Chapter 11
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Biosynthesis of Oxysterols in Plants, Animals, and Microorganisms Edward J. Parish Department of Chemistry, Auburn University, Auburn, AL 36849
As a class of compounds, oxysterols have demonstrated a wide variety of important biological properties. The specific inhibition of sterol biosynthesis is of special interest since it may prove useful in the prevention of reversal of certain cardiovascular diseases and may also prove to be useful in the control of normal and abnormal cell growth. Under certain conditions biological systems have been induced to produce oxysterols which adds support to the hypothesis that oxysterols may be natural regulators of sterol biosynthesis in the intact cell. Little attention has been directed to the occurrance and biosynthesis of oxysterols in the total biosphere. In this report, we have focused on the evolutionary origins of these biosynthetic pathways which have been reported to occur in plants, animals, and microorganisms. Steroids containing multiple oxygen functionality are widely distributed in nature. As a class of compounds, oxysterols can be defined as sterols bearing a second oxygen function, in addition to that of carbon-3, and having an iso-octyl or modified iso-octyl side chain (1-5). These compounds have demonstrated a variety of diverse biological properties, which include cytotoxicity, atherogenicity, carcinogenicity, mutagenicity, hypocholesterolemia, and effects on specific enzymes (1-11). They have been found in animal tissues and food stuffs (9) and have been isolated from drugs used in folk medicine for the treatment of cancer (12-14). Other studies have shown that certain oxysterols have demonstrated significant activity in the inhibition of D N A synthesis in cultured cells (15,16). A number of oxygenated derivatives of cholesterol and sterol intermediates in cholesterol biosynthesis have been found to be potent inhibitors of
0097-6156/92/0497-0146S06.00/0 © 1992 American Chemical Society
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11.
PARISH
Oxysterols in Plants, Animals, and
Microorganisms
sterol biosynthesis in animal cells in culture (10,11,17). The specific inhibition of cholesterol biosynthesis in mammalian cells by oxygenated derivatives of cholesterol and lanosterol has been shown in many cases to decrease cellular levels of 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase activity. This reported inhibitor response has been attributed to a decreased rate of HMG-CoA reductase synthesis (17-19) and in some instances to an increase in enzyme degradation (17,19). Other oxysterols are known to depress the rate of cholesterol synthesis from lanosterol i n rat liver homogenates and may inhibit the 14-demethylation of lanosterol (20-22). An important property of many oxysterols is their ability to repress HMG-CoA reductase activity in cultured mammalian cells (9,10,22). This suppression of activity has been found to vary over a wide range depending on the structural features of the oxysterol. As a general trend, inhibitory activity increases as the distance between carbon-3 and the second oxygen group becomes greater. Sterols with an additional oxygen function in ring D and the side chain have been shown to have the greatest activity. An intact side chain is a requirement for potent activity; a decrease in the length of the (iso-octyl) side chain results in decreased activity (23). Other noticeable trends indicate a relationship between inhibitory activity and the extent to which the second oxygen function is sterically hindered. In general, axial hydroxyl groups are more hindered and possess lower activities than the less hindered equatorial conformation (10,23,24,). Steric hindrance from other parts of the steroid molecule also can result in diminished activity (i.e., effect of carbon-14 alkyl substituents on the carbon-15 hydroxyl group) (25). It has been suggested that oxygen functions in conformationally flexible positions, such as those in ring D and in the side chain, produce more inhibitory steroids due to increased effectiveness of hydrogen bonding or hydrophilic interactions with receptor molecules (11). The experimental evidence described above suggests a regulatory mechanism which, by analogy to steroid hormone receptors and bacterial induction-repression systems, requires a binding protein to recognize oxysterols and mediate subsequent cellular events. There is evidence for the existence of a specific cytosolic receptor protein for oxysterols (23,26). After the activities of a number of sterols were evaluated, a good correlation was found between the actions of certain oxysterols on HMG-CoA reductase in L cells and their affinity for an oxysterol binding protein (23). Moreover, the actions of oxysterols which depress the rate of cholesterol biosynthesis from lanosterol and possibly inhibit the 14-demethylation of lanosterol are also postulated to exert their actions by an oxysterol binding protein (20-22).
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REGULATION OF ISOPENTENOID METABOLISM
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Discussion This report discusses oxysterols resulting from primary metabolism. These will include sterols with an additional oxygen function at either the C-7, C-22, C-24, C-25, or C-32 carbon atom of the sterol nucleus and side chain (Figure 1). It is well known that mammalian systems produce oxysterols. Derivatives of cholesterol hydroxylated in the 7α-, or 25-, or 26- positions are produced in liver during bile acid biosynthesis and in side-chain hydroxylation at the 2a- and 22Rpositions in the initial step in the conversion of cholesterol to steroid hormones in endocrine organs (27-29). In addition, all cells produce 32-hydroxylanosterol during the conversion of lanosterol to cholesterol (30,31). The oxygenated intermediates lanost-8-en-3B,32-diol and 3B-hydroxylanost-8-en-32-al have been isolated from rat liver microsomes incubated with 24,25dihydrolanosterol (32). A number of C-32 hydroxylated derivatives of cholesterol and lanosterol were shown to be potent inhibitors of HMG-CoA reductase, sterol biosynthesis, and possess a high affinity for the oxysterol binding protein in mammalian systems (23,33,34). Another mode of oxysterol biosynthesis has been described which utilizes the isopentenoid pathway to produce side-chain oxygenated derivatives of cholesterol and lanosterol (34,35). Such compounds are derived from squalene 2,3-epoxide by the introduction of a second oxygen function to form squalene 2,3;22,23-dioxidosqualene prior to cyclization. Thus, this intermediate has been shown to form 24(S),25-epoxylanosterol, 24(S),25-epoxycholesterol, and 25hydroxycholesterol in mammalian systems (35-38). 24(S),25Epoxycholesterol has been isolated from cultured mouse L cells, Chinese hamster lung fibroblasts, and human liver (39). These oxygenated side-chain derivatives have been shown to be potent inhibitors of HMG-CoA reductase, sterol biosynthesis, and possess a high affinity for the oxysterol binding protein (35-39). These results add support to the hypothesis that oxysterols may be natural regulators of cholesterol biosynthesis in mammalian cells (5). The biosynthesis of sterols seems to be a ubiquitous property of all vertebrate animals and photosynthetic plants from the prokaryotic blue-green algae to climax angiosperms (40-46). In addition, biosynthesis also occurs in most fungi and some protozoa (44). Not enough attention has been given to the biosynthesis of oxysterols in plants and microorganisms. The goal of the present review is to demonstrate the evolutionary basis of the oxysterol biosynthetic pathways which have been discovered in mammalian models. A variety of oxygenated sterols are known to exist in and have been isolated from plants and microorganisms; these may be precursors to the sterols
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11. PARISH
Oxysteroh in Plants, Animals, and Microorganisms
required for growth and/or reproduction or may be secondary plant metabolites. Many of the most frequently encountered oxysterols are those with a keto or hydroxyl function at the C-7 position. Sterols oxygenated at C-7 and containing a Δ5 double bond are among the most commonly found products of autoxidation (9). Sterols oxygenated at C-7 have been found in animal tissues and in foodstuffs and may play a role as physiological regulators of HMG-CoA reductase, sterol synthesis, and cell replication in the cells in which they are found (6,9,10,47,48). Hydroxylation at C-7 (7
