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Effect of cigarette smoke on DNA damage, oxidative stress, and morphological alterations in mouse testis and spermatozoa Sebastiano La Maestra, Silvio De Flora ∗ , Rosanna T. Micale Department of Health Sciences, University of Genoa, 16132 Genoa, Italy
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Article history: Received 12 June 2014 Received in revised form 19 August 2014 Accepted 29 August 2014 Keywords: Cigarette smoke Mouse sperm DNA damage Oxidative stress Sperm abnormalities Male infertility
a b s t r a c t Although the adverse effects of active smoking on sperm quality and fertilization ability are well established, little is known about possible effects of involuntary exposures to cigarette smoke (CS). We designed an experimental study aimed at evaluating the induction of possible noxious effects on testicular morphology and functions in A/J mice exposed whole-body to CS during the first 70 days of life, from birth to early adulthood. Twenty-five sham-exposed neonatal mice and 23 CS-exposed neonatal mice were used. Exposure to CS caused a variety of interconnected alterations in male gonads, including loss of weight and histomorphological alterations of testis, accompanied by a significant increase in abnormalities affecting epidydimal spermatozoa. Induction of oxidative stress was demonstrated by significantly increased concentrations of both reactive oxygen species and lipid peroxidation products in sperm cells. Occurrence of DNA damage in the same cells was documented by using the single cell gel electrophoresis (comet) assay, which showed a remarkable increase in DNA single- and double-strand breaks in CS-exposed mice, as compared with sham-exposed mice. Since biochemical and molecular alterations of sperm cells are known to be associated with impaired sperm quality, our findings suggest that involuntary smoking is potentially able to impair fertility in subjects exposed early in life. © 2014 Elsevier GmbH. All rights reserved.
Introduction Secondhand tobacco smoke is classified as a group 1 carcinogen (International Agency for Research on Cancer, 2004) and exposure to this complex mixture has been associated with a variety of pathological conditions (U.S. Department of Health and Human Services, 2006). It has been evaluated that secondhand tobacco smoke causes 53,000 deaths per year in the USA, about one non-smoker for every 8 smokers (Schick and Glantz, 2005). The health consequences of CS are particularly severe during critical periods of life. For instance, pregnant women who are exposed to secondhand smoke have a 20% higher odds of giving birth to low birth weight babies than women who are not exposed to secondhand smoke during pregnancy (U.S. Centers for Disease Control, 2007), and exposure of
Abbreviations: CS, cigarette smoke; FTC, Federal Trade Commission; TPM, total particulate matter; TBARS, thiobarbituric acid reactive substances; MDA, malondialdehyde; ROS, reactive oxygen species; DCFH-DA, 2 ,7 -dichlorofluorescein diacetate; DCF, dichlorofluorescein; FU, fluorescence unit; JC-1, 5,5 ,6,6 -tetrachloro1,1 ,3,3 tetraethylbenzimidazolocarbocyanine iodide; SCGE, single cell gel electrophoresis. ∗ Corresponding author. Tel.: +39 010 3538500; fax: +39 010 3538504. E-mail address: [email protected] (S. De Flora).
pregnant mice to CS resulted in a number of genomic and transcriptional alterations in fetus liver (Izzotti et al., 2003b). It is also well known that secondhand smoke can cause serious health problems in children (U.S. Department of Health and Human Services, 2006). Smoke components, such as nicotine and its major metabolites, cross the blood–testis barrier and are detectable in the seminal plasma both of active smokers (Pacifici et al., 1993) and of individuals exposed to CS (Pacifici et al., 1995). Active cigarette smoking increases oxidative damage, DNA adducts, DNA strand breaks, chromosomal aberrations, and heritable mutations in sperm (DeMarini, 2004; Marchetti et al., 2011). Moreover, paternal exposure to secondhand smoke may have reproductive consequences (Marchetti et al., 2011), and the hypothesis has been raised that tobacco consumption may act as an endocrine disruptor on the male hormone ˜ profile (Blanco-Munoz, 2012). Although the adverse effects of active smoking on sperm quality and fertilization ability are well established (Robbins et al., 2005; Agarwal et al., 2006; Practice Committee of the American Society for Reproductive Medicine, 2012), little is known about possible effects of involuntary exposures to CS. These premises prompted us to design an experimental study aimed at evaluating possible noxious effects on testicular morphology and functions in mice exposed
http://dx.doi.org/10.1016/j.ijheh.2014.08.006 1438-4639/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: La Maestra, S., et al., Effect of cigarette smoke on DNA damage, oxidative stress, and morphological alterations in mouse testis and spermatozoa. Int. J. Hyg. Environ. Health (2014), http://dx.doi.org/10.1016/j.ijheh.2014.08.006
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to CS from birth to early adulthood. The results obtained show that high dose CS causes histopathological, biochemical, and molecular alterations in testis and spermatozoa.
Materials and methods Mice Eight pregnant A/J mice were obtained from Harlan Italy. The mice were housed individually in Makrolon cages on sawdust bedding and maintained on standard rodent chow (Teklad 2018, Harlan Italy) and tap water ad libitum. The cages were kept in a cabinet where filtered air was circulated. The animal room had a temperature of 23 ± 2 ◦ C, a relative humidity of 55%, and a 12 h day/night cycle. After birth, the 49 mice born from four dams were kept in filtered air for 10 weeks, whereas the 45 mice born from other four dams were exposed daily to ECS for 10 consecutive weeks, starting within 12 h after birth. After weanling, the mice were divided by gender, and treatment continued for 25 male sham-exposed mice and 23 ECS-exposed mice.
