Molecular Human Reproduction, Vol. 5, No. 2, 125-131,
February 1999
© 1999 European Society of Human Reproduction and Embryology
Detection of benzo[a]pyrene diol epoxide-DNA adducts in embryos from smoking couples: evidence for transmission by spermatozoa
1 Department of Obstetrics and Gynaecology, and 2 Departments of Zoology and Anthropology, University of Toronto, Toronto, Ontario, Canada
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Tobacco smoking is deleterious to reproduction. Benzo[a]pyrene (B[a]P) is a potent carcinogen in cigarette smoke. Its reactive metabolite induces DNA-adducts, which can cause mutations. We investigated whether B[a]P diol epoxide (BPDE) DNA adducts are detectable in preimplantation embryos in relation to parental smoking. A total of 17 couples were classified by their smoking habits: (i) both partners smoke; (ii) wife non-smoker, husband smokes; and (iii) both partners were non-smokers. Their 27 embryos were exposed to an anti-BPDE monoclonal antibody that recognizes BPDE–DNA adducts. Immunostaining was assessed in each embryo and an intensity score was calculated for embryos in each smoking group. The proportion of blastomeres which stained was higher for embryos of smokers than for non-smokers (0.723 versus 0.310). The mean intensity score was also higher for embryos of smokers (1.40 ± 0.28) than for non-smokers (0.38 ± 0.14; P = 0.015), but was similar for both types of smoking couples. The mean intensity score was positively correlated with the number of cigarettes smoked by fathers (P = 0.02). Increased mean immunostaining in embryos from smokers, relative to non-smokers, indicates a relationship with parental smoking. The similar levels of immunostaining in embryos from both types of smoking couples suggest that transmission of modified DNA is mainly through spermatozoa. We confirmed paternal transmission of modified DNA by detection of DNA adducts in spermatozoa of a smoker father and his embryo.
benzo[a]pyrene/cigarette smoking/cotinine/DNA adducts/embryos
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cigarette smoking has deleterious effects on reproduction (Harrison et al., 1990
; Pattinson et al., 1991; Zenzes, 1995; Van Voorhis et al., 1997), but the mechanism producing these effects is still unclear. Polycyclic aromatic hydrocarbons (PAH) are a group of widespread environmental pollutants produced by combustion of fuels and other substances (International Agency for Research on Cancer, 1986). Benzo[a]pyrene (B[a]P) is one of the most extensively studied PAH. It is found in the air, water and soil, and occurs also in cigarette smoke in amounts of 5–200 ng per cigarette. B[a]P is highly mutagenic and carcinogenic. Its carcinogenic metabolite, 7ß,8
-dihydroxy-9,10-epoxy-7,8,10-tetrahydro-benzo[a]pyrene (BPDE-I), is a diol epoxide derivative of B[a]P. This very reactive metabolite binds predominantly to the 2-amino group of DNA guanosine and forms adducts, designated BPDE–I–dG–DNA (Jeffrey et al., 1977). This is a major DNA adduct produced when cells are exposed to B[a]P. There is evidence for a direct aetiological link between BPDE–DNA adducts and a lung cancer gene (p53) mutational spots (Dennisenko et al., 1996; Perera, 1997).
BPDE–DNA adducts have been detected by immunocytochemistry in human placenta (Manchester et al., 1988), cervix, ovary, and lung (Shamsudin and Gan, 1988), and oral mucosa cells (Hsu et al., 1997) of smoking individuals. In ovarian tissue of smokers, BPDE immunoreactivity was reported in oocytes, luteal cells, and stromal arteries of post-mortem ovaries (Shamsudin and Gan, 1988), and in granulosa–lutein cells of women in assisted reproduction (Zenzes et al., 1998).
BPDE–DNA adducts have been detected in spontaneously aborted fetal tissues (liver and lung; Hatch et al., 1990), confirming that human fetuses are targets for DNA damage, but there is no information on human embryos. The availability of spare embryos from assisted reproduction now offers this possibility. In-vitro fertilization (IVF) may be useful for tracing maternal and/or paternal gametic transmission of modified DNA to early preimplantation embryos. The aim of the present study was to analyse whether BPDE–DNA adducts are detectable in human preimplantation embryos in relation to parental smoking habits.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Subjects
A total of 17 couples (with 27 spare embryos) from our IVF and embryo transfer programme participated. Each patient signed a consent form approved by the Committee for Research in Human Subjects of The Toronto Hospital, Canada. Couples were classified by their smoking habits into three groups: (i) both wife and husband smoked (n = 5, with eight embryos at the 3–7-cell stage); (ii) only husband smoked (n = 6, with 12 embryos, eight at the 3–8-cell stage, and four fragmented); and (iii) neither wife or husband smoked (n = 6, with seven embryos, six at the 3–8-cell stage, and one fragmented). Couples where only the wife smoked were few (10% in our IVF programme) and embryos from this type of couple were not available for this study.
