Growth and Angiogenesis Are Inhibited in Vivo in Developing Tissues by Pyrazine and Its Derivatives

Abstract

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.

Hypoxic level and duration differentially affect embryonic organ system development of the chicken (Gallus gallus)

“…In chickens, the CAM is formed on d 4 of incubation after the merging of the chorion and growing allantois with the branching of 2 chorioallantoic arteries and one vein in the mesodermal layer (DeFouw et al, 1989). The CAM is sensitive to hypoxic exposure during incubation, showing stimulated growth after 10 d (Melkonian et al, 2003). The CAM weight changes induced by hypoxia have been reported several times (e.g., Wagner-Amos and Seymour, 2003;Chan and Burggren, 2005).…”

The effect of cigarette smoke on fertilization and pre-implantation development: assessment using animal models, clinical data, and stem cells

“…These include, for example, tar, heavy metals such as cadmium and lead, polycyclic hydrocarbons (PAHs), phenol, benzene, carbon monoxide, nitrosamines, and hydrogen cyanide. In addition, other chemicals that have not traditionally been recognized as toxic, such as pyridines, pyrazines, and phenols, have recently been shown in various biological assays to produce adverse effects at relatively low doses (Ji et al, 2002;Melkonian et al, 2003;. Given the complexity of the mixture of chemicals in burning tobacco, it is not surprising that most chemicals in smoke have not been studied thoroughly and the interaction of these chemicals is not well understood.…”

In vitro assessment of reproductive toxicity of cigarette smoke and deleterious consequences of maternal exposure to its constituents

“…Similarly, cadmium has been demonstrated to concentrate in both follicular fl uid and ovarian tissue (Varga et al, 1993). Although the serum concentrations of smoke components tested in this study in active and passive smokers have not been determined, these chemicals are reported to be reproductive toxic at picomolar dose in several studies (Ji et al, 2002;Melkonian et al, 2003;Riveles et al, 2003). Therefore, it is reasonable to assume that these molecules are present in vivo at high enough concentrations to exert toxicological eff ects.…”

“…Prior studies using solid phase extraction cartridges and gas chromatography-mass spectrometry have identifi ed pyridine and pyrazine derivatives in cigarette smoke, which are demonstrated to have adverse eff ects on human cultured cell proliferation, normal reproductive functioning in rodents, as well as embryo growth and angiogenesis in chick model (Ji et al, 2002;Melkonian et al, 2003;Riveles et al, 2003;Riveles et al, 2004;Yu et al, 2006). Among these characterized compounds, pyrazine and 2-ethylpyridine, which are found exlusively in SS and 3-ethylpyridine (which is present in both MS and SS) are the most potent chemicals with the greatest inhibitory activity on cell proliferation in vitro and in vivo bioassay (Ji et al, 2002;Melkonian et al, 2003;Riveles et al, 2003). The serum concentrations of pyrazine, 2-ethylpyridine, and 3-ethylpyridine in active and passive smokers have not been determined; however, these three compounds are toxic at picmolar doses in vitro, as shown previously and it is likely that these molecules are present in vivo at high enough concentrations to cause toxicological eff ects on reproductive health.…”