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In the United States, as many as 1 in 6 couples may be infertile. Both active and passive smoking increase the risk of infertility, spontaneous abortion, fetal growth retardation, and placental malfunction. Moreover, fetuses have no control over their exposure to mainstream (MS) or sidestream (SS) smoke. During the current award period, we identified three classes of chemicals (pyridines, pyrazines, and phenols) in SS smoke that impair oviductal functioning in an in vitro bioassay and that inhibit growth and blood vessel development in a chick egg assay. In both assays, some of the chemicals were inhibitory at very low doses. Pyridines were further shown to induce cell death in cultured cells from the lining of human blood vessels at ultra low doses. Our data identify three groups of chemicals in tobacco smoke that were not previously recognized as highly toxic to developing tissue. These chemicals were further found in the SS smoke of commercial brands, including harm reduction cigarettes. We have preliminary data showing that 3-ethylpyridine, which was highly toxic in both the oviductal and blood vessel development assays, increased the incidence of spontaneous abortion and decreased both fetal and placental weight when injected into a rodent model. These observations have lead to the goals of this proposal. Proof of concept experiments: We will conduct a complete series of in vivo dose response experiments in which mice will be exposed to varying doses of one of three of the identified toxicants (3-ethylpyridine, pyrazine or phenol) between days 7-19 of pregnancy using small pumps implanted in their abdominal cavity. Growth and blood vessel development in the fetus and placenta will be evaluated after exposure to determine the inhibitory concentration for each chemical for each parameter studied. These data will establish baseline toxicological information for each test chemical in a mammal system in vivo. We will then perform dose response experiments in which different amounts of each test chemical will be spiked into research grade cigarettes, and pregnant females will inhale smoke from these cigarettes between days 7-19 of pregnancy. The same growth and blood vessel development parameters will be examined. This experiment will establish how each toxicant effects fetal and placental growth when administered in vivo by inhalation in combination with the other chemicals contained in cigarette smoke. Serum levels of the identified toxicants and inhibition of fetal and placental growth by harm reduction cigarettes: We will measure the amounts of each of the three identified toxicants in the serum of mice following inhalation of smoke from various types of cigarettes (traditional commercial, mentholated, research, and harm reduction). These data will be compared to data from the preceding in vivo experiment to determine how serum levels of each toxicant correlate with the observed effects on growth and blood vessel development. We will also do an experiment to determine if inhalation of smoke from harm reduction cigarettes retards fetal and placental growth. Mechanism of action of the identified toxicants in vivo: We will compare gene expression in fetuses and placentas at three prenatal times after in vivo exposure of pregnant females to each of the three identified toxicants. These data will reveal the processes and pathways affected by each toxicant in vivo. We will also determine, using microscopic methods, which cells in fetal organs and in the placenta are affected by treatment with each chemical. Significance: These data will establish proof of concept, i.e. that the identified toxicants inhibit in vivo development in a mammalian model. Eight of the chemicals we identified are added to consumer products in the USA. Once additional data are collected on these chemicals, tobacco companies may stop adding them to cigarettes, and our data may lead to a more cautious use of these chemicals in consumer products in general, thereby reducing human exposure to them. Our studies may influence smoking behavior in pregnant women, particularly passive smokers. People are more likely to stop smoking if they know effects are imminent (childbearing) rather than remote (death). It may be possible to remove toxicants from smoke, thereby decreasing the toxicity of smoke to women who are unable to stop smoking during their reproductive years. Since the identified chemicals inhibited a broad spectrum of cell processes, it is likely they affect other organ systems, so our data may be of general interest to medicine. It is also probable that our data would help determine policy and law-making on smoking in public places. Finally, we will gain new information on the chemical composition of harm reduction cigarettes and may obtain data showing that these cigarettes are not as safe as the advertisements suggest. |