Exposure to CS A whole-body exposure of mice to CS was achieved by using a smoking machine (model TE-10c, Teague Enterprises, Davis, CA) burning 3R4F reference cigarettes (College of Agriculture, the Reference Cigarette Program, University of Kentucky, Lexington, KY), having a declared content of 9.4 mg tar and 0.73 mg nicotine each. This machine, which is regulated to produce a mixture of sidestream smoke (89%) and mainstream smoke (11%), is operated under Federal Trade Commission (FTC) guidelines at a rate of one 35 mL puff of 2 s duration, once every minute. Five cigarettes were burnt at one time, 3 h/day, 7 days/week. This accounted for a daily exposure of mice to the ECS generated by 22.5 cigarettes in each one of the 4 exposure chambers. The average total particulate matter (TPM) in each exposure chamber, measured at weekly intervals, was 96.5 ± 1.38 mg/m3 (mean ± SE of 10 analyses), and the average CO in the exposure chambers was 605 ppm (693 mg/m3 ). A continuous air flow was produced by the machine in order to maintain a constant TPM level throughout the 3 h exposure period, after which the mice were removed from the exposure chambers and kept in a cabinet with filtered air for the rest of the day.
Morphological analysis of spermatozoa Slides were prepared by spreading 5–10 L of sperm suspension. After drying, the slides were fixed in 5% acetic acid in 95% ethanol for 3 min, air dried, stained in 5% Eosin Y (Sigma) for 4 min and rinsed in 70% ethanol. The dried slides were scanned under oil immersion (100×). A total of 1000 sperms for sample were classified according to their morphology. Lipid peroxidation products and reactive oxygen species Lipid peroxidation was evaluated in both testis and epididymal sperm by measuring the formation of thiobarbituric acid reactive substances (TBARS), using malondialdehyde (MDA) as a standard (Ohkawa et al., 1979). The data were expressed as MDA equivalents either per mg protein (testis) or 106 spermatozoa. Determination of reactive oxygen species (ROS) used a modified fluorimetric assay (Driver et al., 2000) using 2 ,7 dichlorofluorescein diacetate (DCFH-DA) as the probe. Briefly, aliquots of epididymal sperm were preincubated with DCFH-DA in PBS, pH 7.4, containing D-glucose, at room temperature for 15 min in order to allow the probe to be incorporated into membranebound vesicles and the DA group cleaved by esterases. The conversion of DCFH to the fluorescent product dichlorofluorescein (DCF) was measured by using a fluorescence spectrophotometer with excitation at 485 nm and emission at 530 nm. The results were expressed as fluorescence units (FU)/mg protein. Mitochondrial transmembrane potential The lipophilic cationic probe 5,5 ,6,6 -tetrachloro-1,1 ,3,3 tetraethylbenzimidazolocarbocyanine iodide (JC-1) (Mitochondria Staining Kit, Sigma) was used to differentially label mitochondria (Liu et al., 2007). When JC-1 remains in the monomeric form in mitochondria with low potential (m low), it will fluoresce in green, whereas when JC-1 forms multimers known as J-aggregates, following accumulation in mitochondria with high membrane potential (m high), it will fluoresce in orange. The results were read by using a fluorescence spectrophotometer with excitation at 490 nm and emission at 530 nm (green fluorescence monomers) and excitation at 525 nm and emission at 590 nm (red fluorescence aggregates). Valinomycin, a potassium-specific transporter through lipid membranes, was used as a positive control. The results were expressed as red/green fluorescence ratio. DNA damage in spermatozoa
Collection of testes and sperm After 10 weeks, all 48 mice were weighed and killed by CO2 asphyxiation. The testicles were collected and weighed. The left testes were fixed with Bouin’s solution for 24 h and embedded in paraffin, processed by standard histological techniques, stained with hematoxylin and eosin, and examined by light microscopy. The right testes were homogenized and frozen at −80 ◦ C for lipid peroxidation analyses. The caudal epididymis from each mouse was collected, excised, and minced into 2.5 mL of pre-warmed M16 medium (Sigma, St. Louis, MO). The tissue suspension was incubated at 37 ◦ C in 5% CO2 to allow for sperm dispersal. The sperm solutions were filtered through a 70 m cell strainer (BD Biosciences, Bedford, MA) and either frozen at −80 ◦ C or smeared on clean greasefree slides or immediately challenged with various fluorochromes for the analysis of freshly prepared cells. Counts of epididymal spermatozoa were performed by using a standard Bürker hemocytometer.
The comet assay, also known as single cell gel electrophoresis (SCGE) assay, is based on the principle that, when the sperm DNA breaks, DNA supercoils become loose and the negative charges are exposed. Alkaline SCGE was performed as described by Cordelli et al. (Cordelli et al., 2003) with some modification. Briefly, spermatozoa were suspended in 1% (w/v) low-melting-point agarose at a concentration of 1 × 104 cells/mL, applied to the surface of a microscope slide to form a microgel and allowed to set at 4 ◦ C for 5 min. The slides were submerged in cell lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, pH 10, containing 1% Triton X-100 and 40 mM dithiothreitol) for 1 h at room temperature and protected from light. Thereafter, proteinase K was added to the lysis solution (final concentration 10 g/mL) and additional lysis was achieved at 37 ◦ C for 2.5 h. The slides were washed through three changes of deionized water at 20 min intervals in order to remove salt and detergent from the microgels. The slides were then placed in a horizontal electrophoresis unit and allowed to equilibrate for 20 min with TAE buffer (300 mM NaOH, 1 mM Na2EDTA; HCl was added to reach pH 12.1) before electrophoresis (25 V, 0.01 A) for 20 min.
Please cite this article in press as: La Maestra, S., et al., Effect of cigarette smoke on DNA damage, oxidative stress, and morphological alterations in mouse testis and spermatozoa. Int. J. Hyg. Environ. Health (2014), http://dx.doi.org/10.1016/j.ijheh.2014.08.006
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Fig. 1. Representative photomicrographs of hematoxylin and eosin staining of testes, showing at 3 different magnitudes, the seminiferous tubules from sham-exposed mice (left) and CS-exposed mice (right). The arrows show Leydig cells adjacent to intratubular spaces.
