J. Endocrinol. Invest. 34: e139-e143, 2011
DOI: 10.3275/7298
Environmental car exhaust pollution damages human sperm
chromatin and DNA
A.E. Calogero1, S. La Vignera1, R.A. Condorelli1, A. Perdichizzi1, D. Valenti1, P. Asero1, U. Carbone2,
B. Boggia2, N. De Rosa3, G. Lombardi3, R. D’Agata1, L.O. Vicari1, E. Vicari1, and M. De Rosa3
1Section of Endocrinology, Andrology and Internal Medicine and Master in Andrological, Human Reproduction and Biotechnology
Sciences, Department of Internal Medicine and Systemic Diseases, University of Catania, Catania; 2Departments of Preventive
Medical Sciences; 3Molecular and Clinical Endocrinology and Oncology, University of Naples “Federico II”, Naples, Italy
ABSTRACT. Objective: The adverse role of traffic pollutants
on male fertility is well known. Aim of this study was to evaluate their effects on sperm chromatin/DNA integrity. Methods: To accomplish this, 36 men working at motorway tollgates and 32 unexposed healthy men (controls) were enrolled.
All of them were interviewed about their lifestyle. Hormone,
semen samples, and environmental and biological markers of
pollution were evaluated. Sperm chromatin and DNA integrity were evaluated by flow cytometry following propidium iodide staining and TUNEL assay, respectively. Results: LH, FSH,
and testosterone serum levels were within the normal range in
tollgate workers. Sperm concentration, total sperm count, total and progressive motility, and normal forms were signifi-
cantly lower in these men compared with controls. Motorway
tollgate workers had a significantly higher percentage of spermatozoa with damaged chromatin and DNA fragmentation, a
late sign of apoptosis, compared with controls. A significant
direct correlation was found between spermatozoa with damaged chromatin or fragmented DNA and the length of occupational exposure, suggesting a time-dependent relationship.
Conclusion: This study showed that car exhaust exposure has
a genotoxic effect on human spermatozoa. This may be of relevant importance not only for the reproductive performance
of the men exposed, but also for the offspring health.
(J. Endocrinol. Invest. 34: e139-e143, 2011)
©2011, Editrice Kurtis
INTRODUCTION
mode of action is a disruption of the hormonal regulation of spermatogenesis, a direct toxicity to the seminiferous tubules or both. A direct effect on testicular function is supported by the ability of Pb and Cd to accumulate in reproductive organs including mature spermatozoa whose K+ and Ca2+ channels, respectively, offer an
entry path for these heavy metals (see 8, for review). It
has also been shown that Pb competes or even replaces
the Zn of human protamine, thus causing a conformational change in the protein (8). This interaction seems
to negatively interfere with sperm chromatin condensation (9, 10). In addition, several lines of evidence support
the contention that heavy metals are able to cause an
oxidative stress by both increasing radical oxygen species
(ROS) generation and decreasing seminal plasma antioxidant capacity (11-15). This mechanism may damage
spermatozoa since they are very susceptible to ROS exposure because of their elevated content of polyunsaturated fatty acid, the major components of cellular and intracellular membranes. Furthermore, an increased oxidative stress may fragment sperm DNA. The small cytoplasmic volume of these cells and the low concentrations
of scavenging enzymes contribute to the highly toxic effects of ROS (see 16, for review). Heavy metals are also
able to bind selenium and thus to reduce the activity of
anti-oxidant enzymes such as the selenoprotein PHPGx.
The reduced activity of this enzyme can alter sperm parameters (17). In addition to heavy metals, car exhaust
contains also volatile organic compounds (VOC), such as
benzene, formaldehyde, polycyclic aromatic hydrocarbons, which exert hematotoxic, genotoxic, and carcinogenic effects (18).
Given the potential negative effects of VOC and heavy
metals on sperm chromatin and/or DNA integrity, we hy-
Over the past decades, many studies have shown that
environmental pollution is implicated in poor sperm
quality (1-3). Occupational exposure to traffic pollutants
has also been reported to alter sperm parameters (4-5).
A few years ago, De Rosa and colleagues reported that
motorway tollgate workers had significantly lower total
and progressive sperm motility. In a subset of them,
with sperm motility below normal, methemoglobin inversely correlated with total motility, viability, hypo-osmotic swelling test, acridine orange test, cervical mucus penetration test, linearity and amplitude of lateral
movement of the sperm head, whereas blood Pb (BPb)
levels inversely correlated with sperm count and viability (6). More recently, these authors confirmed the detrimental effects of nitrogen dioxide (NO2), a marker of
traffic pollution, on sperm parameters even at concentrations below the limits established by the Italian legislation (7). Thus, these findings suggest that continuous exposure to traffic pollutants impairs sperm quality
in young/middle-aged men and that Pb is probably the
cause of spermatogenic impairment.
