Age
Total
t
NDI
Fuel tanker drivers
p
3.10 0.003
t
p
4.37
Total
t
0.001
Fuel tanker drivers
p
t
p
-
-
BMI
-
-
-2.58 0.012
-
N. cigarettes/day
-
-
-
-
Alcohol / day
-
-
Airborne benzene
-
2.42
2.76
0.015
0.018
2.88
-
0.011
-
F
Model
R2
F
R2
F
R2
F
R2
9.6b
0.11
10.4b
0.53
5.6b
0.12
8.3a
0.30
ap<0.01; bp<0.001;
When replacing age by duration of employment in the fuel tanker drivers and filling
station attendants, the latter variable did not result significant.

14. CONCLUSIONS
The frequency of chromosome aberrations was not found to be able
to distinguish between fuel tanker drivers, filling station attendants
and controls, and does not therefore seem to be a valid cytogenetic
effect biomarker in workers exposed to low and very low
concentrations of benzene.
The frequency of micronuclei, by contrast, was shown to be higher
in older age fuel tanker drivers than in older age filling station
attendants and controls, whereas no difference was observed among
subjects of younger age. Age, therefore, a variable that by itself
has an influence on the frequency of MN, could also promote the
genotoxic effect of benzene, but only in conditions of exposure to
concentrations not less than one order of magnitude below the TLVTWA.
The duration of employment in fuel tanker drivers could in part
explain the results we obtained.

15. CONCLUSIONS
None of the effect biomarkers studied showed any difference
between filling station attendants and controls, and there did not
seem to be an exposure-response relationship at the very low
concentrations of benzene observed in these two groups. Our results
do not therefore provide confirmation of a genotoxic effect of
benzene at these levels of exposure.
Further studies would be useful to clarify this point, in particular by
assessing, at very low concentrations of benzene, the frequency of
the chromosome aberrations considered to be specific to its effect
(by FISH technique).