When electrophoresis was complete, the slides were rinsed with distilled water, and stained with ethidium bromide (20 g/mL) and mounted with a coverslip. Images were acquired at a 400× magnification by using a fluorescence microscope equipped with a digital camera. Images of at least 150 randomly selected nuclei were acquired from each mouse and analyzed by using an automated imaging system (CASP or Comet Assay Software Project, http://www.casp.sourceforge.net). DNA damage (alkali–labile sites and both double- and single-stranded DNA breaks) was quantified in terms of % tail DNA.
Histomorphological alterations of testes Fig. 1 shows, at 3 different magnifications, an example of damage induced by CS in some seminiferous tubules. Compared to shamexposed mice, CS-exposed mice exhibited smaller seminiferous tubules accompanied by atrophy and degeneration of tubules and loss of spermatogenesis, decrease of the spermatogenic cell layer, and widened interstitial areas with marked reduction in Leydig cells. Number and abnormalities of spermatozoa
Statistical analysis All results were expressed as mean ± SE within the mice composing each experimental group. The statistical significance of the differences between groups was evaluated by ANOVA and Student’s t test.
Results Body weight and testis weight All mice survived until the end of the experiment. At 10 weeks of life, the body weight in CS-exposed mice (39.2 ± 0.73 g) was significantly lower (P < 0.001) than that of sham-exposed mice (44.9 ± 1.55 g). Likewise, the testis weight was significantly decreased (P < 0.05) following exposure to CS (0.14 ± 0.003 g) compared to sham-exposed mice (0.16 ± 0.005 g).
The number of epididymal spermatozoa was 3.4 ± 0.30 × 106 /mL in sham-exposed mice and 2.6 ± 0.23 × 106 /mL in CS-exposed mice. This difference was statistically significant (P < 0.05). The most frequent abnormality of spermatozoa was the absence of tail, which was detected in the 20.8 ± 1.75‰ of sham-exposed mice and in the 29.1‰ of CS-exposed mice. This difference was statistically significant (P < 0.05). The appearance of other abnormal spermatozoa and their frequencies as related to exposure to CS are shown in Fig. 2. CS caused significant increases of spermatozoa having either two tails, no hook, or pinhead. Oxidative and mitochondrial parameters Table 1 summarizes the results of biochemical analyses evaluating oxidative stress and mitochondrial alterations. CS did not affect the levels of TBARS in testis homogenates but significantly increased these lipid peroxidation products in spermatozoa, where
Please cite this article in press as: La Maestra, S., et al., Effect of cigarette smoke on DNA damage, oxidative stress, and morphological alterations in mouse testis and spermatozoa. Int. J. Hyg. Environ. Health (2014), http://dx.doi.org/10.1016/j.ijheh.2014.08.006
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Fig. 2. Appearance and frequencies (mean ± SE) of abnormal spermatozoa in sham-exposed mice (empty columns) and CS-exposed mice (full columns). * Significant difference (P < 0.05).
Table 1 Evaluation of oxidative and mitochondrial changes in A/J mice, as related to exposure to exposure to CS during the first 10 weeks of life. End-point
Organ or cells
Unit
Exposure of mice
TBARS TBARS ROS Mt membrane potential ( m)
Testis Spermatozoa Spermatozoa Spermatozoa
nmol/mg protein nmol/106 sperms FU/mg protein Red/green fluorescence ratio
Sham
CS
14.2 ± 2.06 2.1 ± 0.23 223.9 ± 28.29 4.0 ± 0.70
14.03 ± 1.01 3.43 ± 0.38a 300.2 ± 37.38a 3.6 ± 0.63
The data are means ± SE of the results obtained in 25 sham-exposed mice and 23 CS-exposed mice. a P < 0.05, as compared with Sham.
it also increased ROS generation. In the same cells, exposure to CS did not affect the mitochondrial membrane potential.
DNA damage in spermatozoa The results of the comet assay showed an appreciable (1.32-fold) and statistically significant increase of DNA damage in spermatozoa from CS-exposed mice as compared with sham-exposed mice. Fig. 3 shows an example of the comet appearance and the mean ± SE data obtained in 25 sham-exposed mice and 23 CS-exposed mice, expressed as % tail DNA.
Fig. 3. Appearance of comets and % tail DNA (mean ± SE) in spermatozoa from shamexposed mice and CS-exposed mice. a Significant difference (P < 0.01).
Discussion The present study evaluated morphological, biochemical, and molecular alterations in testis and sperm cells from mice exposed to CS early in life, starting soon after birth. The 70-day exposure period covered the immediate post-natal time, during which the sudden transition from the maternal-mediated respiration to the autonomous pulmonary respiration of neonatal mice causes paraphysiological genomic alterations in the lung (Izzotti et al., 2003a), followed by the lactation period until completion of weaning. This period partially overlaps with adolescence, which in mice has been divided into 3 ages, including pre-adolescence (23–35 days), mid-adolescence (36–48 days), and post-adolescence (49–61 days) (Adriani et al., 2004), leading to sexual maturity and early adulthood. Spermatogenesis in mice is initiated 3 to 7 days after birth, and during the prepubescence period a sequential appearance of spermatogenic cells in the testis can be observed (Bellvé et al., 1977). It is known that children are especially sensitive to the respiratory effects of CS exposure (California Environmental Protection Agency, 2005). Due to a variety of composite mechanisms, mice exposed during the first 5 weeks of life are more susceptible than their dams, exposed under identical conditions, to molecular, biochemical and cytogenetic alterations induced by CS in the respiratory tract (De Flora et al., 2008). As shown in mice exposed