The mechanism of male reproductive toxicity has not
been fully characterized. It is not known whether the main
Key words: Car exhaust, chromatin damage, DNA fragmentation, propidium iodide
staining, spermatozoa, TUNEL assay.
Correspondence: A.E. Calogero, Sezione di Endocrinologia, Andrologia e Medicina Interna, Dipartimento di Medicina Interna e Patologie Sistemiche, Università di Catania,
Policlinico “G. Rodolico”, Via S. Sofia 78, Bldg 4, Room 2C19, 95123 Catania, Italy.
Email: acaloger@unict.it
Accepted July 22, 2010.
First published online October 15, 2010.
e139
A.E. Calogero, S. La Vignera, R. Condorelli, et al.
pothesized that tollgate workers might be at increased
risk of having sperm chromatin damage and/or DNA fragmentation. Therefore, the present study was undertaken
to test this hypothesis. To accomplish this, sperm chromatin packaging and DNA fragmentation were evaluated by flow cytometry in 36 motorway tollgate workers
and in 32 unexposed age-matched controls.
PATIENTS AND METHODS
A total of 36 men working at motorway tollgates were enrolled
in this study. A complete medical history was collected and all of
them underwent physical examination, laboratory evaluation,
and endocrine screening. Men with systemic diseases, male accessory gland infection, history positive for cryptorchidism or
varicocele, microrchidism, alcohol or drug abuse, recent hormonal treatment were excluded. Thirty-two age-matched men,
not occupationally exposed to pollutants (employers, etc.) working in the same government agency, were randomly recruited
and served as controls. Each subject gave his informed consent
to the study.
All men were interviewed about their lifestyle. They were asked
the number of months of unprotected intercourse before the
couple’s first pregnancy [time to pregnancy (TTP)]. Serum LH,
FSH, and total testosterone (T) levels and semen analysis were
measured to evaluate the gonadal function. The concentrations
of nitrogen oxide (NO), sulphur oxide (SO), and Pb were measured at all tollgates and at the sites in the urban area where
the 36 subjects live. Blood levels of methemoglobin (MHb),
sulphemoglobin (SHb), carboxyhemoglobin (COHb), Pb, biological markers of environmental pollution, were assayed in all
subjects.
Environmental pollutant assay
The gaseous pollutants (NOx, sulphur compounds, and SOx)
were measured with specific analyzers (BABUC, L.S.I., Milan,
Italy) 24 h/day for 30 days during summer (15 June-15 July) and
winter (1-31 January) at 8 tollgates where the subjects worked
and at 8 sites where they lived, as previously reported (6). Because of the volcanic character of soil, due to active volcanic
mouths that let sulphur in the atmosphere, we measured also
the total quantity of sulphur compounds. The amount of H2S
levels was calculated by subtracting the SO from sulphur compounds. The values of environmental pollutants were attributed to each subject as mean of all measurements in his own work
place. For the control groups the gaseous pollutants were measured in the same manner and for the same period. Atmospheric
Pb was concentrated on porous filters by a suction pump (BRAVO, TCR Tecpora, Milan, Italy) and the filters were analyzed by
atomic absorption (19). The maximal occupational exposure to
these pollutants according to the Italian legislation, is <80
μg/m3/24 h or <200 μg/m3 for 1 h/24 h for NOx; <40 mg/m3/24
h for Pb; <2 μg/m3/24 h for SOx.
Toxicological evaluation
Absorption of environmental pollutants was evaluated by specific dose-and-effect gauges: MHb for NO2, SHb for sulphur
dioxide (SO2) and circulating BPb for environmental Pb (20). The
percentage concentrations of MHb (normal range <1.5% Hb),
SHb (normal range <1.2% Hb), and COHb (normal range: for
smokers <8%, for non-smokers <2.5%) was measured by the
oxymetric method (OSM3, Radiometer, Denmark) (21). BPb lev-
els (normal range <20 μg/dl) were measured by spectral analysis in atomic absorption.
Hormone assays
Serum levels of LH, FSH, and T were measured by chemiluminescent enzyme immunometric assays, using commercially available kits and the IMMULITE® automated analyzer (Diagnostic
Products Corporation, Los Angeles, USA; Medical System,
Genoa, Italy). Intra- and inter-assay coefficients of variation were
respectively: LH: 4.5% and 7.3%; FSH: 4.5% and 6.2%; T: 4.6%
and 6.2%.
