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.