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to CS, oxidative stress and the resulting DNA damage provide a major contribution to the high susceptibility of mice exposed early in life (Micale et al., 2013). Our previous studies showed that, under experimental conditions similar to those of the present study, CS causes early genomic and microRNA alterations in mouse lung (Izzotti et al., 2009; D’Agostini et al., 2009). Adolescent male mice (28-day old) and adult male mice (70-day old) also differ in several aspects of nicotine dependence (Kota et al., 2007). The doses of CS to which our mice were exposed, which accounted for an average TPM of 96.5 mg/m3 air, were within the ranges reported in studies using the same smoking machine available for the present study. In fact, a recent review of 28 studies showed that the TPM in the exposure chambers was between 70 and 150 mg/m3 air (Leberl et al., 2013). Using the same smoking machine available to our laboratory, at similar TPM concentrations, and in the same mouse strain, plasma cotinine levels were found on an average to be 175 ng/mL and were unrelated to the time of exposure to CS (Witschi et al., 2004). This cotinine level is comparable to the one measured in subjects smoking 5–9 cigarettes per day (Seccareccia et al., 2003). The CS dose used in the present study was high but not unrealistic for human exposure. In fact, the Scientific Review Panel of the California Environmental Protection Agency has reported that the respirable particulate matter in certain entertainment venues are estimated to range from less than 15 mg/m3 where smoking is prohibited up to 350 mg/m3 where smoking is allowed. In the home environment, peaks up to 300 mg/m3 have been found, and inside vehicles concentrations are estimated to range from about 90 mg/m3 and well over 1,000 mg/m3 (California Environmental Protection Agency, 2005). Even at relatively low levels (3.7 mg/m3 air), exposure of 4 fertile adult rhesus macaques (Macaca mulatta) for 6 months caused metabolic changes in the sperm, as assessed by NMR analysis. However, CS did not affect semen quality and sperm function (Hung et al., 2009). Under our experimental conditions, a high dose exposure of mice to CS for 10 weeks, starting at birth, resulted in a significant decrease of testis weight, in evident histomorphological alterations of testes, in a significant decrease in the number of epidydimal spermatozoa, and in a variety of abnormalities of these cells. The most important CS-related abnormality was the absence of tail, which is regarded as a sign of sperm fragility and is associated with fertilization failure (Marmor and Grob-Menendez, 1991). In parallel, CS caused an oxidative damage that was not detectable in whole testis but was well documented by an increase of both ROS and lipid peroxidation products in epidydimal spermatozoa. On the other hand, the sperm m was not decreased in CS-exposed mice to a significant extent. This finding suggests that the increased ROS generation in spermatozoa of CS-exposed mice is amenable to enzymatic systems located in the plasma membrane, utilizing NAD(P)H as a substrate (Vernet et al., 2001), rather than to mitochondrial mechanisms. The observed sperm alterations were accompanied by a well-appreciable generation of ROS and induction of DNA damage. It has been reported that the mechanism by which smoking can affect semen quality does not exclude direct involvement of toxic substances in smoke, such as CO, nicotine, tar, etc., affecting male gametes (Taha et al., 2012). However, even under extreme in vitro conditions, CO does not affect human sperm motility (Makler et al., 1993). Interestingly, morphological and biochemical alterations were accompanied by a remarkable DNA damage, which was detected in the sperm of ECS-exposed mice by using the comet assay, a highly sensitive method for evaluating the DNA fragmentation due to single- and double-strand breaks. Previously, we demonstrated that exposure of adult rats to CS for 4 weeks caused a significant, 1.9-fold increase in bulky DNA adduct levels in testis (Izzotti et al.,
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1999). DNA adducts are promutagenic lesions that, unless repaired, can trigger the DNA damage process that was documented in the present study. Measurement of sperm DNA damage is a useful tool in the evaluation of male infertility, as the sperm nucleus lacks protection against oxidative stress and is vulnerable to oxidationmediated DNA damage (Simon and Carrell, 2013). Generation of ROS in germ cells is a physiological mechanism required for maturation, capacitation, acrosome reaction of spermatozoa, binding with the zona pellucida, and oocyte function (De Lamirande and Gagnon, 1993). However, excessive ROS levels attack bases in nucleic acids, amino acid side chains in proteins and double bonds in unsaturated fatty acids, and cause oxidative stress, which can damage DNA, RNA, proteins, and lipids thereby resulting in an increased risk for a variety of diseases (Lü et al., 2010). It has been shown that ROS can affect the structural and functional parameters of sperm (Henkel, 2005). Experimental studies have suggested that exposure of rats to CS results in oxidative imbalances in sperm cells, as demonstrated either by an increase in TBARS and/or a decrease in antioxidant mechanisms (Peltola et al., 1994; Rajpurkar et al., 2000a,b; Ozyurt et al., 2006; Mohamed et al., 2011; Sankako et al., 2012). The sperm plasma membrane contains high concentrations of polyunsaturated fatty acids, which is an essential requirement for the male germ cell to maintain sperm functions. Also due to the very low content of protective systems in spermatozoa, polyunsaturated fatty acids are highly susceptible to oxidative damage leading to a loss of integrity in the sperm membrane (Aitken et al., 1989). This has been shown to be a cause of impaired sperm function and interference with its fertilization ability (Agarwal et al., 2005; Ghaffari and Rostami, 2012). It is noteworthy that, in addition to DNA damage, exposure of mice to CS has been shown to cause epigenetic alterations in testis (Xu et al., 2013), which have been associated with male infertility (Rajender et al., 2011). This kind of alterations is compatible with the strong oxidative damage produced in testis by CS, as shown in the present study. The histomorphological alterations that we observed in the testes of mice exposed to CS since birth are in line with similar findings detected in the testes of CS-exposed rats, either during the pubertal period (Rajpurkar and Dhabuwala, 2000) or in adult life (Mohamed et al., 2011; Güven et al., 1999; Ahmadnia et al., 2007).
Conclusion The results herein reported show for the first time that exposure of mice to high doses of CS, starting at birth and continuing until achievement of sexual maturity, causes a variety of interconnected alterations in male gonads, including loss of weight and histopathological alterations of testis, morphological abnormalities of spermatozoa, oxidative stress, and DNA damage in sperm cells.
Limitations of the study The results reported in the present study should be interpreted with caution due to the high doses of CS that have to be used in experimental models. Moreover, we did not measure cotinine levels in the plasma of exposed mice but relied on literature data generated under comparable experimental conditions.
Conflict of interest The authors declare they have no conflict of interests.
