||Dr. Hollenberg's laboratory is primarily concerned with investigations of the microsomal cytochrome P450-dependent mixed-function oxidases found in most mammalian tissues. These enzymes catalyze the metabolism of a wide variety of xenobiotics including drugs, anesthetics, pesticides, chemical carcinogens, and organic solvents as well as endogenous compounds of great physiological importance including steroids, fatty acids, retinoids, eicosanoids, and lipid hydroperoxides. They also serve as nitric oxide (NO) synthases. Understanding the catalytic mechanisms, the relationship between the three-dimensional structure and the catalytic function, regulation, and roles of the P450s in the metabolism of endogenous and exogenous compounds is of great importance to the fields of pharmacology, endocrinology, toxicology, and oncology.
Epidemiological studies suggest most human cancers are due to exposure to chemicals in the environment which cause cancer. In general, these chemicals are biologically inert and require activation by the drug-metabolizing enzymes in the target tissue to express this carcinogenic potential. Compounds known to cause cancer as a result of metabolic activation by P450 include polycyclic aromatic hydrocarbons, nitrosamines, and aflatoxins. Thus, a primary objective of the laboratory is to gain a better understanding of mechanisms by which cells activate environmental chemicals to reactive forms that cause cancer and ultimately to develop approaches which could be used to protect humans against potentially carcinogenic or toxic chemicals in the environment.
The Human Genome Project has identified the genes for 57 P450s in the human genome. Of those 57, the genes for approximately 40 of those cytochromes P450 have been found to be expressed in human tissues. Each form of P450 exhibits its own unique substrate specificity and even when two different forms metabolize the same substrate, they generally give very different product profiles. For some time, a major interest in our laboratory has been concerned with the relationships between the structures of the active sites of various forms of P450 and their catalytic functions. Earlier studies in our lab focused on the purified cytochrome P450s 2B1, 2B4, and 2E1, forms which were purified from rats and rabbits. However, with the availability of the appropriate cDNAs for various human P450s and the development of systems for expressing and purifying human P450s from bacteria we have switched our studies to focus on human P450s which play a major role in the metabolism of various drugs and chemical carcinogens.
Recently, we have successfully expressed in our laboratory several human P450s and we are now studying human P450 3A4, the human liver P450 which appears to play a major role in the metabolism of up to 50% of all drugs currently used in the United States; P450 3A5, which is very similar to 3A4 in structure and plays a major role in drug metabolism in some individuals but has a different substrate specificity from 3A4; P450 2B6, another human P450 which plays an important role in the metabolism of a variety of drugs and toxins, and P450 2E1, which is known to play an important role in the metabolic activation and detoxication of many low molecular weight carcinogens and other toxins. We are using mechanism-based inactivators to investigate the structures of active sites of these P450s and to identify the specific amino acid residues in the active sites involved in catalysis or substrate-binding. Mechanism-based inactivators are substrates of an enzyme which, during the course of catalysis, are converted to highly reactive forms which react with moieties in the active site to form covalent adducts, thereby preventing the enzyme from traversing further catalytic cycles.
We are also trying to identify and develop mechanism-based inactivators which are specific for the forms of P450 such as 2E1 that are responsible for the activation of carcinogens, and thus have potential to be used as chemopreventive agents to protect against environmental carcinogens. We are working with a variety of mechanism-based inactivators including mifepristone (RU486), tamoxifen, 17 alpha-ethinylestradiol, phencyclidene (angel dust), benzyl isothiocyanate, tert-butylisothiocyanate, 7-ethynylcoumarin, and bergamottin. We have identified critical peptide regions and, in some cases, specific amino acid residues in the active sites of P450s 2B1, 2B4, 2B6, 2E1, 3A4, and 3A5.
Current studies are concerned with: identification of additional active site amino acid(s) and peptide(s) of the enzymes modified during inactivation; determination of the mechanism(s) by which inactivation occurs, and determination of the specificity of the inactivators for the various forms of human P450. These studies will provide valuable information for designing inactivators to selectively manipulate the catalytic activity of P450s and may ultimately lead to the development of approaches which may be used to protect people against developing cancer due to carcinogen exposure.