IVF and embryo transfer
For ovarian stimulation patients had gonadotrophin suppression by gonadotrophin-releasing hormone (GnRH) agonist (Lupron, Abbott, Montreal, Canada): 1 mg administered s.c. daily in a long protocol with a luteal phase start. The final stage of follicular maturation was initiated by injection of 10 000 IU human chorionic gonadotrophin (HCG, Profasi; Serono). Follicles were monitored by ultrasound (Bruel & Kjaar, Naerum, Denmark) and were aspirated 36 h after HCG administration using transvaginal guidance and local anaesthesia. Sperm preparation, oocyte insemination in vitro, and embryo transfer were carried out using conventional procedures (Gonen et al., 1990).
Cotinine assessments
Cotinine is used as a reliable marker for recent cigarette smoke exposure and dose (Benowitz et al., 1983). Follicular fluid (FF) and seminal plasma (SP) samples were stored at –20°C. After thawing, samples were centrifuged at 800 g, and the supernatants were assessed for cotinine concentrations by radioimmunoassay using the method of Langone et al. (1973). Briefly, we used isogel Tris buffer, tritiated cotinine rabbit antiserum, and goat anti-rabbit g-globulin to separate the antibody-bound cotinine. Concentrations of cotinine were expressed as ng/ml. The sensitivity of the assay (lowest detectable value) was 0.25 ng/ml.
Exposure of bovine embryos to B[a]P
Bovine embryos (n = 60) from IVF at the 4-cell stage were exposed to serial concentrations (15 embryos each at 0, 1, 5 and 10 µg/ml) of B[a]P (Sigma, St Louis, MO, USA), for 24 h at 38°C in an atmosphere of 95% humidity and 5% CO2. Then, the embryos (a majority at 8-cell stage) were washed twice in culture medium (TCM 199; Gibco, Burlington, Ontario, Canada).
Preparation of embryos and spermatozoa for immunocytochemistry
The zona pellucida around each human and bovine embryo was removed by incubation in pronase (Boehringer Mannheim, Mannheim, Germany), 0.5% in phosphate-buffered saline (PBS) at 37°C, for 1–3 min under microscopic observation. Human and bovine embryos were left to recuperate in the incubator for 20 min, in human tubal fluid medium (HTF; Irvine Scientific, Santa Ana, CA, USA) and TCM-199 respectively. Embryos were washed twice in PBS and fixed in methanol at –20°C for 3 min, followed by acetone at –20°C for 10 s; embryos were immediately put on slides and air-dried. The preparations were then permeabilized with 0.1% of Triton X100 (Sigma), and incubated overnight at 4°C with #5D11 monoclonal antibody (from Dr Regina Santella, Columbia University, New York, USA; 1:50). This antibody recognizes BPDE–I-DNA adducts and also cross-reacts with diolepoxide-DNA adducts of other PAH (Santella et al., 1970, 1984). To detect the bound antibody, a biotinylated secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA) and streptavidin-conjugated peroxidase (1:200) complex were used. The peroxidase reaction was developed with 3,3' diaminobenzidine (Sigma). As a negative control, an extra embryo was treated with mouse serum (1:50; Sigma) instead of the primary antibody.
Spermatozoa
The sperm pellets were washed once and prepared by swim-up in HTF containing 0.5% human serum albumin (Miles Inc, Elkhart, IN, USA). The motile spermatozoa in the upper layer were then centrifuged at 800 g, washed in PBS, and fixed in methanol/acetic acid (3:1). Sperm cells were put on slides and air-dried. Sperm cells were then decondensed using SSC (0.3 M NaCl, 30 mM sodium citrate) twice for 5 min, followed by treatment with 2.5 mM dithiothreitol (Sigma) in 1 M Tris–HCl, and by two washes in sodium chloride/sodium citrate (SSC). Sperm cell preparations were then washed in PBS, dehydrated in serial concentrations of ethanol (from 70 to 100%) and air-dried. The preparations were immunostained with peroxidase as for human and bovine embryos. For negative controls one preparation was treated with DNAse (100 µg/ml; Pharmacia LKB Biotech, Piscataway, NJ, USA) for 1 h at 37°C prior to the method; another preparation was treated with mouse serum (1:500) instead of primary antibody.
Microscopic evaluations of immunostaining
Presence, distribution and intensity of cell immunostaining was determined by careful microscopic evaluations. These were done by two different observers without knowledge of the smoking status. Immunostaining intensity was scored as negative (0), weak (1), moderate (2) or strong (3) in a total of 155 cells (blastomeres) from 45 bovine embryos, 112 cells (blastomeres) from 22 human embryos and five fragmented human embryos, and in 200 sperm cells. The staining intensity of each embryo, using the stained blastomeres, was calculated. For couples with multiple embryos, a mean staining intensity of their embryos was calculated. The proportion of blastomeres that stained (with any intensity) was calculated for the embryos of each couple. An intensity score (IS; e.g. the sum of products of proportions of blastomeres having a given rating times that rating) was calculated for each embryo of each couple (excluding the five fragmented embryos), and for each sperm sample.