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Effect of cigarette smoke on DNA damage, oxidative stress, and morphological alterations in mouse testis and spermatozoa Sebastiano La Maestra, Silvio De Flora ∗ , Rosanna T. Micale Department of Health Sciences, University of Genoa, 16132 Genoa, Italy
a r t i c l e
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Article history: Received 12 June 2014 Received in revised form 19 August 2014 Accepted 29 August 2014 Keywords: Cigarette smoke Mouse sperm DNA damage Oxidative stress Sperm abnormalities Male infertility
a b s t r a c t Although the adverse effects of active smoking on sperm quality and fertilization ability are well established, little is known about possible effects of involuntary exposures to cigarette smoke (CS). We designed an experimental study aimed at evaluating the induction of possible noxious effects on testicular morphology and functions in A/J mice exposed whole-body to CS during the first 70 days of life, from birth to early adulthood. Twenty-five sham-exposed neonatal mice and 23 CS-exposed neonatal mice were used. Exposure to CS caused a variety of interconnected alterations in male gonads, including loss of weight and histomorphological alterations of testis, accompanied by a significant increase in abnormalities affecting epidydimal spermatozoa. Induction of oxidative stress was demonstrated by significantly increased concentrations of both reactive oxygen species and lipid peroxidation products in sperm cells. Occurrence of DNA damage in the same cells was documented by using the single cell gel electrophoresis (comet) assay, which showed a remarkable increase in DNA single- and double-strand breaks in CS-exposed mice, as compared with sham-exposed mice. Since biochemical and molecular alterations of sperm cells are known to be associated with impaired sperm quality, our findings suggest that involuntary smoking is potentially able to impair fertility in subjects exposed early in life. © 2014 Elsevier GmbH. All rights reserved.
Introduction Secondhand tobacco smoke is classified as a group 1 carcinogen (International Agency for Research on Cancer, 2004) and exposure to this complex mixture has been associated with a variety of pathological conditions (U.S. Department of Health and Human Services, 2006). It has been evaluated that secondhand tobacco smoke causes 53,000 deaths per year in the USA, about one non-smoker for every 8 smokers (Schick and Glantz, 2005). The health consequences of CS are particularly severe during critical periods of life. For instance, pregnant women who are exposed to secondhand smoke have a 20% higher odds of giving birth to low birth weight babies than women who are not exposed to secondhand smoke during pregnancy (U.S. Centers for Disease Control, 2007), and exposure of
Abbreviations: CS, cigarette smoke; FTC, Federal Trade Commission; TPM, total particulate matter; TBARS, thiobarbituric acid reactive substances; MDA, malondialdehyde; ROS, reactive oxygen species; DCFH-DA, 2 ,7 -dichlorofluorescein diacetate; DCF, dichlorofluorescein; FU, fluorescence unit; JC-1, 5,5 ,6,6 -tetrachloro1,1 ,3,3 tetraethylbenzimidazolocarbocyanine iodide; SCGE, single cell gel electrophoresis. ∗ Corresponding author. Tel.: +39 010 3538500; fax: +39 010 3538504. E-mail address: [email protected] (S. De Flora).
pregnant mice to CS resulted in a number of genomic and transcriptional alterations in fetus liver (Izzotti et al., 2003b). It is also well known that secondhand smoke can cause serious health problems in children (U.S. Department of Health and Human Services, 2006). Smoke components, such as nicotine and its major metabolites, cross the blood–testis barrier and are detectable in the seminal plasma both of active smokers (Pacifici et al., 1993) and of individuals exposed to CS (Pacifici et al., 1995). Active cigarette smoking increases oxidative damage, DNA adducts, DNA strand breaks, chromosomal aberrations, and heritable mutations in sperm (DeMarini, 2004; Marchetti et al., 2011). Moreover, paternal exposure to secondhand smoke may have reproductive consequences (Marchetti et al., 2011), and the hypothesis has been raised that tobacco consumption may act as an endocrine disruptor on the male hormone ˜ profile (Blanco-Munoz, 2012). Although the adverse effects of active smoking on sperm quality and fertilization ability are well established (Robbins et al., 2005; Agarwal et al., 2006; Practice Committee of the American Society for Reproductive Medicine, 2012), little is known about possible effects of involuntary exposures to CS. These premises prompted us to design an experimental study aimed at evaluating possible noxious effects on testicular morphology and functions in mice exposed
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Please cite this article in press as: La Maestra, S., et al., Effect of cigarette smoke on DNA damage, oxidative stress, and morphological alterations in mouse testis and spermatozoa. Int. J. Hyg. Environ. Health (2014), http://dx.doi.org/10.1016/j.ijheh.2014.08.006
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to CS from birth to early adulthood. The results obtained show that high dose CS causes histopathological, biochemical, and molecular alterations in testis and spermatozoa.
Materials and methods Mice Eight pregnant A/J mice were obtained from Harlan Italy. The mice were housed individually in Makrolon cages on sawdust bedding and maintained on standard rodent chow (Teklad 2018, Harlan Italy) and tap water ad libitum. The cages were kept in a cabinet where filtered air was circulated. The animal room had a temperature of 23 ± 2 ◦ C, a relative humidity of 55%, and a 12 h day/night cycle. After birth, the 49 mice born from four dams were kept in filtered air for 10 weeks, whereas the 45 mice born from other four dams were exposed daily to ECS for 10 consecutive weeks, starting within 12 h after birth. After weanling, the mice were divided by gender, and treatment continued for 25 male sham-exposed mice and 23 ECS-exposed mice.