Sperm analysis
Semen samples were collected by masturbation after 3-5 days of
sexual abstinence. After liquefaction, they were analyzed according to the World Health Organization criteria (22). The remaining spermatozoa were coded and used for flow cytometry
analysis in a blinded fashion.
Flow cytometry analysis
Flow cytometry to evaluate sperm chromatin integrity and DNA
fragmentation was performed using the flow cytometer EPICS XL
(Coulter Electronics, IL, Italy) equipped with a 488 nm argon
laser as light source. Two fluorescent detectors were used to
measure the fluorescence corresponding to the green color (FL1 detector 525 nm wavelength band) and the red color (FL-3
detector 620 nm wavelength band); 20,000 events were measured for each sample at a flow rate of 50-100 events/second
and analyzed using the Sistem II™ Software, 3.0 Version. As previously reported (23), the debris was gated out, by drawing a
region on forward vs side scatter dot plot which encloses the
population of cells of interest. The compensations and the settings was adapted according to the assay utilized.
Evaluation of sperm chromatin integrity
The evaluation of sperm chromatin integrity was evaluated by
propidium iodide (PI) staining performed after sperm membrane
permeabilization, as previously reported (23). Spermatozoon
permeabilization allows PI to gain access to nuclear DNA. Mature spermatozoa have a very compact structure that has a low
number of free legation sites, whereas spermatozoa which bind
a larger amounts of fluorochrome contain endogenous DNA
nicks, thus highlighting anomalies in chromatin packaging.
To accomplish this, semen samples from motorway tollgate
workers and controls were centrifuged at 500 g for 10 min at
room temperature, the supernatant removed and spermatozoa
collected. An aliquot of about 1×106 spermatozoa was incubated in PBS containing 50 μg/ml of PI (Sigma Chemical, Milan,
Italy), 0.1% sodium citrate, and 0.1% Nonidet P40 (Sigma Chemical), 100 Kunits/ml of RNAse type A (Sigma Chemical) in the
dark, at room temperature for 30 min. At the end of the incubation, flow cytometry analysis was performed using the detector FL3. In order to gate out, and thus exclude from the analysis doublets and cell aggregates, a “doublet discrimination module” was used.
Evaluation of sperm DNA fragmentation
Sperm chromatin integrity was evaluated by TUNEL assay as
previously reported (23). Briefly, spermatozoa were labelled with
the Apoptosis Mebstain kit (Beckman Coulter, IL, Milan, Italy).
The negative control was obtained by not adding TdT at the reaction mixture; the positive control was obtained by pre-treating
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Car exhaust and sperm genotoxicity
Table 1 - Mean±SD and range (in parentheses) hormonal concentration, sperm parameters and toxicological markers in 36 motorway
tollgate workers and in 32 controls.
Motorway tollgate workers
Controls
LH (IU/l)
2.9±0.7 (1.9-4.5)
3.2±1.1 (1.5-6.5)
FSH (IU/l)
4.2±1.1 (1.9-7.1)
4.3±1.5 (2.4-7.1)
Total testosterone (μg/l)
4.0±1.9 (1.3-8.0)
4.1±1.3 (1.9-7.0)
Sperm concentration (mil/ml)a
24.1±15.4 (6.3-70)*
99.2±56.7 (27-260)
Total sperm count (mil/ejaculate)a
64.9±43.3 (12.6-175)*
240.6±111.4 (94.5-520)
Total motility (%)
29.6±12.8 (11-65)*
51.8±10.2 (26-70)
Progressive motility (%)a
12.4±8.7 (2-30)*
27.7±6.9 (18-54)
Normal form (%)
17.2±0.8 (8-28)*
20.1±0.6 (15-27)
Methahaemoglobin (%)
1.00±0.1 (0.78-1.23)
Not measured
Sulphahaemoglobin (%)
0.87±0.10 (0.69-1.11)
Not measured
Carboxyhaemoglobin (%)
4.16±0.72 (2.7-5.5)
Not measured
Blood lead (μg/dl)
19.9±3.8 (12-25)
Not measured
Atmospheric lead (μg/dl)
2.8±0.4 (2.3-3.6)
2.7±0.1 (2.0-3.3)
Reference values: LH=0.8-7.6 IU/l; FSH=0.7-11.1 IU/l; Total testosterone=2.8-15.1 μg/l; Methemoglobin <1.5%; Sulphemoglobin <1.2%; Carboxyhemoglobin: smokers <8%, non-smokers <2.5%; Blood lead <20 μg/dl. aWHO 1999 criteria; *p<0.05 vs controls.
