Reproductive Toxicology 15 (2001) 479 – 486
Resveratrol, a natural aryl hydrocarbon receptor antagonist, protects sperm from DNA damage and apoptosis caused by benzo(a)pyrene Ariel Revela, Hila Raanania, Edward Younglaib, Jing Xua, Robin Hana, Jean-Francois Savouretc, Robert F. Caspera,* a
Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Toronto, and Samuel Lunenfeld Research Institute. Mount Sinai Hospital, Toronto, Ontario, Canada b Department of Obstetrics and Gynecology, McMaster University, Hamilton, Ontario, Canada c INSERM unit 135, Hopital Bicetre, Le Kremlin-Bicetre, France Received 9 January 2001; received in revised form 11 April 2001; accepted 1 May 2001
Abstract Benzo(a)pyrene (BaP), an aryl hydrocarbon receptor (AhR) ligand present in cigarette smoke and car exhaust, is thought to have negative effects on male reproduction. We hypothesized that BaP damages sperm through AhR activation, phase 1 enzyme induction, DNA adduct formation, and increased germ cell apoptosis in the testis, and that resveratrol, a natural competitive inhibitor of the AhR found in some red wines, could prevent the adverse effects of BaP on sperm. Male Balb C mice were injected subcutaneously (s.c.) for 5 weeks with a range of BaP doses (0.5 mg/kg to 50 mg/kg). Live sperm were obtained from the vas deferens, counted, and stained to measure annexin-V positive (apoptotic) cells. In a subsequent study, mice were injected for 5 weeks with corn oil (control), BaP (5 mg/kg/week), or BaP plus resveratrol (50 mg/kg/week) (n ⫽ 3 per group). Immunohistochemistry (IHC) was performed on testis sections for the determination of CYP1A1, BaP diol epoxide (BPDE) DNA adducts, and apoptosis and the results quantified by using the HSCORE, a semiquantitative scoring system. Our results demonstrated that sperm counts after 5 weeks were inversely correlated to BaP dosage. BaP (0.5 to 5 mg/week) positively correlated with sperm apoptosis while higher doses increased sperm necrosis. CYP1A1 protein was observed mainly in interstitial cells of some testis sections, but there was no significant induction by BaP. BPDE DNA adducts were induced in all components of the seminiferous tubules by BaP and suppressed by resveratrol: median HSCORE (interquartile range) control 61 (52—71.5); BaP 213 (192—248), P ⫽ 0.01 compared to control; BaP plus resveratrol 83 (70 —90). BaP significantly increased apoptosis, mainly in spermatogonia: medain HSCORE (interquartile range) BaP 189 (161—223) versus control 83 (57—93), P ⬍ 0.01; and this effect was abrogated by resveratrol. Median HSCORE for BaP plus resveratrol was 112 (range 99 –121). In summary, BaP caused increased sperm cell BPDE DNA adduct formation and apoptosis in the mouse. The natural AhR antagonist, resveratrol diminished BaP-induced DNA adducts and apoptosis in seminiferous tubules. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Benzo(a)pyrene; AhR; spermatogenesis; BPDE; DNA adducts; apoptosis; CYP1A1; mice
1. Introduction Environmental toxicants that can affect reproductive function in both men and women include organochloride pesticides (DDT), vinyl chloride, polycyclic aromatic hy-
* Corresponding author. Tel.: ⫹416-586-5930; fax: ⫹416-972-0036. E-mail address: [email protected]
(R.F. Casper). This research was supported by grants from the Toxic Substances Research Initiative (TSRI), Health Canada, and The Medical Research Council of Canada, Ottawa, Canada. Presented in part at the Society for Gynecologic Investigation meeting March 2000, Chicago IL.