Exposure to CS A whole-body exposure of mice to CS was achieved by using a smoking machine (model TE-10c, Teague Enterprises, Davis, CA) burning 3R4F reference cigarettes (College of Agriculture, the Reference Cigarette Program, University of Kentucky, Lexington, KY), having a declared content of 9.4 mg tar and 0.73 mg nicotine each. This machine, which is regulated to produce a mixture of sidestream smoke (89%) and mainstream smoke (11%), is operated under Federal Trade Commission (FTC) guidelines at a rate of one 35 mL puff of 2 s duration, once every minute. Five cigarettes were burnt at one time, 3 h/day, 7 days/week. This accounted for a daily exposure of mice to the ECS generated by 22.5 cigarettes in each one of the 4 exposure chambers. The average total particulate matter (TPM) in each exposure chamber, measured at weekly intervals, was 96.5 ± 1.38 mg/m3 (mean ± SE of 10 analyses), and the average CO in the exposure chambers was 605 ppm (693 mg/m3 ). A continuous air flow was produced by the machine in order to maintain a constant TPM level throughout the 3 h exposure period, after which the mice were removed from the exposure chambers and kept in a cabinet with filtered air for the rest of the day.
Morphological analysis of spermatozoa Slides were prepared by spreading 5–10 L of sperm suspension. After drying, the slides were fixed in 5% acetic acid in 95% ethanol for 3 min, air dried, stained in 5% Eosin Y (Sigma) for 4 min and rinsed in 70% ethanol. The dried slides were scanned under oil immersion (100×). A total of 1000 sperms for sample were classified according to their morphology. Lipid peroxidation products and reactive oxygen species Lipid peroxidation was evaluated in both testis and epididymal sperm by measuring the formation of thiobarbituric acid reactive substances (TBARS), using malondialdehyde (MDA) as a standard (Ohkawa et al., 1979). The data were expressed as MDA equivalents either per mg protein (testis) or 106 spermatozoa. Determination of reactive oxygen species (ROS) used a modified fluorimetric assay (Driver et al., 2000) using 2 ,7 dichlorofluorescein diacetate (DCFH-DA) as the probe. Briefly, aliquots of epididymal sperm were preincubated with DCFH-DA in PBS, pH 7.4, containing D-glucose, at room temperature for 15 min in order to allow the probe to be incorporated into membranebound vesicles and the DA group cleaved by esterases. The conversion of DCFH to the fluorescent product dichlorofluorescein (DCF) was measured by using a fluorescence spectrophotometer with excitation at 485 nm and emission at 530 nm. The results were expressed as fluorescence units (FU)/mg protein. Mitochondrial transmembrane potential The lipophilic cationic probe 5,5 ,6,6 -tetrachloro-1,1 ,3,3 tetraethylbenzimidazolocarbocyanine iodide (JC-1) (Mitochondria Staining Kit, Sigma) was used to differentially label mitochondria (Liu et al., 2007). When JC-1 remains in the monomeric form in mitochondria with low potential (m low), it will fluoresce in green, whereas when JC-1 forms multimers known as J-aggregates, following accumulation in mitochondria with high membrane potential (m high), it will fluoresce in orange. The results were read by using a fluorescence spectrophotometer with excitation at 490 nm and emission at 530 nm (green fluorescence monomers) and excitation at 525 nm and emission at 590 nm (red fluorescence aggregates). Valinomycin, a potassium-specific transporter through lipid membranes, was used as a positive control. The results were expressed as red/green fluorescence ratio. DNA damage in spermatozoa
Collection of testes and sperm After 10 weeks, all 48 mice were weighed and killed by CO2 asphyxiation. The testicles were collected and weighed. The left testes were fixed with Bouin’s solution for 24 h and embedded in paraffin, processed by standard histological techniques, stained with hematoxylin and eosin, and examined by light microscopy. The right testes were homogenized and frozen at −80 ◦ C for lipid peroxidation analyses. The caudal epididymis from each mouse was collected, excised, and minced into 2.5 mL of pre-warmed M16 medium (Sigma, St. Louis, MO). The tissue suspension was incubated at 37 ◦ C in 5% CO2 to allow for sperm dispersal. The sperm solutions were filtered through a 70 m cell strainer (BD Biosciences, Bedford, MA) and either frozen at −80 ◦ C or smeared on clean greasefree slides or immediately challenged with various fluorochromes for the analysis of freshly prepared cells. Counts of epididymal spermatozoa were performed by using a standard Bürker hemocytometer.
The comet assay, also known as single cell gel electrophoresis (SCGE) assay, is based on the principle that, when the sperm DNA breaks, DNA supercoils become loose and the negative charges are exposed. Alkaline SCGE was performed as described by Cordelli et al. (Cordelli et al., 2003) with some modification. Briefly, spermatozoa were suspended in 1% (w/v) low-melting-point agarose at a concentration of 1 × 104 cells/mL, applied to the surface of a microscope slide to form a microgel and allowed to set at 4 ◦ C for 5 min. The slides were submerged in cell lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, pH 10, containing 1% Triton X-100 and 40 mM dithiothreitol) for 1 h at room temperature and protected from light. Thereafter, proteinase K was added to the lysis solution (final concentration 10 g/mL) and additional lysis was achieved at 37 ◦ C for 2.5 h. The slides were washed through three changes of deionized water at 20 min intervals in order to remove salt and detergent from the microgels. The slides were then placed in a horizontal electrophoresis unit and allowed to equilibrate for 20 min with TAE buffer (300 mM NaOH, 1 mM Na2EDTA; HCl was added to reach pH 12.1) before electrophoresis (25 V, 0.01 A) for 20 min.
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Fig. 1. Representative photomicrographs of hematoxylin and eosin staining of testes, showing at 3 different magnitudes, the seminiferous tubules from sham-exposed mice (left) and CS-exposed mice (right). The arrows show Leydig cells adjacent to intratubular spaces.
When electrophoresis was complete, the slides were rinsed with distilled water, and stained with ethidium bromide (20 g/mL) and mounted with a coverslip. Images were acquired at a 400× magnification by using a fluorescence microscope equipped with a digital camera. Images of at least 150 randomly selected nuclei were acquired from each mouse and analyzed by using an automated imaging system (CASP or Comet Assay Software Project, http://www.casp.sourceforge.net). DNA damage (alkali–labile sites and both double- and single-stranded DNA breaks) was quantified in terms of % tail DNA.
