A long-standing goal of the Eckhert Lab has been to understand the biology of boron and its role in human health at the molecular level. The argument for boron's essentiality in humans is weakened by the fact that no molecular mechanism has been identified. Boron is an essential nutrient for plants, and the human consumption of boron from dietary plants equals an intake of approximately 1 mg/day. Boron from plants is nearly completely absorbed as boric acid (BA) and circulates unchanged with a half-life of about 23 hours. This work focuses on three unsolved problems in the area of human boron biology: 1) characterization of the molecular target of BA and how it interacts with protein targets, 2) the signal transduction consequences of BA induced Ca2+ signaling dysregulation, and 3) a molecular explanation of how BA can inhibit cell proliferation without resulting in apoptosis. Collectively, the work presented herein defines a mechanism for BA in human cells.

The Eckhert Lab has previously reported that the molecular target of BA is cyclic ADP-ribose (cADPR), the endogenous agonist of the ryanodine receptor (RyR), an endoplasmic reticulum (ER) Ca2+ channel. There were two questions remaining. First, it was unknown if cADPR binds to RyR or one of its accessory proteins. Second, it was unclear how BA interacted with the RyR complex to inhibit cADPR induced Ca2+ release from the ER. In the first chapter, I provide evidence that demonstrates cADPR binds directly to the accessory protein FKPB12. In this chapter, I also provide evidence that demonstrates that in the presence of BA, the binding of cADPR to FKBP12 is inhibited.

The Eckhert Lab has also reported that BA induces mild ER stress, a consequence of ER Ca2+ dysregulation. Building on the work of Kimberly Henderson and Sarah Kobylewski, I describe in chapter 2 the molecular pathways that are activated by BA treatment of prostate cancer cells at physiological concentrations. I describe evidence that BA activates the eIF2alpha/ATF4 and ATF6 pathways. These results raise two additional questions. EIF2alpha; phosphorylation can occur through one of four kinases, each sensitive to different environmental stresses, but it was not known which kinase is activated by BA. In addition, model activators of the eIF2alpha; pathway usually activate CHOP, an apoptotic protein. However, BA does not cause apoptosis or increase levels of CHOP protein or mRNA and we did not have a molecular explanation for this observation.

The question of which kinase was responsible for BA induction of ph- eIF2alpha; needed to be addressed. In the third chapter, I identify PERK as the kinase sensitive to BA. PERK has two known substrates, eIF2alpha; and Nrf2. I also provide evidence that demonstrates that BA treatment activates Nrf2, which is a transcription factor for the antioxidant response element and induces transcription of several antioxidant genes. This data indicates that BA treatment can increase levels of antioxidant proteins in the cell and provides evidence for why BA does not induce CHOP .

The work described in these three chapters make a large contribution to the field of boron biology. Future studies should explore the link between the molecular mechanisms outlined herein with the specific effects observed with boron supplementation in animal models.

Nutritional chemoprevention is a growing area in the field of toxicology. What we do and do not eat has a major impact on the development of cancer. However, it is difficult to show a causal relationship between a natural product and cancer prevention because mechanistic biochemical data are often missing and animals studies can be inconclusive. Both determining and elucidating molecular mechanisms that modulate pathological endpoints are necessary components in the risk assessment process used to determine if chemicals that show chemoprevention properties in the laboratory are safe for public use. The research presented in this dissertation focuses on chemoprevention through the consumption of a nutrient or the avoidance of toxic food additives. Part I presents research that elucidated a molecular pathway activated by boric acid (BA), an essential plant nutrient, which may provide insight into the inhibition of cell proliferation of prostate cancer cells and reduced risk of prostate cancer. Part II consists of molecular toxicology research in application to public interest and health. It is a critical analysis of two commonly consumed FDA-approved food additives, rebaudioside A, an artificial sweetener, and artificial food dyes. In Part I, it was shown that BA is not an isoform-specific antagonist to the ryanodine receptor (RyR), a calcium (Ca2+) channel, but is a RyR antagonist that functions by interacting and competing with the receptor's only known endogenous agonist. This results in altered Ca2+ signaling that induces ER stress and the eIF2alpha/ATF4 and ATF6 branches of the unfolded protein response (UPR) in DU-145 prostate cancer cells. ER stress and the UPR are tightly associated with cell proliferation. The specific pathway that we have unfolded in BA-treated DU-145 cells is correlated with cell survival and an inhibition of cell proliferation. In Part II, we describe how in vivo and in vitro studies on rebaudioside A and food dyes demonstrated their toxicity. The assessment of toxicology studies on food dyes showed they do present an increased health risk and this is important given their widespread use by the public. The research presented here thus presents both the molecular mechanistic and public health sides of molecular toxicology.

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