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Human reproductive health is affected by smoking. We propose to characterize the effects of tobacco smoke on reproduction, identify the toxicants in tobacco smoke that affect reproduction, understand the mechanism of action of these toxicants, and develop methods to control or prevent their effects. Specifically, active smoking increases the risk of infertility, spontaneous abortion, and ectopic pregnancy. These reproductive disorders/diseases have increased in the past several decades. In the United States, as many as 1 in 6 couples are infertile. Ectopic pregnancy, which is the leading cause of maternal death during the first trimester and which is almost always fatal to the fetus, has quadrupled in the United States since 1970. Our previous work has shown that both the oviduct and corpus luteum of females are targets of mainstream (MS) and sidestream (SS) cigarette smoke. The oviduct and corpus luteum are important reproductive organs; impairment of their function by smoke could cause infertility, spontaneous abortion, and ectopic pregnancy.
We propose to characterize the effects of MS and SS smoke on the oviduct and the corpus luteum, determine if the effects can be reversed, identify the toxicants in smoke that produce these effects, and begin to clarify the mechanism of action of the toxicants. Most work will be done using the hamster as a model, some parallel experiments will be done on human oviducts, and some experiments will be done on cultured human endothelial cells.
Oviducts: To characterize the structural and functional effects of MS and SS on the living oviduct, hamsters will be placed in smoking machine experiments, and the target cells in their oviducts will be evaluated using electron microscopy and physiological tests. Any para-meter that is affected by smoke exposure will then be examined in a reversal experiment to determine if “giving up” smoking restores the normal condition. Finally, human oviducts from nonsmokers, active smokers, and passive smokers undergoing elective surgery will be studied to characterize the structural and functional effects of smoke on the human oviduct.
Corpus luteum: Our previous work shows that MS and SS smoke inhibit blood vessel development in corpora lutea. Our second goal is to study this effect in more detail. We will characterize the structural effects of smoke on the hamster corpus luteum and on a chick membrane used to assay blood vessel development. Finally, we will determine how the genes which regulate blood vessel development are affected by smoke. The genes which are affected by the toxicant will be sequenced and their expression will be studied in the corpora lutea of hamsters in smoking machine experiments.
Results from these studies may influence smoking behavior in young women of child-bearing age. Young people are more likely to stop smoking if they know its effects are imminent (childbearing) rather than remote (death). Our video tapes showing the rapid response of the reproductive organs to smoke inhalation will be made available for educational purposes and may deter young women from smoking. Our video laparoscopy technique may become widely established for toxicology testing; this technique has the potential to be applied to other organs, routes of delivery, toxicants, and to males. The toxicants in smoke, once identified, could be removed form cigarette smoke by appropriate filters, thereby decreasing the toxicity of smoke to women unable to stop smoking during their reproductive years.
Passive smokers are often unaware of the dangers of smoke exposure. Our experimental designs compare MS and SS smoke and thus will provide much needed data on the influence of passive smoking on reproductive events.
Finally, our studies will benefit other branches of medicine. Results on the oviduct may apply to other organs having ciliated cells and smooth muscle cells, such as the respiratory system, and studies on the corpus luteum will contribute to understanding blood vessel development at other sites, such as in wounds, the endometrium, the placenta, and the embryo/fetus. In addition our studies on blood vessel development may augment our understanding of diseases affected by blood vessels such as solid tumor growth, and rheumatoid arthritis. In summary, data obtained in this study will lead to health improvements for pregnant women and their fetuses, and will have a positive impact on the reproductive health of our species. |
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Our overall objectives are to understand how cigarette smoke components affect the female reproductive track and to identify components in smoke that produce these effects with the aim of alleviating reproductive problems in humans due to smoking. Our focus has been on characterizing the effects of cigarette smoke on functioning of the oviduct using the hamster as a model and on the process of angiogenesis using the chick chorioallantoic membrane (CAM) assay. Assays were developed to measure oocyte cumulus complex (OCC) pick-up rate by the oviduct and adhesion of the OCC to the surface of the oviduct. Mainstream (MS) or sidestream (SS) cigarette smoke solutions inhibited OCC pick-up rate, and oviducts did not recover from this treatment after wash out of the smoke solutions. Interestingly OCC pick-up rate remained inhibited even when ciliary beat frequency recovered to control values, indicating OCC pick-up depends on processes in addition to ciliary beating. The mechanism of OCC pick-up was found to involve adhesion between the OCC matrix and the tips of the oviductal cilia. Experiments are currently in progress to determine if smoke solutions alter adhesion and thereby preclude OCC pick-up. An in vivo experiment was done in which transport of preimplantation embryos through the oviduct was compared in control and MS and SS smoke treated groups. Both MS and SS smoke inhalation inhibited embryo transport through the oviduct at doses similar to those in the serum of smokers. This inhibition was due at least in part to inhibition of contraction of the oviductal muscles. The in vitro and in vivo data support that conclusion that both the cilia and muscle of the oviduct are targets of MS and SS cigarette smoke.
To determine if smoke solutions inhibit angiogenesis, MS or SS smoke solutions were placed on CAMs and their effects evaluated 5 days later. Both MS and SS cigarette smoke altered normal pattern formation in CAM blood vessels and altered the composition of the extracellular matrix of the CAM. Both MS and SS smoke increased collagen and decreased hyaluronic acid in the matrix but only SS smoke increased fibronectin. An improved CAM assay is currently being used to evaluate the effects of smoke components on CAM growth and angiogenesis.
Chemicals that impair oviductal functioning and angiogenesis were identified in MS and SS smoke solutions using a solid phase extraction purification step followed by GC-MS. Pyridine derivatives were a major group of chemicals identified using this procedure. We have screened these pyridine derivatives using both the oviductal and angiogenesis assays. While most derivatives were effective only at relatively high doses (uM) several derivatives were identified that inhibited oviductal functional, CAM growth, and angiogenesis at picomolar doses. At least one of these chemicals is added to cigarettes as well as food and cosmetic products. These findings appear to be highly significant and may provide important public health information on a chemical generally regarded as safe but which is inhibits several biological processes at picomolar doses. |