Histomorphological alterations of testes Fig. 1 shows, at 3 different magnifications, an example of damage induced by CS in some seminiferous tubules. Compared to shamexposed mice, CS-exposed mice exhibited smaller seminiferous tubules accompanied by atrophy and degeneration of tubules and loss of spermatogenesis, decrease of the spermatogenic cell layer, and widened interstitial areas with marked reduction in Leydig cells. Number and abnormalities of spermatozoa
Statistical analysis All results were expressed as mean ± SE within the mice composing each experimental group. The statistical significance of the differences between groups was evaluated by ANOVA and Student’s t test.
Results Body weight and testis weight All mice survived until the end of the experiment. At 10 weeks of life, the body weight in CS-exposed mice (39.2 ± 0.73 g) was significantly lower (P < 0.001) than that of sham-exposed mice (44.9 ± 1.55 g). Likewise, the testis weight was significantly decreased (P < 0.05) following exposure to CS (0.14 ± 0.003 g) compared to sham-exposed mice (0.16 ± 0.005 g).
The number of epididymal spermatozoa was 3.4 ± 0.30 × 106 /mL in sham-exposed mice and 2.6 ± 0.23 × 106 /mL in CS-exposed mice. This difference was statistically significant (P < 0.05). The most frequent abnormality of spermatozoa was the absence of tail, which was detected in the 20.8 ± 1.75‰ of sham-exposed mice and in the 29.1‰ of CS-exposed mice. This difference was statistically significant (P < 0.05). The appearance of other abnormal spermatozoa and their frequencies as related to exposure to CS are shown in Fig. 2. CS caused significant increases of spermatozoa having either two tails, no hook, or pinhead. Oxidative and mitochondrial parameters Table 1 summarizes the results of biochemical analyses evaluating oxidative stress and mitochondrial alterations. CS did not affect the levels of TBARS in testis homogenates but significantly increased these lipid peroxidation products in spermatozoa, where
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Fig. 2. Appearance and frequencies (mean ± SE) of abnormal spermatozoa in sham-exposed mice (empty columns) and CS-exposed mice (full columns). * Significant difference (P < 0.05).
Table 1 Evaluation of oxidative and mitochondrial changes in A/J mice, as related to exposure to exposure to CS during the first 10 weeks of life. End-point
Organ or cells
Unit
Exposure of mice
TBARS TBARS ROS Mt membrane potential ( m)
Testis Spermatozoa Spermatozoa Spermatozoa
nmol/mg protein nmol/106 sperms FU/mg protein Red/green fluorescence ratio
Sham
CS
14.2 ± 2.06 2.1 ± 0.23 223.9 ± 28.29 4.0 ± 0.70
14.03 ± 1.01 3.43 ± 0.38a 300.2 ± 37.38a 3.6 ± 0.63
The data are means ± SE of the results obtained in 25 sham-exposed mice and 23 CS-exposed mice. a P < 0.05, as compared with Sham.
it also increased ROS generation. In the same cells, exposure to CS did not affect the mitochondrial membrane potential.
DNA damage in spermatozoa The results of the comet assay showed an appreciable (1.32-fold) and statistically significant increase of DNA damage in spermatozoa from CS-exposed mice as compared with sham-exposed mice. Fig. 3 shows an example of the comet appearance and the mean ± SE data obtained in 25 sham-exposed mice and 23 CS-exposed mice, expressed as % tail DNA.
Fig. 3. Appearance of comets and % tail DNA (mean ± SE) in spermatozoa from shamexposed mice and CS-exposed mice. a Significant difference (P < 0.01).
Discussion The present study evaluated morphological, biochemical, and molecular alterations in testis and sperm cells from mice exposed to CS early in life, starting soon after birth. The 70-day exposure period covered the immediate post-natal time, during which the sudden transition from the maternal-mediated respiration to the autonomous pulmonary respiration of neonatal mice causes paraphysiological genomic alterations in the lung (Izzotti et al., 2003a), followed by the lactation period until completion of weaning. This period partially overlaps with adolescence, which in mice has been divided into 3 ages, including pre-adolescence (23–35 days), mid-adolescence (36–48 days), and post-adolescence (49–61 days) (Adriani et al., 2004), leading to sexual maturity and early adulthood. Spermatogenesis in mice is initiated 3 to 7 days after birth, and during the prepubescence period a sequential appearance of spermatogenic cells in the testis can be observed (Bellvé et al., 1977). It is known that children are especially sensitive to the respiratory effects of CS exposure (California Environmental Protection Agency, 2005). Due to a variety of composite mechanisms, mice exposed during the first 5 weeks of life are more susceptible than their dams, exposed under identical conditions, to molecular, biochemical and cytogenetic alterations induced by CS in the respiratory tract (De Flora et al., 2008). As shown in mice exposed
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to CS, oxidative stress and the resulting DNA damage provide a major contribution to the high susceptibility of mice exposed early in life (Micale et al., 2013). Our previous studies showed that, under experimental conditions similar to those of the present study, CS causes early genomic and microRNA alterations in mouse lung (Izzotti et al., 2009; D’Agostini et al., 2009). Adolescent male mice (28-day old) and adult male mice (70-day old) also differ in several aspects of nicotine dependence (Kota et al., 2007). The doses of CS to which our mice were exposed, which accounted for an average TPM of 96.5 mg/m3 air, were within the ranges reported in studies using the same smoking machine available for the present study. In fact, a recent review of 28 studies showed that the TPM in the exposure chambers was between 70 and 150 mg/m3 air (Leberl et al., 2013). Using the same smoking machine available to our laboratory, at similar TPM concentrations, and in the same mouse strain, plasma cotinine levels were found on an average to be 175 ng/mL and were unrelated to the time of exposure to CS (Witschi et al., 2004). This cotinine level is comparable to the one measured in subjects smoking 5–9 cigarettes per day (Seccareccia et al., 2003). The CS dose used in the present study was high but not unrealistic for human exposure. In fact, the Scientific Review Panel of the California Environmental Protection Agency has reported that the respirable particulate matter in certain entertainment venues are estimated to range from less than 15 mg/m3 where smoking is prohibited up to 350 mg/m3 where smoking is allowed. In the home