Statistical analysis
All P values are two-tailed. We used logarithms (base 10) of cotinine values for statistical calculations because of the extreme non-normality of its distribution (Zenzes et al., 1996). The 4.5 version of StatView (Abacus Concepts, Berkeley, CA, USA) for the Power Macintosh was used for performing statistical calculations. Comparisons between smoking groups, for staining intensity, IS values, and FF cotinine values, were done by analysis of variance (ANOVA). Linear regression and correlation were done to test relationships between variables, and the 
2 contingency test was used for comparing proportions.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The mean age (±SE) of women was 33.6 ± 4.7 years and the range was 27–43 years; age did not differ among the three groups. The smoking women smoked 5–20 (mean 13.0 ± 3.0) cigarettes per day (c/d) and smoking men smoked 10–20 (16.8 ± 1.5) c/d. Table I
summarizes data on the couples by smoking status, smoking level, mean cotinine values in FF samples, mean embryo staining IS, and proportion of staining blastomeres.
|
FF cotinine
Correlation of log10 FF cotinine values with number of cigarettes smoked per day was significant both for women (R = 0.76, P = 0.0004) and for their husbands (R = 0.65; P = 0.005). The mean FF cotinine concentrations in women from couples where both partners smoked, in women of couples where only the husband smoked, and in women from couples where neither partner smoked were all significantly different (P < 0.0001). Pairwise comparisons using Fisher's protected least significant difference (Snedecor and Cochran, 1980
) showed higher values when both partners smoked than when only the husband smoked (P = 0.0003) or when neither smoked (P = 0.0001). The values were not significantly different when couples in which only the husband smoked were compared with non-smokers (P = 0.07).
Bovine embryos
Suitability of our method for detecting BPDE–DNA adducts was determined in bovine embryos. Regression of immunostaining intensity on B[a]P dose in 155 blastomeres of 45 bovine embryos (the 10 µg/ml dose was cytotoxic) revealed a significant and reliable dose-related increase in intensity with increasing concentrations of B[a]P (P = 0.0087).
Human embryos
Embryos
A total of 22 embryos had three to eight blastomeres, some embryos with a few cytoplasmic fragments. Five embryos (four from couples where the husbands smoked and one from a non-smoking couple) were very fragmented. Immunostaining was visualized in the nuclei; in some embryos it was also visualized in the cytoplasm. This is shown in Figure 1. This figure depicts four embryos in 4–8-cell stages; two of them (Figures 1A and 1D) show also cytoplasmic fragments. Removal of the zona pellucida sometimes produced artefacts in the embryos, for example the separated blastomeres in the embryo of Figure 1A, or the overlapping of blastomeres in the embryos of Figures 1B and 1D. In very fragmented embryos, where blastomeres were not distinguishable, staining was visualized within the cytoplasmic fragments (not shown).
|
The mean intensity score (IS) for all 22 embryos (five fragmented not included) was 1.00 ± 0.22. The mean IS for embryos of smoking couples of either type (1.40 ± 0.28) was higher (P = 0.015) than for embryos of non-smokers (0.38 ± 0.14). The mean IS for embryos of couples where both partners smoked (1.34 ± 0.30), and for embryos where only the father smoked (1.48 ± 0.55) were similar (see Table I
). The IS was positively correlated with number of cigarettes smoked by the husbands (P = 0.02), but not with the number of cigarettes smoked by the wives. The correlation between IS and FF cotinine was not significant. All 20 embryos from smoking couples, where either both members smoked or only husbands smoked, had a positive staining reaction; in contrast, two of the seven embryos from non-smokers were negative. The mean IS of embryos for all smoking couples was 1.78 ± 0.20; for all non-smoking couples it was 0.80 ± 0.22. These values were significantly different (P = 0.007).
In Figure 1, representative micrographs of embryos from the three types of couples, and a negative control, are shown. In the embryo of Figure 1A, both members of the couple smoked (10 cigarettes each per day; the woman's FF cotinine concentration was 44.3 ng/ml). In Figure 1B, only the husband smoked (15–20 cigarettes per day; the woman's FF cotinine concentration was 144.2 ng/ml; a passive smoker, Zenzes et al., 1996). In Figure 1C, both partners were non-smokers (the woman's FF cotinine value was 1.33 ng/ml). The embryo in Figure 1D was a sibling of the embryo in Figure 1C and represented the negative control treated with mouse serum instead of primary antibody. As shown in Figure 1, the IS of the embryos from smoker parents (Figures 1A and 1B) was stronger than that in the embryo of non-smoker parents (Figure 1C).
Blastomeres
In some of the stained embryos (of any intensity) not all blastomeres were stained, as shown in the embryo of Figures 1A and 1B. The mean proportion of blastomeres which stained in the 22 embryos (five fragmented excluded) was 0.60 ± 0.08. The overall proportion of stained blastomeres (total no. staining blastomeres)/(total no. blastomeres) was higher in embryos of smoking couples of either type (60/83 = 0.72) than in embryos of non-smoking couples (nine out of 29 = 0.31) (Fisher's exact 2x2 test, P = 0.0001). The same comparison in embryos of couples where both members smoked compared with those where only the husband smoked was not significant (33/40 = 0.83 versus 27/43 = 0.63; P = 0.053), see Table I.
Spermatozoa
In sperm cells the immunostaining intensity ranged from negative to strong (0 to 3) both in a smoker father (of the embryo of Figure 1B) and in a non-smoker father, but many more cells from this smoker father had strong or moderate scores. In this smoker father, 19% were scored as 3, 40% were 2, 37% were 1 and 4% were 0; the mean IS based on 100 cells was 1.74; his seminal plasma (SP) cotinine value was 369.6 ng/ml. In the non-smoker father, 0% were 3, 2% were 2, 25% were 1 and 73% were 0; the mean IS (100 cells) was 0.29; his SP cotinine value was 0.95 ng/ml. The difference in score distributions between the smoking and non-smoking fathers was significant (2x4 contingency 2; P < 0.0001).
