Our objective is to more fully understand the metabolic and physiological consequences of the excentric cleavage mechanism of beta-carotene. This objective, however, must be placed in the context of very strong epidemiological evidence, both observation and through intervention studies, indicating that carotenoids in general, and, beta-carotene in particular, can function as dietary anticarcinogens. In addition, more than two decades of clinical experience has demonstrated that the administration of high doses of beta--carotene (up to 300 mg/d) for extended periods of time results in no toxic side effects; thus we are driven to understand better the mechanism of carotenoid anticarcinogenesis. The accumulated data that the major metabolic products of carotenoids (the retinoids, and retinoic acid in particular) are effective anticancer agents, although associated with high risks of side-effects, indicate that understanding the process whereby beta- carotene is metabolized to retinoic acid is a key area of investigation. As described in our Progress Report, we have documented that a process of excentric cleavage occurs, in which beta-carotene is cleaved at several double bonds, in addition to the central double bond (the latter activity referred to as 15, 15'-dioxygenase). These reactions have been demonstrated in vitro in intestinal tissue from humans, monkeys, ferrets, rats and rabbits, as well as in liver, adipose and kidney tissue of rats, ferrets, and monkeys. In addition, we have utilized the intestinal perfusion of beta-carotene in ferrets to demonstrate absorption of intact beta-carotene into the lymph, and absorption of retinoic acid and more polar metabolites into the portal circulation. All of these experiments have been carried out using the all-trans isomer of beta-carotene; now we are interested in carrying out similar experiments with 9-cis-beta- carotene. The identification of 9-cis-retinoic acid as the specific RXR ligand is an intriguing observation, and immediately raises the question as to the source of 9-cis-retinoic acid in the body. It may arise through isomerization of the all-trans-retinoic acid in the body, or through conversion of 9-cis-beta-carotene to this biologically active retinoid, via the excentric cleavage mechanism. In this context, our specific aims are to localize the excentric cleavage activity in intestinal tissues of humans and rabbits, [[purify the enzyme(s),]] investigate whether a process similar to fatty acid beta-- oxidation operates on the various beta-apo-carotenoic acid intermediates, using both all-trans-beta-carotene and 9-cis-beta-carotene. In addition, we will perform in vivo experiments on 9-cis-beta-carotene metabolism to see if we can identify the formation of 9-cis-retinoic acid in the intestine, and its mechanism of absorption. We will also attempt to demonstrate excentric cleavage directly in our in vivo system. Finally, we will determine which if any of the intermediates, derived from either all-trans-beta-carotene or 9-cis-beta-carotene metabolism, can trans- activate the nuclear receptors for either RAR or RXR.
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