(range: 6-27 months) which was significantly higher compared with controls [12.1±0.9 months (range: 6-20
months)]. The hormonal concentration, main sperm parameters and the toxicological markers are reported in
Table 1. Serum levels of LH, FSH, and T did not differ
significantly within the two groups, whereas the following
sperm parameters were significantly different in motorway tollgate workers: sperm concentration, sperm total
count, total and progressive motility, and normal forms
(p<0.05 vs controls, Student’s t test).
Motorway tollgate workers had a significantly higher
(p<0.001, Student’s t test) percentage of spermatozoa
with damaged chromatin (18.3±1.4%) compared with
controls (11.3±1.2%) (Fig. 1). Likewise, the percentage
of spermatozoa with fragmented DNA, a late sign of
spermatozoa with 1 μg/ml of RNAse-free deoxyribonuclease I
(Sigma Chemical) at 37 C for 60 min before labeling. The debris was eliminated following the same procedure described
above. The percentage of fluorescein isothiocyanate-labeled
spermatozoa was determined by FL-1.
Statistical analysis
Factors and outcomes are reported as mean±SD and range
throughout the study. The data were analyzed by unpaired Student’s t test for identifying the statistical mean differences between factors and outcomes in the control group vs the case
study group. The multiple linear regression analysis was applied
for detecting linear relationships between factors and outcomes
within the control group and within the motorway tollgate workers. The following parameters were selected as independent
variables (outcomes): age, length of occupational exposure,
smoking habit, CO, COHb, NOx, SOx, atmospheric lead, blood
lead, MHb, SHb and LH, FSH, and T serum levels; whereas the
following were chosen as dependent variables (factors): spermatozoa with abnormal chromatin, spermatozoa with fragmented DNA, sperm volume, sperm concentration, sperm total count, and total and progressive motility. The software SPSS
9.0 for Windows was used for statistical evaluation (SPSS Inc.,
Chicago IL, USA). A statistically significant difference was accepted when the p-value was <0.05.
Spermatozoa (%) with damaged chromatin
40
RESULTS
Motorway tollgate workers had a mean age of 37.1±5.5
yr (range: 28-47 yr) which did not differ significantly from
that of controls (mean age 35.0±7.4 yr, range: 20-47 yr).
The tollgate workers were on this job from a mean of
11.8±0.7 yr (range: 7-23 yr). About half of them (54.3%)
smoked a mean of 13.6±1.0 cigarettes/day (range: 1020 cigarette/day) and 29 (82.9%) were married. A similar
percentage of smokers (53.1%) was found among controls who smoked 12.5±1.1 cigarettes/day (range: 10-20
cigarette/day). They had a mean of 1.4±0.2 children
(range: 0-3 children) and a TTP of 14.3±1.0 months
35
30
25
20
15
10
5
*p<0.001 vs Controls
0
Controls
Tollgate workers
Fig. 1 - Percentage of spermatozoa with damaged chromatin in
36 motorway tollgate workers chronically exposed to car exhaust and in 32 unexposed normal healthy men (controls). The
line represents the mean value for each group.
e141
A.E. Calogero, S. La Vignera, R. Condorelli, et al.
Spermatozoa (%) with fragmented DNA
25
*p<0.001 vs Controls
20
15
10
5
0
Controls
Tollgate workers
Fig. 2 - Percentage of spermatozoa with DNA fragmentation in
36 motorway tollgate workers chronically exposed to car exhaust and in 32 unexposed normal healthy men (controls). The
line represents the mean value for each group.
apoptosis, was also significantly (p<0.001, Student’s t
test) higher in tollgate workers (9.3±0.9%) compared with
controls (4.5±0.4%) (Fig. 2). Multiple linear regression
analysis showed no linear relationship in the control
group between independent (age, length of occupational
exposure, smoking habit, CO and LH, FSH, and T serum
levels) and dependent variables (spermatozoa with abnormal chromatin, spermatozoa with fragmented DNA,
sperm volume, sperm concentration, total sperm count,
sperm total and progressive motility). On the other hand,
tollgate workers showed a statistically significant linear
relationship between: a) percentage of DNA fragmented spermatozoa and length of occupational exposure
and FSH serum levels; b) sperm concentration and LH
serum levels; c) sperm total motility and LH serum levels,
COHb and MHb; d) sperm progressive motility and age
and SHb; and e) seminal volume and smoking habit.
DISCUSSION
The results of this study showed that prolonged exposure to traffic pollution is associated with lower conventional sperm parameters and with a negative impact on
sperm chromatin and DNA integrity. Indeed, motorway
tollgate workers had about twice as much spermatozoa
with damaged chromatin and about 50% more spermatozoa with fragmented DNA compared to unexposed
controls. These findings suggest that human sperm chromatin and DNA are in vivo targets of the toxic compounds present in men exposed to car exhaust. Although
many motor fume components are potentially harmful,
heavy metals have been shown to potentially play a relevant role by interfering with the sperm DNA protamination process.