In Figure 2, representative micrographs of sperm cells from this smoker father (Figure 2A) and this non-smoker father (Figure 2B) clearly showed a greater intensity of immunostaining in the spermatozoa from the smoker. Figure 2C was the negative control.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
This is the first report on the detection of BPDE–DNA adducts in human preimplantation embryos. Furthermore, we also showed a relationship between parental smoking and these adducts. Using three measures of immunostaining intensity, we showed that embryos of smoking couples have, on average, higher levels of reactivity to the monoclonal antibody than do embryos of non-smoking couples. There was a 3.6-fold increase for the mean score of immunostaining intensity. These results indicate that parental smoking, through gametic transmission of modified DNA to the embryo, results in detectable BPDE–DNA adducts in the embryo. Our approach using immunoperoxidase for monitoring exposure to environmental pollutants in cigarette smoke is therefore useful when relatively small numbers of cells are available (Hsu et al., 1997
; Zenzes et al., 1998), as in the present study.
The similar proportions of staining blastomeres and the similar mean intensity scores of immunostaining, in embryos where both parents smoke, compared to those where only the father smokes, suggests that the contribution of modified DNA to the embryo is mainly through the spermatozoa. Data on embryos from couples of smoker mothers with non-smoker fathers could provide further confirmation, but were not available. We provided, however, direct evidence for sperm contribution of modified DNA to the embryo through detection of BPDE–DNA adducts in spermatozoa from one such father. A greater contribution of modified DNA by the father than the mother was expected because of the much larger number (about six times) of cell divisions (when a cell is most susceptible to mutagens) between zygote and spermatozoa than between zygote and oocyte (Vogel and Motulsky, 1986; Crow, 1993; Lessells, 1997). Unlike oocytes, spermatid cells have minimal DNA repair capacity; once their chromatin has condensed they do not repair DNA damage, thus increasing the risk of heritable mutations (Bentley and Working, 1988; Wyrobek, 1993). There is some evidence suggesting that heavy paternal smoking may increase the risk of childhood cancer in offspring (Ames et al., 1994; Ji et al., 1997).
There is evidence of pre-zygotic damage from smoking on human gametic cells. A study on chromosome status in 286 oocytes from women in IVF therapy (Zenzes et al., 1995) found increased proportions (P < 0.01) of diploid oocytes (with 46 chromosomes instead of the normal 23) in smokers compared to non-smokers, with a significant (P = 0.0003) dose-effect. The increased rates of diploid oocytes, which result from non-extrusion of first polar body, suggests that smoking disturbs the equilibrium of microtubules (i.e. assembly–disassembly of tubulin) in the meiotic spindle, leading to digynic oocytes. We also found in this study that the proportion of oocytes which were mature (in metaphase II and, therefore, suitable for cytogenetic analysis) was significantly higher (P = 0.0003) in smokers than in non-smokers (Zenzes et al., 1995). Furthermore, in a recent study which analysed 2020 oocytes for maturity status in relation to FF cotinine concentrations (Zenzes et al., 1997), we found that, after correcting for age, the proportion of mature oocytes increased significantly (P = 0.002) with increasing concentrations of FF cotinine. The similar results of the two different studies suggest that smoking selects out oocytes of poor quality from the pool of retrieved oocytes, resulting in increased proportions of mature oocytes in smokers. This effect of smoking on the quality of oocytes was further detected in embryos also in relation to FF cotinine (Zenzes and Reed, 1996). In this analysis of 1094 embryos graded for quality (e.g. grades 1 to 4), mean values of FF cotinine were positively correlated, and in a dose-dependent manner, with better embryo quality (grades 1 and 2). The proportion of low-quality embryos (grades 3, 4) decreased significantly (P < 0.01) with increasing concentrations of cotinine. This significant trend in the proportion of good-quality embryos, however, may not increase the number of successful pregnancies. It is known that women who smoke have an increased risk for spontaneous abortions (Zenzes, 1995).
The fact that smoking is associated with high steroid hormone concentrations (James, 1997), may explain an increased probability of multiple births found in smoking mothers. Yerushalmy (1965) and Olsen et al. (1988) reported significantly increased multiple births in smoking mothers. The latter authors reported a significant correlation (P < 0.01) between the amount of smoking and dizygotic (DZ) twinning, but not monozygotic twinning. Thus, the oestrogen concentration is claimed to be a variable in a causal chain linking smoking with DZ twinning. This relationship may have repercussions for other several diseases partially caused by hormonal concentrations (James, 1997).