drocarbons (PAH), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyls (PCBs), and related dioxin-like chemicals . Concerns about increased environmental pollution have been expressed in regard to declining sperm counts. Carlsen et al.  reviewed data from 61 publications and reported that the average sperm concentration had decreased from 113,000,000/mL to 66,000,000/mL over the last half-century . However, other studies have suggested the reported decline in sperm count could be due to observational bias and overinterpretation of linear regression . Swan et al. (1997)  reported a decrease in sperm density in the United States and Europe but not in nonwestern countries. A recent study in young Czech men suggested
0890-6238/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 0 - 6 2 3 8 ( 0 1 ) 0 0 1 4 9 - 6
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that alterations in sperm quality may occur after exposure to periods of elevated air pollution, without changes in sperm numbers . Polycyclic aromatic hydrocarbons such as benzo(a)pyrene (BaP) are produced as byproducts of garbage incineration, combustion of diesel and gasoline fuel, and furnace gases. Importantly, cigarette smoke contains high concentrations (20 to 40 ng per cigarette) of PAH, in particular BaP, one of the most highly mutagenic and carcinogenic compounds known [6,7], and to which much of the population is actively or passively exposed. Deleterious effects of cigarette smoking on male fertility have been reported to include reduced sperm concentration, increased abnormal cells, and chromosomal abnormalities [8 –10]. A study in the Czech republic has reported that teenage smokers showed elevated frequencies of sperm aneuploidy, reduced sperm motility, and decreased normal morphology with potential adverse effects on future fertility . We have recently demonstrated increased DNA damage in sperm from smokers compared to nonsmokers . Toxic effects of PAH have also been demonstrated in male animals including inhibition of spermatocyte meiosis in vitro , decreased sperm count, decreased epididymis weight, and decreased seminiferous tubule diameter [14 –16] . The biologic effects of PAH are mediated by the aryl hydrocarbon receptor (AhR). The AhR is a 110 kDa protein of the basic helix loop helix/PAS family of transcription factors . AhR is present in the cytosol of mammalian cells of almost all organs and tissues bound to heat shock protein 90 . Upon binding to ligand, AhR dissociates from HSP-90 and the ligand-AhR complex is translocated to the nucleus through association with a structurally related protein, the AhR nuclear translocator (ARNT). Inside the nucleus, the heterodimeric AhR/ARNT complex regulates gene transcription by binding to DNA at dioxin-responsive elements (DRE) located within, or upstream of, a number of genes for phase 1 (cytochrome P-450) enzymes such as CYP1A1, 1A2, and 1B1, and several other genes . Phase 1 enzymes are responsible for the production of aryl hydrocarbon hydrolase (AHH) and the oxidative metabolism of AhR ligands. BaP is metabolized by AHH to a procarcinogenic compound, BPDE [(⫹-)-anti7␤, 8␣-dihydroxy-9␣,10a-epoxy-7,8,9,10-tetrahydropyrene], which binds to DNA and forms predominantly covalent (⫹) trans adducts at the N2 position of guanine . Phase 1 enzymes also increase the production of reactive oxygen species (ROS) , which have been shown to be associated with lipid peroxidation, oxidative DNA damage and other pathologic effects [20,21]. We recently demonstrated that resveratrol (3,5,4⬘-trihydroxystilbene), a plant antifungal agent or phytoalexin, present in some red wines, is a competitive inhibitor of AhR ligands . Resveratrol binds AhR, elicits nuclear translocation but inhibits the induction of DRE-driven transcription by AhR ligands and prevents initiation of the transcription of CYP1A1 and other phase 1 enzymes both ex vivo
and in vivo . Another recent publication has also shown inhibition of AhR ligand-induced CYP1A1 by resveratrol . In the present study, we focused on the potential adverse male fertility effects of BaP. The objectives of the present study were to evaluate whether exposure to BaP results in sperm DNA damage and apoptosis, and to assess if the natural AhR antagonist, resveratrol, could prevent such damage.
2. Materials and methods 2.1. Animals Balb C mice were purchased from Charles River Laboratories (Montreal, Quebec). The mice were caged in groups of three, received food and water ad libidum and after one week were started on weekly injections of benzo(a)pyrene (Sigma-Aldrich Canada, B1760, Oakville, Ontario, Canada) in corn oil. In order to determine the BaP dosage causing sperm apoptosis, we injected Balb C mice (n ⫽ 3 per group) for 5 weeks (length of spermatogenesis in the mouse) with various dosages [0,0.5,1,3,5,10,30, and 50 mg/kg/week] of BaP. BaP aliquots were dissolved in DMSO (dimethyl sulphoxide CAS 67– 68-5, Caledon Laboratories LTD Georgetown, Ontario, Canada), sonicated, and kept at – 4 C. Prior to injection, the aliquots were thawed, mixed thoroughly with corn oil and injected s.c., thus serving as a depot preparation. The final concentration of DMSO was ⬍ 1%. The mice were sacrificed 3 days after the last injection by cervical dislocation. The vas deferens was dissected, immediately incubated in warmed modified (HEPES-buffered) HTF (mHTF) medium (Irvine Scientific, Santa Ana, CA) and cut into 