It is well known that, during spermatogenesis, histones
are replaced by protamines (P1 and P2 families) which
tightly pack and, thus, protect sperm DNA. P2, a Zn-pro-
tein, has been reported to bind both Pb and Zn in vitro
and that Pb causes a dose-dependent decrease of P2
binding to DNA (9, 10). Therefore, these findings suggest that Pb is also able to alter chromatin stability which
in turn may lead to DNA damage. Accordingly, a significant effect of Pb on semen chromatin structure has been
reported in cynomolgus monkeys which were given Pb
without any effect on their endocrine function or conventional sperm parameters (24). These findings were
corroborated by a more recent study, conducted in urban men with mean BPb concentration of about 10
μg/dl. About half of the men enrolled had high values
of nuclear chromatin condensation (NCD) which directly correlates with the concentrations of Pb in the seminal
fluid and Zn in spermatozoa. Given that sperm Zn content is higher in fertile men, these results suggest that
Pb may affect sperm chromatin by altering sperm Zn
availability (25). Accordingly, Zn supplementation has
been shown to ameliorates Pb-induced testicular damage both at the cellular and subcellular level (26, 27).
In contrast with these data, Bonde and colleagues did
not find any susceptibility to acid-induced denaturation of
sperm chromatin evaluated by SCSA in a cross sectional
survey of the semen of 503 men employed by 10 companies in the United Kingdom, Italy, and Belgium, at least
for blood concentration of Pb <45 μg/dl. On the other
hand, the median sperm concentration was reduced by
49% in men with BPb concentration >50 μg/dl and, interestingly, the Authors identified a likely threshold BPb
concentration of 44 μg/dl (28).
We are not aware of studies showing an increased sperm
DNA fragmentation in human beings exposed to traffic
pollution. Two different mechanisms may explain the increased number of spermatozoa with fragmented DNA
that we found in motorway tollgate workers. The first may
be the consequence of the above-discussed alteration of
sperm chromatin condensation process (29, see 30 for review). In this regard, Aoki and colleagues reported that individual spermatozoa displaying the lowest levels of protamine have an increased susceptibility to DNA damage
(31). Accordingly, we found a direct correlation between
the percentage of spermatozoa with chromatin abnormality and the percentage of spermatozoa with fragmented DNA (r=0.53, p<0.001) (data not shown). A second mechanism may relate to an increased oxidative stress
(32) which may be due to heavy metal intoxication (11-15).
By multiple regression analysis, we did not find any significant linear relationship between blood Pb, NOx or
SOx concentrations and sperm chromatin and/or DNA
damage. It may be hypothesized that intra-sperm Pb
content may have a closer relationship with sperm genotoxicity. Accordingly, it has been reported that BPb concentration is not associated with semen quality or NCD
(25). This suggests that Pb in semen compartments (seminal plasma or spermatozoa) may give a better assessment of the amount of Pb present in the reproductive
tract. Alternatively, it may be hypothesized that an array
of pollutants, including VOC, rather than a single compound may be responsible for the genotoxicity observed. This is underlined by the significant direct relationship between sperm DNA fragmentation and length
of occupational exposure, by multiple regression analy-
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Car exhaust and sperm genotoxicity
15.
sis. In addition, it is noteworthy that SO2 has been reported to be able to alter spermatozoa and Sertoli cells
in mice (33). Thus, this compound may contribute to the
sperm genotoxicity observed in tollgate workers. On the
other hand, an association between exposure to high
levels of air pollution and increased sperm DNA damage has been reported (34-36). These pollution-related
effects are associated with the seasonality (higher in the
winter than in the summer) and with the null polymorphisms of glutathione-S-transferase M1 gene (GSTM1-).
Selevan and colleagues pointed out that sperm of subjects living in the Teplice district of the Czech Republic
showed an abnormal chromatin. These men were inconstantly exposed to CO during the year. On the contrary, the tollgate workers included in this study were exposed continuously for 8 h/day during the year under a
constant pollution level (measurements performed several times during the whole year).
In conclusion, the results of this study suggest that car
exhaust exposure has a detrimental effect on sperm chromatin and DNA integrity. This may explain the reduced
fertilizing potential reported by many Authors on subjects exposed to traffic pollution (4, 5). Our study indicates that the urban population exposed to traffic pollution has an increased risk not only of developing lung
cancer (37), but also of experiencing infertility.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
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