Studies on smoking effects on sperm quality found reduced concentrations (13–17%) and increased proportions of abnormal cells with increased concentrations of seminal plasma (SP) cotinine (Pacifici et al., 1993; Vine, 1996; Vine et al., 1993). A trend for higher proportions of spermatozoa with chromosomal aneuploidy or DNA fragmentation (Wyrobek et al., 1995; Sun et al., 1997) was detected in smokers. Increased levels of a major form of oxidative DNA damage, 8-hydroxydeoxyguanosine (8-OHdG), a lesion of guanine, were reported in spermatozoa of smokers of 20 cigarettes a day, compared with non-smokers (Fraga et al., 1991, 1996), and in dose-related association with the concentrations of cotinine in seminal plasma (Shen et al., 1997). Also, increased amounts of BPDE–DNA adducts were detected in the spermatozoa of smokers in dose-related association with concentrations of cotinine in seminal plasma (Zenzes and Bielecki, 1998). Spermatozoa containing DNA lesions may still reach and fertilize the ovum (Sun et al., 1997). The reproductive outcome will depend upon the capacity of the fertilized egg to repair tobacco-induced lesions in the spermatozoa (Genescá et al., 1982).
There was considerable variation in immunostaining intensity scores among the embryos from the same smoking group of couples. This variation may be due to different environmental exposures to B[a]P of the parents of these embryos and/or to differences in metabolism. Previous studies demonstrated that post-implantation mouse embryos showed genetic differences in metabolism of B[a]P in vitro, detected by sister chromatid exchange; there were also differences in DNA repair capacity or other genetic differences (Galloway et al., 1980; Perera, 1997).
In the present study, unstained blastomeres visualized in some stained embryos may represent anucleated blastomeres; these occur in human embryos from IVF (Zenzes et al., 1992; Zenzes and Casper, 1992). Also, these may be the result of corrective repair of pre-zygotic DNA damage (Genescá et al., 1982) and may represent inter- and intra-individual variations in repair capacity. It has been estimated that ~50–80% of BPDE–DNA adducts are removed within 24 h following exposure (Oesch et al., 1987), but there are significant inter-individual differences in the ability to repair this damage. Some individuals seem to have complete inability to repair this adduct (Oesch et al., 1987; Dickey et al., 1997).
The appreciable high mean IS of embryos from couples where neither partner smokes, ~28% of the score for the two groups of smoking couples, is noteworthy. This is much higher than the similar comparison for FF cotinine concentrations (<1%; see Table I). A similar finding comes from a study of BPDE–DNA adducts in ovarian granulosa–lutein cells: the intensity score of such cells from non-smoking women was found to be 32% of the score from smoking women, while the comparable ratio for FF cotinine values was 1.1% (Zenzes et al., 1998). These great differences are likely to be due to the different half-lives of B[a]P and cotinine. While the half-life of cotinine is ~20 h (Benowitz et al., 1983), in B[a]P it was estimated to be ~10 weeks (Mooney et al., 1995). The relatively high levels of staining of BPDE–DNA adducts in the embryos of non-smokers probably results from a longer, cumulative exposure of parental gametes to B[a]P. A positive reaction is also shown in the spermatozoa of a non-smoker, albeit at significantly lower levels than in the spermatozoa of a smoker. BPDE–DNA adducts are postulated to be a result of both recent and long-term exposure to active and passive smoking, as well as to other sources of PAH in the ambient air and food (Perera, 1997). Unspecific immunostaining in embryos and sperm cells of non-smokers can also be a contributing factor. The relatively high levels in these embryos of non-smoker parents, in the sperm cells of the non-smoker father, and in ovarian granulosa–lutein cells of non-smoking women (Zenzes et al., 1998), also indicates the great environmental contribution of B[a]P. This finding is also supported by a previous study on fetal tissues of spontaneous abortions; adducts were found irrespective of parental smoking (Hatch et al., 1990).
The major finding of this study is the demonstration that parental smoking results in BPDE–DNA adducts in preimplantation embryos. We further provide evidence for a greater paternal than maternal contribution to these embryonal adducts. If these pre-zygotic DNA lesions in the embryos are not repaired, they may be an important cause of the deleterious effects of tobacco smoking on reproduction, including delayed conception and increased rates of abortion in women (Harrison et al., 1990; Pattison et al., 1991; Zenzes, 1995; Van Voorhis et al., 1997).
|
Acknowledgments |
|---|
We thank the patients in the IVF/embryo transfer programme at the Toronto Hospital for donating their spare embryos for research; Robert F.Casper, Director of the IVF/embryo transfer programme at the Toronto Hospital, Toronto, Ontario, Canada for providing IVF materials; S.P.Leibo, Director of the Bovine IVF/embryo transfer programme, University of Guelph, Guelph, Ontario, for providing bovine embryos and Ester Semple, for assistance in the procedure; Regina M.Santella, Cancer Center, Division of Environmental Sciences, Columbia University, New York, for the kind donation of 5D11 monoclonal antibody; Julia Klein, Hospital for Sick Children, Toronto, for performing the cotinine assay; and the technologists of the IVF/embryo transfer programme at the Toronto Hospital, Toni DiBernardino, Rose Marie D'Onofrio and Kelly Doherty, for their valuable assistance. This work was supported by a grant of the Medical Research Council of Canada to MTZ (#MT 1247, Ottawa, Canada).
|
Notes |
|---|
3 To whom correspondence should be addressed at: The Toronto Hospital Research Centre, Room CCRW 1–813, 101 College Street, Toronto, Ontario M5G 1L7, Canada
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Ames, B.N., Motchnik, P.A., Fraga, C.G. et al. (1994) Antioxidant Prevention of Birth Defects and Cancer. In Mattison, D.R. and Olshan, A. (eds), Male-Mediated Developmental Toxicology. Plenum Press, New York, USA, pp. 243–259.