3 mm pieces enabling sperm to leak into the medium. After one hour at 37 C, the sperm were washed in PBS and counted. Samples of approximately 106 sperm cells were used for detection of apoptosis. In the next experiment Balb C male mice were injected for 5 weeks with corn oil, BaP (5 mg/kg/week), or BaP plus 50 mg/kg/week of resveratrol (3,4⬘,5- trihydroxy-trans-stilbene, Sigma-Aldrich Canada, R5010) (n ⫽ 3 per group). The dose of resveratrol was determined from our previous studies in a rat model (unpublished) in which a 10-fold excess of resveratrol was required to block the effects of BaP in vivo. The resveratrol was also made up in DMSO as a stock solution and dissolved in corn oil for injection as described above for BaP. The mice were weighed weekly, and examined for normal activity and behavior, coat condition, and normal food and water intake. After 5 weeks of treatment, mice were sacrificed and testes were resected and fixed in 4% paraformaldehyde (Sigma Chemical Co., St. Louis, MO). 2.2. Flow cytometry for apoptosis detection in sperm Annexin V- enhanced green fluorescent protein (EGFP)  was used (ApoAlert Annexin V-EGFP Apoptosis Kit, CLONTECH Laboratories, Inc. Palo Alto, CA). Sperm cells
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were rinsed and then resuspended in 200 l of binding buffer. Sperm were incubated (37 C) for 15 min in the dark with 5 l of Annexin V-EGFP and 10 l of propidium iodide (Sigma-Aldrich). Analysis by flow cytometry was performed using a single laser emitting excitation light at 488 nm. Flow cytometry enabled the detection and counting of three fractions of sperm: 1) annexin V negative and propidium iodide (PI) negative (healthy), 2) annexin V positive and PI negative (apoptotic), and 3) annexin V positive and PI positive (necrotic). 2.3. Tissue processing The testes were fixed in 4% paraformaldehyde overnight and then washed in PBS for 8 h (changing PBS every 2 h). The tissues were put in cassettes (Omnicassete tissue cassette, Fisher Scientific, Pittsburgh, PA) and passed through an ethanol series (70% to 100%). After incubation in xylene (X5–1, FL 05 0896 Fisher Scientific, Fairlawn, NJ) for 2 h, the cassettes were put in warmed (60 C) wax (tissue prep, Fisher Scientific, Fairlawn, NJ) overnight, after which the tissues were embedded, sectioned, and dried on microscope slides (Superfrost plus, Fisher Scientific). 2.4. Immunohistochemistry Immunohistochemistry was performed on two slides from each mouse. Polyclonal antibody against CYP1A1 was obtained from Daiichi Pure Chemicals, LTD (Tokyo, Japan). Polyclonal antibody against BPDE was kindly donated by Prof. PG Natali, Scientific Director, Instituto Regina Elena, Roma, Italy. Slides were dewaxed and endogenous peroxidase was quenched in 1% H2O2 in methanol for 30 min. Sections were treated sequentially with three permeabilization solutions. The first solution was RNase 100 g/mL in TE buffer, pH 8.0 for 1 h at 37 C. After washing in PBS at room temperature, the second solution used was Proteinase K 10 g/mL in TE buffer, pH 7.4 for 10 min at room temperature. Sections were then treated with 0.1% Triton X (v/v) in PBS for 15 min at room temperature. This treatment was followed by DNA denaturation, using 1M HCl solution at 37 C for 30 min. Sections were washed in PBS and 10% normal goat serum (v/v) in PBS was used to block nonspecific binding. Sections were incubated with polyclonal rabbit anti-BPDE antibody (or polyclonal Cyp1A1 antibody) overnight at 4 C. Diaminobenzidine (DAB) was used as the color substrate. Carazzis’ haematoxylin was used as a nuclear counterstain. 2.5. Apoptosis assay The Apoptag Kit (Oncor, Gaithersburg, MD) was used for identification of apoptotic nuclei. In brief, 5-mm thick paraffin sections were dewed with xylene and rehydrated with graded dilutions of ethanol in water. Subsequently, the
tissues were digested with 20 mg/mL proteinase-K (SigmaAldrich Canada) for 15 min at room temperature. Slides were then treated with a 3% solution of H2O2, washed with PBS, and immersed in equilibration buffer for 5 min. After tapping off the excess solution, the sections were incubated with the reaction mixture containing TDT enzyme and digoxigenin-dUTP for 1 h in a humid atmosphere at 37 C. Washing in stop/wash buffer for 30 min at 37 C following the manufacturer’s instructions ended the reaction. For negative controls, TDT was eliminated from the reaction mixture. The specimens were washed in PBS and incubated with antidigoxigenin -peroxidase for 30 min at room temperature. The end products were visualized by treating the sections with freshly prepared solution of DAB and counterstaining was carried out with methyl green. 2.6. HSCORE Three immunohistochemical slides from each animal were blindly read and staining intensity was estimated using a semiquantitative score (HSCORE) as previously described . The HSCORE was calculated for each slide by application of the following algorithm: HSCORE ⫽ Pi(i⫹1), where i is the intensity of staining (0 ⫽ no staining, 1 ⫽ weak, 2 ⫽ moderate, 3 ⫽ strong) and Pi is the percentage of stained cells for each intensity (0 to 100%). 2.7. Statistics Sperm concentration data were analysed using simple linear regression. The HSCORE results were assessed using a Kruskal-Wallis one way ANOVA on ranks for nonparametric data. Both tests were performed on Sigmastat for Windows (Jandel Scientific, San Rafael, CA).