environment, peaks up to 300 mg/m3 have been found, and inside vehicles concentrations are estimated to range from about 90 mg/m3 and well over 1,000 mg/m3 (California Environmental Protection Agency, 2005). Even at relatively low levels (3.7 mg/m3 air), exposure of 4 fertile adult rhesus macaques (Macaca mulatta) for 6 months caused metabolic changes in the sperm, as assessed by NMR analysis. However, CS did not affect semen quality and sperm function (Hung et al., 2009). Under our experimental conditions, a high dose exposure of mice to CS for 10 weeks, starting at birth, resulted in a significant decrease of testis weight, in evident histomorphological alterations of testes, in a significant decrease in the number of epidydimal spermatozoa, and in a variety of abnormalities of these cells. The most important CS-related abnormality was the absence of tail, which is regarded as a sign of sperm fragility and is associated with fertilization failure (Marmor and Grob-Menendez, 1991). In parallel, CS caused an oxidative damage that was not detectable in whole testis but was well documented by an increase of both ROS and lipid peroxidation products in epidydimal spermatozoa. On the other hand, the sperm m was not decreased in CS-exposed mice to a significant extent. This finding suggests that the increased ROS generation in spermatozoa of CS-exposed mice is amenable to enzymatic systems located in the plasma membrane, utilizing NAD(P)H as a substrate (Vernet et al., 2001), rather than to mitochondrial mechanisms. The observed sperm alterations were accompanied by a well-appreciable generation of ROS and induction of DNA damage. It has been reported that the mechanism by which smoking can affect semen quality does not exclude direct involvement of toxic substances in smoke, such as CO, nicotine, tar, etc., affecting male gametes (Taha et al., 2012). However, even under extreme in vitro conditions, CO does not affect human sperm motility (Makler et al., 1993). Interestingly, morphological and biochemical alterations were accompanied by a remarkable DNA damage, which was detected in the sperm of ECS-exposed mice by using the comet assay, a highly sensitive method for evaluating the DNA fragmentation due to single- and double-strand breaks. Previously, we demonstrated that exposure of adult rats to CS for 4 weeks caused a significant, 1.9-fold increase in bulky DNA adduct levels in testis (Izzotti et al.,
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1999). DNA adducts are promutagenic lesions that, unless repaired, can trigger the DNA damage process that was documented in the present study. Measurement of sperm DNA damage is a useful tool in the evaluation of male infertility, as the sperm nucleus lacks protection against oxidative stress and is vulnerable to oxidationmediated DNA damage (Simon and Carrell, 2013). Generation of ROS in germ cells is a physiological mechanism required for maturation, capacitation, acrosome reaction of spermatozoa, binding with the zona pellucida, and oocyte function (De Lamirande and Gagnon, 1993). However, excessive ROS levels attack bases in nucleic acids, amino acid side chains in proteins and double bonds in unsaturated fatty acids, and cause oxidative stress, which can damage DNA, RNA, proteins, and lipids thereby resulting in an increased risk for a variety of diseases (Lü et al., 2010). It has been shown that ROS can affect the structural and functional parameters of sperm (Henkel, 2005). Experimental studies have suggested that exposure of rats to CS results in oxidative imbalances in sperm cells, as demonstrated either by an increase in TBARS and/or a decrease in antioxidant mechanisms (Peltola et al., 1994; Rajpurkar et al., 2000a,b; Ozyurt et al., 2006; Mohamed et al., 2011; Sankako et al., 2012). The sperm plasma membrane contains high concentrations of polyunsaturated fatty acids, which is an essential requirement for the male germ cell to maintain sperm functions. Also due to the very low content of protective systems in spermatozoa, polyunsaturated fatty acids are highly susceptible to oxidative damage leading to a loss of integrity in the sperm membrane (Aitken et al., 1989). This has been shown to be a cause of impaired sperm function and interference with its fertilization ability (Agarwal et al., 2005; Ghaffari and Rostami, 2012). It is noteworthy that, in addition to DNA damage, exposure of mice to CS has been shown to cause epigenetic alterations in testis (Xu et al., 2013), which have been associated with male infertility (Rajender et al., 2011). This kind of alterations is compatible with the strong oxidative damage produced in testis by CS, as shown in the present study. The histomorphological alterations that we observed in the testes of mice exposed to CS since birth are in line with similar findings detected in the testes of CS-exposed rats, either during the pubertal period (Rajpurkar and Dhabuwala, 2000) or in adult life (Mohamed et al., 2011; Güven et al., 1999; Ahmadnia et al., 2007).
Conclusion The results herein reported show for the first time that exposure of mice to high doses of CS, starting at birth and continuing until achievement of sexual maturity, causes a variety of interconnected alterations in male gonads, including loss of weight and histopathological alterations of testis, morphological abnormalities of spermatozoa, oxidative stress, and DNA damage in sperm cells.
Limitations of the study The results reported in the present study should be interpreted with caution due to the high doses of CS that have to be used in experimental models. Moreover, we did not measure cotinine levels in the plasma of exposed mice but relied on literature data generated under comparable experimental conditions.
Conflict of interest The authors declare they have no conflict of interests.
Please cite this article in press as: La Maestra, S., et al., Effect of cigarette smoke on DNA damage, oxidative stress, and morphological alterations in mouse testis and spermatozoa. Int. J. Hyg. Environ. Health (2014), http://dx.doi.org/10.1016/j.ijheh.2014.08.006
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ARTICLE IN PRESS S. La Maestra et al. / International Journal of Hygiene and Environmental Health xxx (2014) xxx–xxx
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Please cite this article in press as: La Maestra, S., et al., Effect of cigarette smoke on DNA damage, oxidative stress, and morphological alterations in mouse testis and spermatozoa. Int. J. Hyg. Environ. Health (2014), http://dx.doi.org/10.1016/j.ijheh.2014.08.006