Benowitz, N.L., Kuyt, F., Jacob, P. III and Jones, R.T. (1983) Cotinine disposition and effects. Clin. Pharmacol. Ther., 34, 604–611.[Web of Science][Medline]
Bentley, K.S. and Working, P.K. (1988) Use of seminiferous tubule segments of study stage specificity of unscheduled DNA synthesis in rat spermatogenic cells. Environ. Mol. Mutagen., 12, 285–297.[Web of Science][Medline]
Crow J. (1993) How much do we know about spontaneous mutation rates? Environ. Mol. Mutagen., 21, 122–129.[Web of Science][Medline]
Dennisenko, M.F., Pao, A., Tang, M. and Pfeifer, G.P. (1996) Preferential formation of benzo(a)pyrene adducts at lung cancer mutational hot spots in p53. Science, 274, 430–432.
Dickey, C., Santella, R.M., Hattis, D. et al. (1997) Variability of PHA-DNA adduct measurements in peripheral mononuclear cells: implications for quantitative cancer risk assessment. Risk Analysis, 17, 649–656.[Web of Science][Medline]
Fraga, C.G., Motchnik, P.A., Shigenaga, M.K. et al. (1991) Ascorbic acid protects against endogenous oxidative damage in human sperm. Proc. Natl. Acad. Science. USA, 88, 11003–11006.
Fraga, C.G., Motchik, P.A., Wyrobek, A.J. et. al. (1996) Smoking and low antioxidant levels increase oxidative damage to sperm DNA. Mut. Res., 35, 199–203.
Galloway, S.M., Perry, P.E., Meneses, J. et al. (1980) Cultured mouse embryos metabolize benzo(a)pyrene during early gestation: genetic differences detectable by sister chromatid exchange. Proc. Natl. Acad. Sci. USA, 77, 3524–3528.
Genescá, A., Caballín, M.R., Miró, R. et al. (1982) Repair of human sperm chromosome aberrations in the hamster egg. Hum. Genet., 89, 181–186.
Gonen, J., Jacobson, W. and Casper, R.F. (1990) Gonadotropin suppression with oral contraceptives before in vitro fertilization. Fertil. Steril., 53, 282–287.[Web of Science][Medline]
Harrison, K.L., Breen, T.M. and Hennessey, J.F. (1990) The effect of patient smoking habit on the outcome of IVF and GIFT treatment. Aust. N.Z. J. Obstet. Gynaecol., 30, 340–342.[Web of Science][Medline]
Hatch, M.C., Warburton, D. and Santella, R.M. (1990) Polycyclic aromatic hydrocarbon-DNA adducts in spontaneously aborted fetal tissue. Carcinogenesis, 11, 1673–1755.
Hsu, T-A., Zhang, Y-J. and Santella, R.M. (1997) Immunoperoxidase quantitation of 4-aminobiphenyl- and polycyclic aromatic hydrocarbon-DNA adducts in exfoliated oral and urothelial cells of smokers and non-smokers. Cancer Epidem. Biomarkers. Prev., 6, 193–199.[Abstract]
International Agency for Research on Cancer (1986) Tobacco smoking. IARC Monographs on the Evaluation of Carcinogenic Risks of Chemicals in Humans. Lyon, France, pp. 36–38.
James, W.H. (1997) An hypothesis on the association between maternal smoking and dizygotic twinning. Hum. Reprod., 12, 1391–1392
Jeffrey, A.M., Weinstein I.B., Jennette, K.W. et al. (1977) Structures of benzo(a)pyrene nuclei acid adducts formed in human and bovine bronchial explants. Nature, 269, 348–350.[Medline]
Ji, B-T., Shu, X-O., Linet, M.S. et al. (1997) Paternal cigarette smoking and the risk of childhood cancer among offspring of nonsmoking mothers. J. Natl. Cancer. Inst., 89, 238–244.
Langone, J., Gijka H.B. and Van Vunakis, H. (1973) Nicotine and its metabolites: radioimmunoassay for nicotine and cotinine. Biochemistry, 12, 5015–5030.
Lessells, K. (1997) More mutations in males. Nature, 390, 236.[Medline]
Manchester, D.K., Weston, A., Choi, J.-S. et al. (1988) Detection of benzo(a)pyrene diol epoxide-DNA adducts in human placenta. Proc. Natl. Acad. Sci. USA, 85, 9245–9247.
Mooney, La.V., Santella, R.M., Covey, L. et al. (1995) Decline of DNA damage and other biomarkers in peripheral blood following smoking cessation. Cancer Epidemiol. Biomarkers Prev., 4, 627–634.[Abstract]
Oesch, F., Aulmann, Platt, K.L. and Doerjer, G. (1987) Individual differences in DNA repair capacities in man. Arch. Toxicol., 10 (Suppl.), 172–179.