3. Results 3.1. Sperm counts Sperm counts were inversely correlated to BaP dosage (P ⬍ 0.0001; Fig. 1A). We observed no other obvious adverse physical effects on the mice at any of the doses of BaP. No differences in the weights, activity, coat, or food and water intake of the mice were observed throughout the study (weekly intervals) or at its completion (data not shown). 3.2. Annexin V labeling and flow cytometry Annexin V and propidium iodide (PI) enabled flow cytometry sorting of sperm cells into live, apoptotic, and necrotic cells. All PI positive stained sperm were classified as necrotic regardless of their annexin V staining. Sperm with no PI staining were sorted to annexin V positive (apoptotic) and annexin V negative (normal). There was a positive correlation between BaP dosage and apoptosis at
A. Revel et al. / Reproductive Toxicology 15 (2001) 479 – 486
Fig. 1. (A) Mean (⫾ SEM) sperm concentration (vas deferens) in mice (n ⫽ 3 per group) treated for 5 weeks with increasing doses of benzo(a)pyrene (BaP) from 0 (control) to 50 mg/kg/week. There was a significant (P ⬍ 0.0001) negative correlation between sperm concentration and BaP dose. (B) Flow cytometry of sperm (vas deferens) labeled with annexin V and propidium iodide (PI) from mice (n ⫽ 3 per group) treated for 5 weeks with increasing doses of BaP. Percentage of total sperm that were apoptotic was determined by sorting those labeled with annexin V but negative for PI labeling. Percentage of total sperm that were necrotic was determined by sorting those labeled with PI regardless of annexin V labeling.
low to mid-range doses. Higher BaP doses resulted in increased sperm necrosis (Fig. 1B). 3.3. Immunohistochemistry Based on the results of sperm apoptosis, which appeared to be maximal at intermediate BaP doses, we assessed testicular tissue from mice injected for 5 weeks with corn oil (control), BaP (5 mg/kg/week), or BaP plus resveratrol (50 mg/kg/week) respectively (n ⫽ 3 per group). We performed immunohistochemistry for CYP1A1, BPDE DNA adducts, and apoptosis. Staining for CYP1A1 was observed to variable degrees in most of the testis sections, predominantly in the interstitial cells. Compared to controls in which the median HSCORE was 100 (interquartile range 88 —123), the median HSCORE for CYP1A1 in the BaP treated animals was 167 [149 —178], which did not reach significance. Mice co-treated with BaP and resveratrol also had no change in CYP1A1 compared to controls with a median HSCORE of 162 (interquartile range 116 —172). We verified the lack of significant induction of CYP1A1 protein by western blot, which demonstrated a low level of constitutive CYP1A1 protein in all treatment groups (data not shown). Testes from mice exposed to BaP demonstrated nuclear,
nucleolar, and cytoplasmic staining for BPDE DNA adducts in all compartments of the testis (Fig. 2). Median HSCORE (interquarile range) for control was 61 [52—71.5]. HSCORE results for BPDE DNA adducts were significantly increased by BaP exposure (Median HSCORE [interquartile range] 213 [192—248]; P ⫽ 0.01). In contrast, mice exposed to BaP plus resveratrol had an IHC pattern similar to control mice (median HSCORE [interquartile range] 83 [70 —99.5]; Fig. 2). The exposure of the mice to BaP resulted in apoptosis of germ cells, mostly affecting spermatogonia. Resveratrol had a protective effect when coadministered with BaP as shown by the decreased apoptag staining (Fig. 3). Median HSCORE (interquartile range) of apoptag immunohistochemistry in controls was 83 [957—93]. BaP exposure significantly (P ⫽ 0.01) increased the median HSCORE to 189 (interquartile range 161—223) while BaP plus resveratrol resulted in a median HSCORE (interquartile range) of 112 (99 —121, not significant compared to control; Fig. 3).
