Growth and angiogenesis are inhibited in vivo in developing tissues by pyrazine and its derivatives.

Sidestream cigarette smoke solution was previously screened to identify the groups of chemicals in smoke that inhibit growth and angiogenesis in the chick chorioallantoic membrane (CAM). Pyrazine and several pyrazine derivatives were identified as a major chemical group in this screen. In the current study, purified pyrazine and six pyrazine derivatives identified in the screen were tested in dose response experiments to measure their effects on CAM growth, embryo growth, and angiogenesis. Chemicals or control medium were placed on CAMs in ovo on day 5 of development, and results were evaluated on day 6. Of the chemicals tested, pyrazine was the most potent and inhibited both CAM and embryo growth at picomolar doses. 2-Ethylpyrazine and 2,3-dimethylpyrazine were inhibitory at nanomolar doses. Inhibition of growth by pyrazine was correlated with inhibition of DNA synthesis. The pattern of blood vessel development in CAMs was disturbed by micromolar doses of pyrazine and 2,3-dimethylpyrazine. Migration of mesodermal blood vessels to the ectoderm of CAMs and their subsequent differentiation into the capillary plexus was impaired by nanomolar doses of pyrazine. In summary, these data show that pyrazine and some of its derivatives inhibit growth and certain processes important in angiogenesis at very low doses. Since pyrazine and some of its derivatives are considered safe food additives, further toxicological testing of pyrazine, in particular on developing tissues, should be done to fully evaluate its safety as a consumer product additive.

Mainstream and sidestream cigarette smoke inhibit growth and angiogenesis in the day 5 chick chorioallantoic membrane.

The purpose of this study was to test the hypothesis that components in mainstream (MS) and sidestream (SS) cigarette smoke inhibit growth and angiogenesis using the chick chorioallantoic membrane (CAM). Varying doses of whole or gas-phase MS and SS smoke solutions were placed on day 5 CAMs, and their effects on angiogenesis were evaluated on day 6. All parameters evaluated (CAM area, major blood vessel area, major blood vessel diameter, blood vessel pattern formation, and capillary plexus formation) were inhibited to different degrees in a dose-dependent manner by both MS and SS smoke treatment. Inhibition of growth and vessel development was correlated with inhibition of cell proliferation. Inhibition of capillary plexus formation was caused by failure of mesodermal blood vessels to migrate to the ectoderm. SS smoke solution was more inhibitory than MS smoke solution in all assays, except for capillary plexus formation. In all assays, the toxicants in SS smoke partitioned mainly with the gas phase, whereas those in MS smoke were deduced to be mainly in the particulate phase in the proliferation-dependent assays (CAM area, blood vessel area, blood vessel diameter) and in both the gas and particulate phase in the pattern formation and plexus formation assays. Some of the inhibitory doses of MS and SS smoke solutions had nicotine concentrations within the range found in human smokers. Taken together, these data demonstrate that exposure to complex mixtures of chemicals in MS and SS cigarette smoke adversely affect growth, vessel development, vessel migration, and cell proliferation.

Capillary plexus development in the day five to day six chick chorioallantoic membrane is inhibited by cytochalasin D and suramin.

The chick chorioallantoic membrane (CAM) is a valuable model for evaluating angiogenesis and vasculogenesis. Our purpose was to characterize the formation of the CAM vasculature, in particular the capillary plexus, between days five and six after fertilization and to examine the mode of action of cytochalasin D and suramin on vascular development during this interval. The CAM increased 20-fold in size between days five and six, during which time the capillary plexus forms by both migration of mesodermal blood vessels toward the ectoderm and by the formation of new vessels from angioblasts near the ectoderm. Between days five and six, the CAM becomes thinner, and the density of the mesodermal cells decreases. To determine the mode of action of anti-angiogenic drugs on the day five to day six CAM, various concentrations of cytochalasin D or suramin were added directly to day five CAMs, and their effects were evaluated on day six. Both drugs significantly inhibited CAM growth, altered branching patterns of the major vessels, decreased area of the major vessels, and inhibited the formation of the capillary plexus by inhibiting both vasculogenesis and the migration of mesodermal blood vessels to the ectoderm. Cytochalasin D also inhibited compartmentalization of the plexus. Cytochalasin D and suramin were inhibitory at similar doses. This study provides new information on early CAM development, establishes the mode of action and dose dependency of cytochalasin D and suramin on day five to day six CAMs, and demonstrates that the day five to day six CAM provides a useful assay to examine the effect of anti-angiogenic drugs on blood vessel development, including capillary plexus formation.