Olsen, J., Bønnelykke, B. and Nielsen, J. (1988) Tobacco smoking and twinning. Acta Med. Scand., 224, 491–494.[Web of Science][Medline]
Pacifici, R., Altieri, I., Gandini, L. et al. (1993) Nicotine, cotinine and trans-3-cotinine levels in seminal plasma of smokers: effects of sperm parameters. Ther. Drug Monit., 15, 358–363.[Web of Science][Medline]
Pattinson, H.A., Taylor, P.J. and Pattinson, M.H. (1991) The effect of cigarette smoking on ovarian function and early pregnancy outcome of in vitro fertilization treatment. Fertil. Steril., 55, 780–783.[Web of Science][Medline]
Perera, F.P. (1997) Environment and cancer: who are susceptible? Science, 278, 1068–1073.
Santella, R.M., Li, Y., Zhang, Y. et al. (1970) Immunological methods for the detection of polycyclic aromatic hydrocarbon-DNA and protein adducts. In Waters, M.D., Daniel, F.B., Lewlas, J. et al. (eds), Genetic Toxicology of Complex Mixtures. Plenum Press, New York, USA, pp. 291–301.
Santella, R.M., Lin, C.D., Cleveland, W.L. and Weinstein, I.B. (1984) Monoclonal antibodies to DNA modified by a benzo(a)pyrene diol epoxide. Carcinogenesis, 5, 373–377.
Shamsuddin, A.K.M. and Gan, R. (1988) Immunocytochemical localization of benzo(a)pyrene-DNA adducts in human tissues. Hum. Pathol., 19, 309–315.[Web of Science][Medline]
Shen, H.-M., Chia, S.-E., Ni, Z.-Y., New, A.-L. et al. (1997) Detection of oxidative DNA damage in human sperm and the association with cigarette smoking. Reprod. Toxicol., 11, 675–680.[Web of Science][Medline]
Snedecor, G.W. and Cochran, G.W. (1980) Statistical Methods. 7th edn. Iowa State University Press, Ames, Iowa, USA, pp. 234.
Sun, J.G., Jurisicova, A. and Casper, R.F. (1997) Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol. Reprod., 56, 602–607.[Abstract]
Van Voorhis, B.J., Dawson, J.D., Stovall, D.W. et al. (1997) The effects of smoking on ovarian function and fertility during assisted reproduction cycles. Obstet. Gynecol., 88, 785–791.
Vine, M.F. (1996) Smoking and male reproduction: a review. Int. J. Androl., 19, 323–337.[Web of Science][Medline]
Vine, M.F., Tse, C.-K., Hu, P.-C. and Truang, K.Y. (1993) Cigarette smoking and semen quality. Fertil. Steril., 65, 835–842.
Vogel, F. and Motulsky, A. (1986) Human Genetics. Springer Verlag, Berlin, Germany.
Wyrobek, A.J. (1993) Methods and concepts in detecting abnormal reproductive outcomes of paternal origin. J. Reprod. Toxicol., 7, 3–16.
Wyrobek, A.J., Rubes, J., Cassel, M. et al. (1995) Smokers produce more aneuploid sperm than non-smokers. Am. J. Hum. Genet., 57, A131.
Yerushalmy, J. (1965) Letter to the editors. Am. J. Obstet. Gynecol., 91, 883–884.
Zenzes, M.T. (1995) Cigarette smoking as a cause of delay in conception. Reprod. Med. Rev., 4, 189–205.
Zenzes, M.T. and Bielecki, R. (1998) Immunodetection of benzo[a]pyrene, a carcinogen in cigarette smoke, in sperm of men in IVF–ET who smoke. Fertil. Steril., 70, S.463, pp. 1092.
Zenzes, M.T. and Casper, R.F. (1992) Cytogenetics of human oocytes, zygotes and embryos after in vitro fertilization. Hum. Genet., 88, 367–375.[Web of Science][Medline]
Zenzes, M.T. and Reed T.E. (1996) Cigarette smoking may suppress apoptosis in human embryos. Hum. Reprod., 11, 153.
Zenzes, M.T., Wang, P. and Casper, R.F. (1992) Chromosome status of untransferred (spare) embryos and probability of pregnancy after in-vitro fertilisation. Lancet, 340, 391–394.[Web of Science][Medline]
Zenzes, M.T., Wang, P. and Casper, R.F. (1995) Cigarette smoking may affect meiotic maturation of human oocytes. Hum. Reprod., 10, 3213–3217.
Zenzes, M.T., Reed, T.E., Wang, P. and Klein J. (1996) Cotinine, a major metabolite of nicotine, is detectable in follicular fluids of passive smokers in in vitro fertilization therapy. Fertil. Steril., 66, 614–619.[Web of Science][Medline]
Zenzes, M.T., Reed, T. and Casper, R.F. (1997) Effects of cigarette smoking and age on the maturation of human oocytes. Hum. Reprod., 12, 1736–1741.
Zenzes, M.T., Puy, L. and Bielecki, R. (1998) Immunodetection of benzo[a]pyrene adducts in ovarian cells of women exposed to cigarette smoke. Mol. Hum. Reprod., 4, 159–165.