4. Discussion The findings of the present study showed increased apoptosis in male germ cells in mice exposed to BaP using two
A. Revel et al. / Reproductive Toxicology 15 (2001) 479 – 486
Fig. 2. Representative immunohistochemistry of testis sections from mice labeled with anti-BPDE antibody. Diaminobenzidine (DAB) was used as the color substrate (brown). Carazzis’ haematoxylin was used as a nuclear counterstain. Magnification is 400⫻. Section labeled control is from a mouse treated with corn oil for 5 weeks. Section labeled BaP is from a mouse treated with BaP (5 mg/kg/week) for 5 weeks. Section labeled BaP ⫹ resveratrol is from a mouse treated with BaP (5 mg/kg/week) concomitantly with resveratrol (50 mg/kg/week) for 5 weeks. HSCORE results are presented in the lower right hand panel. The data are shown as stacked bar graphs of the percentage of cells calculated for each staining intensity (n ⫽ 3 testis sections from each animal) for each of the treatments. 0 ⫽ no staining, 1 ⫽ weak staining, 2 ⫽ moderate staining, 3 ⫽ strong staining. (* P ⬍ 0.01 compared to control and BaP ⫹ resveratrol).
different techniques (Figs. 1B and 3). Detection of apoptosis in mature sperm, before gross morphologic changes, can be performed on live cells using staining by annexin V. Soon after initiating apoptosis, most mammalian cell types translocate phosphatidylserine (PS) from the inner surface of the plasma membrane to the outer cell surface. Once on the cell surface, PS can be specifically detected by staining with fluorescein isothiocyanate (FITC)-labeled annexin V (annexin V-FITC), a protein with a strong natural affinity for PS . Annexin V has been successfully applied to the evaluation of sperm quality after cryopreservation . The highest fraction of annexin V positive and PI negative sperm, implying early apoptosis, was observed in the intermediate dose (5 mg/kg) range of BaP (Fig. 1B). Higher BaP doses appeared to result in increasing degrees of sperm cell necrosis as seen by the increase in the PI positive fraction. Apoptosis was also demonstrated in the present study by immunohistochemistry on sections of mouse testes using a terminal transferase-mediated DNA nick end labeling (TUNEL) assay kit. Interestingly, the heaviest staining for DNA fragmentation was observed in spermatogonia or primary spermatocytes. This observation is consistent with apoptosis of the immature germ cells leading to cell death and elimination during spermatogenesis and subsequent reduction in mature sperm as we have demonstrated. It is not surprising that mature sperm did not label with TUNEL since DNA fragmentation detected by TUNEL is a late sign of apoptosis. It is possible that sperm membrane changes occurred in response to BaP, consistent with our annexin V results on vas deferens sperm, but were not detectable by the
TUNEL technique in IHC sections. In addition, our dosefinding study demonstrated only between 5% and 15% of mature sperm labeled with Annexin V (Fig. 1B), a marker for both early and late apoptosis. Thus, the percentage of sperm with DNA fragmentation, labeled by the TUNEL technique, would be lower and likely below the sensitivity of immunohistochemistry detection. Apoptosis is a physiologic process that entails the programmed death of a cell. Apoptosis plays a critical role during development and in the maintenance of tissue and organ homeostasis . Apoptosis is also the process by which many genotoxic and chemotherapeutic drugs exert their cytotoxic effects . Apoptosis has been proposed as a mechanism by which testicular germ cells are removed during various pathologic conditions [30,31]. Although cigarette smoking is associated with a reduction in sperm quality, standard parameters of the semen analysis do not provide information on the genetic integrity of sperm cells. We have recently shown that men who smoke have a 5-fold increase in sperm DNA damage compared to men who do not smoke . This observation raises the possibility that sperm DNA damage, besides affecting fertilization directly , may possibly result in later toxic effects on the embryo or genotoxic effects postnatally (see below). Therefore, apoptosis of damaged premeiotic germ cells may serve a critical role in protecting subsequent generations from the diverse genotoxic effects of environmental toxicants. The present study showed that injection of BaP in vivo in mice leads to the significant formation of BPDE-DNA adducts in sperm cells. The formation of DNA adducts in
A. Revel et al. / Reproductive Toxicology 15 (2001) 479 – 486
Fig. 3. Representative immunohistochemistry of testis sections from mice using TUNEL for detection of DNA fragmentation and apoptosis. Diaminobenzidine (DAB) was used as the color substrate (brown) and counterstaining was carried out with methyl green. Section labeled control is from a mouse treated with corn oil for 5 weeks. Section labeled BaP is from a mouse treated with BaP (5 mg/kg/week) for 5 weeks. Section labeled BaP ⫹ resveratrol is from a mouse treated with BaP (5 mg/kg/week) concomitantly with resveratrol (50 mg/kg/week) for 5 weeks. Magnification is 400⫻ for control and BaP and 200⫻ for BaP ⫹ resveratrol. HSCORE results are presented in the lower right hand panel. The data are shown as stacked bar graphs of the percentage of cells calculated for each staining intensity (n ⫽ 3 testis sections from each animal) for each of the treatments. 0 ⫽ no staining, 1 ⫽ weak staining, 2 ⫽ moderate staining, 3 ⫽ strong staining. (*P ⬍ 0.01 compared to control and BaP ⫹ resveratrol).
spermatozoa can lead to DNA damage and provide the trigger for initiation of apoptosis, as has been shown in other cell types . In addition, sperm DNA adducts are a potential source of transmissible prezygotic DNA damage. Zenzes et al.  have demonstrated BPDE DNA adducts in human sperm and oocytes obtained from cigarette smokers, and have found that the sperm contributes DNA adducts to human preimplantation embryos formed through in vitro fertilization . The childhood risk of cancer associated with paternal smoking was shown to be higher than with maternal smoking [36 –38]. Reported smoking habits for the parents of 1549 children who died from cancer, compared by matched pairs analysis, found a positive correlation between paternal daily consumption of tobacco and the risk of childhood cancer (P ⬍ 0.001). About 15% of all childhood cancers in this series could be attributable to paternal smoking [37,38]. Thus, there is evidence to suggest the possibility of fetal genotoxic effects associated with the presence of sperm DNA adducts from exposure to metabolites of tobacco smoke. Although our initial hypothesis in the present study was that apoptosis seen in male germ cells is the result of AhR activation by BaP leading to increased expression of phase 1 P450 enzymes, BaP metabolization and subsequent sperm DNA damage, our findings in the Balb-C mice demonstrated no CYP1A1 induction by BaP. A possible explanation for the apparent lack of induction of CYP1A1 may be because baseline expression of CYP1A1 in testis was already relatively high (median HSCORE of 100) suggesting
that ubiquitous environmental exposure to AhR ligands may be substantial. However, the highly significant increase in BPDE DNA adduct formation in the BaP exposed mice is strongly suggestive that induction of phase 1 enzyme expression was functionally significant. In fact, we noticed extensive anti-BPDE staining involving the cytoplasm as well as the nucleus in the testis sections. Intracytoplasmic staining could originate from adducts targeting mitochondrial DNA  and cytoplasmic proteins . It is likely that other CYP enzymes, possibly CYP1B1, which is also induced by AhR activation [41,42] and which has been shown to be present in testis , could result in significant BPDE adducts and apoptosis in the absence of a large induction of CYP1A1 It is important to develop a method to antagonize the adverse effects of toxic AhR ligands. Our findings suggest a possible protective role for resveratrol in the testis as demonstrated by the almost complete reversal of BaP-induced DNA adduct formation and apoptosis in the male germ cells. We  and others  have demonstrated that resveratrol is able to prevent dioxin response element (DRE)-induced transcription of CYP1A1, as well as other genes containing a DRE in their promoter region . Resveratrol, naturally present in some red wines, has also been demonstrated to have anti-oxidant [44 – 46], anti-inflammatory [46,47] and other activities , which may also be beneficial in preventing adverse environmental effects on spermatogenesis. Further clinical research is necessary to confirm whether these potential protective effects,
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as well as the apparent protection against BPDE DNA adduct-induced toxicity observed in the present study, will be pertinent to humans. In summary, exposure of mice to the AhR ligand BaP results in a toxic effect on spermatogenesis, and a decrease in sperm concentration. BaP exposure did not significantly activate CYP1A1 in this study but did significantly induce the formation of BPDE DNA adducts during spermatogenesis. These adducts may result in DNA damage, apoptosis, and necrosis. Early events in apoptosis could be detected in live sperm by using annexin V labeling. Resveratrol, a naturally occurring AhR antagonist was able to significantly decrease the formation of sperm BPDE DNA adducts and apoptosis, suggesting a possible therapeutic application in prevention of adverse AhR toxicant effects on reproductive function.
The assistance of Dr. MT Zenzes and R. Bielecki in the sperm collection procedures is greatly appreciated. We are grateful to Prof. PG Natali, Scientific Director, Instituto Regina Elena, Roma, Italy for the generous donation of antibody against BPDE-DNA adducts.