Submitted on May 18, 1998; accepted on October 27, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. Paul, S. Teng, and P. T.K. Saunders A Single, Mild, Transient Scrotal Heat Stress Causes Hypoxia and Oxidative Stress in Mouse Testes, Which Induces Germ Cell Death Biol Reprod, May 1, 2009; 80(5): 913 - 919. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.L. Waylen, M. Metwally, G.L. Jones, A.J. Wilkinson, and W.L. Ledger Effects of cigarette smoking upon clinical outcomes of assisted reproduction: a meta-analysis Hum. Reprod. Update, January 1, 2009; 15(1): 31 - 44. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Zhao and R. J. Epstein Programmed Genetic Instability: A Tumor-Permissive Mechanism for Maintaining the Evolvability of Higher Species through Methylation-Dependent Mutation of DNA Repair Genes in the Male Germ Line Mol. Biol. Evol., August 1, 2008; 25(8): 1737 - 1749. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Paul, A. A Murray, N. Spears, and P. T K Saunders A single, mild, transient scrotal heat stress causes DNA damage, subfertility and impairs formation of blastocysts in mice Reproduction, July 1, 2008; 136(1): 73 - 84. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. A. Rybicki, C. Neslund-Dudas, C. H. Bock, A. Rundle, A. T. Savera, J. J. Yang, N. L. Nock, and D. Tang Polycyclic Aromatic Hydrocarbon-DNA Adducts in Prostate and Biochemical Recurrence after Prostatectomy Clin. Cancer Res., February 1, 2008; 14(3): 750 - 757. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Paul, D. W. Melton, and P. T.K. Saunders Do heat stress and deficits in DNA repair pathways have a negative impact on male fertility? Mol. Hum. Reprod., January 1, 2008; 14(1): 1 - 8. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Y. Kim, J.-Y. Chung, J.-E. Park, S. G. Lee, Y.-J. Kim, M.-S. Cha, M. S. Han, H.-J. Lee, Y. H. Yoo, and J.-M. Kim Benzo[a]pyrene Induces Apoptosis in RL95-2 Human Endometrial Cancer Cells by Cytochrome P450 1A1 Activation Endocrinology, October 1, 2007; 148(10): 5112 - 5122. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. L. Nock, D. Tang, A. Rundle, C. Neslund-Dudas, A. T. Savera, C. H. Bock, K. G. Monaghan, A. Koprowski, N. Mitrache, J. J. Yang, et al. Associations between Smoking, Polymorphisms in Polycyclic Aromatic Hydrocarbon (PAH) Metabolism and Conjugation Genes and PAH-DNA Adducts in Prostate Tumors Differ by Race Cancer Epidemiol. Biomarkers Prev., June 1, 2007; 16(6): 1236 - 1245. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Astle, R. Newton, S. Thornton, M. Vatish, and D.M. Slater Expression and regulation of prostaglandin E synthase isoforms in human myometrium with labour Mol. Hum. Reprod., January 1, 2007; 13(1): 69 - 75. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Chang, S. Selvin, C. Metayer, V. Crouse, A. Golembesky, and P. A. Buffler Parental Smoking and the Risk of Childhood Leukemia Am. J. Epidemiol., June 15, 2006; 163(12): 1091 - 1100. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. V. Younglai, A. C. Holloway, and W. G. Foster Environmental and occupational factors affecting fertility and IVF success Hum. Reprod. Update, January 1, 2005; 11(1): 43 - 57. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. A. Rybicki, A. Rundle, A. T. Savera, S. S. Sankey, and D. Tang Polycyclic Aromatic Hydrocarbon-DNA Adducts in Prostate Cancer Cancer Res., December 15, 2004; 64(24): 8854 - 8859. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Cordier, C. Monfort, G. Filippini, S. Preston-Martin, F. Lubin, B. A. Mueller, E. A. Holly, R. Peris-Bonet, M. McCredie, W. Choi, et al. Parental Exposure to Polycyclic Aromatic Hydrocarbons and the Risk of Childhood Brain Tumors: The SEARCH International Childhood Brain Tumor Study Am. J. Epidemiol., June 15, 2004; 159(12): 1109 - 1116. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. H. Phillips Smoking-related DNA and protein adducts in human tissues Carcinogenesis, December 1, 2002; 23(12): 1979 - 2004. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Nelson The miseries of passive smoiong Human and Experimental Toxicology, February 1, 2001; 20(2): 61 - 83. [Abstract] [PDF]
|
|
|
|
|
|
A. Rundle, D. Tang, J. Zhou, S. Cho, and F. Perera The Association between Glutathione S-Transferase M1 Genotype and Polycyclic Aromatic Hydrocarbon-DNA Adducts in Breast Tissue Cancer Epidemiol. Biomarkers Prev., October 1, 2000; 9(10): 1079 - 1085. [Abstract] [Full Text]
|
|
|
|
|
|
A. Rundle, D. Tang, H. Hibshoosh, A. Estabrook, F. Schnabel, W. Cao, S. Grumet, and F. P. Perera The relationship between genetic damage from polycyclic aromatic hydrocarbons in breast tissue and breast cancer Carcinogenesis, July 1, 2000; 21(7): 1281 - 1289. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Santella Immunological Methods for Detection of Carcinogen-DNA Damage in Humans Cancer Epidemiol. Biomarkers Prev., September 1, 1999; 8(9): 733 - 739. [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||