References  United States government accounting office. Reproductive and developmental toxicants. GAO/PEMD 1991; 92.  Carlsen E, Giwercman A, Keiding N, Skakkebaek NE. Evidence for decreasing quality of semen during past 50 years. BMJ 1992;305: 609 –13.  Bahadur G, Ling KL, Katz M. Statistical modelling reveals demography and time are the main contributing factors in global sperm count changes between 1938 and 1996. Human Reproduction 1996; 11:2635–2639.  Swan SH, Elkin EP, Fenster L. Have sperm densities declined? A reanalysis of global trend data. Environ Health Perspect 1997;105: 1228 –32.  Selevan SG, Borkovec L, Slott VL, Zudova Z, Rubes J, Evenson DP, Perreault SD. Semen quality and reproductive health of young Czech men exposed to seasonal air pollution. Environ Health Perspect 2000; 108:887–94.  Denissenko MF, Pao A, Tang M, Pfeifer GP. Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53. Science 1996; 274: 430 –2.  Hengstler JG, Van der Burg B, Steinberg P, Oesch F. Interspecies differences in cancer susceptibility and toxicity. Drug Metab Rev 1999;31:917–70.  Pacifici R, Altieri I, Gandini L, Lenzi A, Pichini S, Rosa M, Zuccaro P, Dondero F. Nicotine, cotinine, and trans-3-hydroxycotinine levels in seminal plasma of smokers: effects on sperm parameters. Ther Drug Monit 1993;15:358 – 63.  Vine MF, Margolin BH, Morrison HI, Hulka BS. Cigarette smoking and sperm density: a meta-analysis. Fertil Steril 1994;61:35– 43.  Vine MF. Smoking and male reproduction: a review. Int J Androl 1996;19:323–37.  Rubes J, Lowe X, Moore D 2nd, Perreault S, Slott V, Evenson D, Selevan SG, Wyrobek AJ. Smoking cigarettes is associated with
increased sperm disomy in teenage men. Fertil Steril 1998;70:715– 23. Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997;56:602–7. Georgellis A, Toppari J, Veromaa T, Rydstrom J, Parvinen M. Inhibition of meiotic divisions of rat spermatocytes in vitro by polycyclic aromatic hydrocarbons. Mutat Res 1990;231:125–35. Mably TA, Moore RW, Peterson RE. In utero and lactational exposure of male rats to 2,3,7,8- tetrachlorodibenzo-p-dioxin. 1. Effects on androgenic status. Toxicol Appl Pharmacol 1992;114:97–107. Mably TA, Moore RW, Goy RW, Peterson RE. In utero and lactational exposure of male rats to 2,3,7,8- tetrachlorodibenzo-p-dioxin. 2. Effects on sexual behavior and the regulation of luteinizing hormone secretion in adulthood. Toxicol Appl Pharmacol 1992;114: 108 –17. Mably TA, Bjerke DL, Moore RW, Gendron-Fitzpatrick A, Peterson RE. In utero and lactational exposure of male rats to 2,3,7,8- tetrachlorodibenzo-p-dioxin. 3. Effects on spermatogenesis and reproductive capability. Toxicol Appl Pharmacol 1992;114:118 –26. Okey AB, Riddick DS, Harper PA. Molecular biology of the aromatic hydrocarbon (dioxin) receptor. Trends Pharmacol Sci 1994;15:226 – 32. Rowlands JC, Gustafsson JA. Aryl hydrocarbon receptor-mediated signal transduction. Crit Rev Toxicol 1997;27:109 –34. Lai ZW, Pineau T, Esser C. Identification of dioxin-responsive elements (DREs) in the 5⬘ regions of putative dioxin-inducible genes. Chem Biol Interact 1996;100:97–112. Lopes S, Jurisicova A, Sun JG, Casper RF. Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum Reprod 1998;13:896 –900. Park JY, Shigenaga MK, Ames BN. Induction of cytochrome P4501A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin or indolo(3,2-b)carbazole is associated with oxidative DNA damage. Proc Natl Acad Sci U S A 1996;93:2322–7. Casper RF, Quesne M, Rogers IM, Shirota T, Jolivet A, Milgrom E, Savouret JF. Resveratrol has antagonist activity on the aryl hydrocarbon receptor: implications for prevention of dioxin toxicity. Mol Pharmacol 1999;56:784 –90. Ciolino HP, Daschner PJ, Yeh GC. Resveratrol inhibits transcription of CYP1A1 in vitro by preventing activation of the aryl hydrocarbon receptor. Cancer Res 1998;58:5707–12. Ernst JD, Yang L, Rosales JL, Broaddus VC. Preparation and characterization of an endogenously fluorescent annexin for detection of apoptotic cells. Anal Biochem 1998;260:18 –23. Lessey BA, Killam AP, Metzger DA, Haney AF, Greene GL, McCarty KS Jr. Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle. J Clin Endocrinol Metab 1988;67:334 – 40. Zhang G, Gurtu V, Kain SR, Yan G. Early detection of apoptosis using a fluorescent conjugate of annexin V. Biotechniques 1997;23: 525–31. Glander HJ, Schaller J. Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage. Mol Hum Reprod 1999;5:109 –15. Jacobson MD, Weil M, Raff MC. Programmed cell death in animal development. Cell 1997;88:347–54. Perez GI, Knudson CM, Leykin L, Korsmeyer SJ, Tilly JL. Apoptosis-associated signaling pathways are required for chemotherapymediated female germ cell destruction. Nat Med 1997;3:1228 –32. Qiu J, Hales BF, Robaire B. Damage to rat spermatozoal DNA after chronic cyclophosphamide exposure. Biol Reprod 1995;53:1465–73. Cai L, Hales BF, Robaire B. Induction of apoptosis in the germ cells of adult male rats after exposure to cyclophosphamide. Biol Reprod 1997;56:1490 –7. Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen
A. Revel et al. / Reproductive Toxicology 15 (2001) 479 – 486 samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril 1998;69:528 –32. Rorke EA, Sizemore N, Mukhtar H, Couch LH, Howard PC. Polycyclic aromatic hydrocarbons enhance terminal cell death of human ectocervical cells. Int J Oncol 1998;13:557– 63. Zenzes MT, Puy LA, Bielecki R. Immunodetection of benzo[a]pyrene adducts in ovarian cells of women exposed to cigarette smoke. Mol Hum Reprod 1998;4:159 – 65. Zenzes MT, Bielecki R, Reed TE. Detection of benzo(a)pyrene diol epoxide-DNA adducts in sperm of men exposed to cigarette smoke. Fertil Steril 1999;72:330 –5. Ji BT, Shu XO, Linet MS, Zheng W, Wacholder S, Gao YT, Ying DM, Jin F. Paternal cigarette smoking and the risk of childhood cancer among offspring of nonsmoking mothers. J Natl Cancer Inst 1997;89:238 – 44. Sorahan T, Lancashire RJ, Hulten MA, Peck I, Stewart AM. Childhood cancer and parental use of tobacco: deaths from 1953 to 1955. Br J Cancer 1997;75:134 – 8. Sorahan T, Prior P, Lancashire RJ, Faux SP, Hulten MA, Peck IM, Stewart AM. Childhood cancer and parental use of tobacco: deaths from 1971 to 1976. Br J Cancer 1997;76:1525–31. Backer JM, Weinstein IB. Interaction of benzo(a)pyrene and its dihydrodiol-epoxide derivative with nuclear and mitochondrial DNA in C3H10T 1/2 cell cultures. Cancer Res 1982;42:2764 –9. Kure EH, Andreassen A, Ovrebo S, Grzybowska E, Fiala Z, Strozyk M, Chorazy M, Haugen A. Benzo(a)pyrene-albumin adducts in humans exposed to polycyclic aromatic hydrocarbons in an industrial area of Poland. Occup Environ Med 1997;54:662– 6.
 Shimada T, Hayes CL, Yamazaki H, Amin S, Hecht SS, Guengerich FP, Sutter TR. Activation of chemically diverse procarcinogens by human cytochrome P- 450 1B1. Cancer Res 1996;56:2979 – 84.  Hakkola J, Pasanen M, Pelkonen O, Hukkanen J, Evisalmi S, Anttila S, Rane A, Mantyla M, Purkunen R, Saarikoski S, Tooming M, Raunio H. Expression of CYP1B1 in human adult and fetal tissues and differential inducibility of CYP1B1 and CYP1A1 by Ah receptor ligands in human placenta and cultured cells. Carcinogenesis 1997; 18:391–7.  Walker NJ, Gastel JA, Costa LT, Clark GC, Lucier GW, Sutter TR. Rat CYP1B1: an adrenal cytochrome P450 that exhibits sex-dependent expression in livers and kidneys of TCDD-treated animals. Carcinogenesis 1995;16:1319 –27.  Zbikowska HM, Olas B. Antioxidants with carcinostatic activity (resveratrol, vitamin E and selenium) in modulation of blood platelet adhesion. J Physiol Pharmacol 2000;51:513–20.  Hung LM, Chen JK, Huang SS, Lee RS, Su MJ. Cardioprotective effect of resveratrol, a natural antioxidant derived from grapes. Cardiovasc Res 2000;47:549 –55.  Martinez J, Moreno JJ. Effect of resveratrol, a natural polyphenolic compound, on reactive oxygen species and prostaglandin production. Biochem Pharmacol 2000;59:865–70.  Jang M, Pezzuto JM. Cancer chemopreventive activity of resveratrol. Drugs Exp Clin Res 1999;25:65–77.  Holmes-McNary M, Baldwin AS Jr. Chemopreventive properties of trans-resveratrol are associated with inhibition of activation of the IkappaB kinase. Cancer Res 2000;60:3477– 83.