Other studies in our laboratory are aimed at understanding the relationship between the structure and function of P450 and they involve site-specific mutagenesis of selected amino acid residues in the P450 active site followed by investigations into changes in the catalytic function of the enzyme. We have performed site-specific mutagenesis studies on P450s 2B1, 2B2, 2B4, 2B6, 2E1, and 3A4. Additional site-specific mutagenesis studies target those amino acid residues which have been identified by the studies using mechanism-based inactivators and will yield valuable information regarding the role(s) of those residues in catalysis and substrate binding.
Isothiocyanates are naturally occurring compounds found in cruciferous vegetables such as broccoli, cabbage, and watercress. These compounds have been shown to be effective cancer chemopreventative agents and some are currently in clinical trials for this activity. One of the mechanisms by which they are thought to act involves inhibition of the cytochromes P450 involved in the metabolic activation of environmental carcinogens such as nitrosamines. We have demonstrated that several isothiocyanates are mechanism-based inactivators of various forms of P450s. Benzyl isothiocyanate (BITC) is a potent mechanism-based inactivator of human 2E1. Human 2E1 is also inactivated by tert-butylisothiocyanate. We have characterized both inactivations in detail. We are currently used the radio-labeled isothiocyanates as probes for the structures of the active sites of these P450s. Studies aimed at identifying other isothiocyanates which are mechanism-based inactivators of P450s and at developing ones which are specific for different forms of human P450s are underway.
Oral coadministration of grapefruit juice significantly increases the oral availability of numerous clinically used drugs including felodipine, nifedipine, cyclosporine A, midazolam, terfenadine, and ethinylestradiol. Since all of these drugs are metabolized primarily by cytochrome P450 3A4, the predominant intestinal and hepatic P450, it has been suggested that the grapefruit juice effect may be due to inhibition of P450 3A4 activity.
We have demonstrated that grapefruit juice contains several furanocoumarins that inhibit human P450 3A4. Two of the most important compounds in grapefruit juice were shown to be bergamottin (BG) and 6',7'-dihydroxybergamottin (DHB). We have demonstrated that both BG and DHB are potent mechanism-based inactivators of P450 3A4. The mechanism of inactivation appears to involve covalent modification of the apo P450 in the active site of the enzyme. We are currently investigating the effects of these compounds on other hepatic P450s.
Over the past several years we have also been studying the role(s) of the cytochrome P450s in the endocannabinoid system. The endocannabinoid system consists of the endocannabinoids anandamide and 2-arachidonoylglycerol, the enzymes involved in the synthesis and degradation of the endocannabinoids, and the cannabinoid CB1 and CB2 receptors. The components of the endocannabinoid system represent novel pharmacological targets in the treatment of many disorders including neurodegeneration, inflammation, cancer, chronic pain, and others. The CB2 receptor is found in the brain and is considered to be a very important therapeutic target for the treatment of central nervous system inflammation.
We have previously demonstrated that anandamide is oxidized by human cytochrome P450s to form hydroxyeicosatetraenoic acid (HETE) and epoxyeicosatrienoic acid (EET) ethanolamide (EA) derivatives. P450 3A4 is the primary P450 in the liver responsible for the epoxyidation of anandamide to give the 5,6-, 8,9- 11,12, and 14,15-EET-EAs. These EET-EAs produced in the liver by the microsomal P450s are then converted to their corresponding dihydroxy derivatives by microsomal epoxide hydrolase. Anandamide is metabolized by P450 4F2 in the liver and the kidneys to give a mono-oxygenated product which has been identified as 20-HETE-EA. Using Chinese hamster ovary cells stably expressing the human CB2 receptor, we have shown that the 5,6-EET-EA metabolite of anandamide is a potent CB2 agonist. Stability studies in mouse brain preparations have revealed that this 5,6-EET-EA is significantly more refractory to enzymatic hydrolysis than the parent anandamide, which is mainly metabolized by the enzyme fatty acid amide hydrolase (FAAH). Since the epoxide of anandamide is more stable than the parent compound and is a better agonist of the CB2 receptor, this suggests that it may be a much more important compound in vivo, and that metabolism of anandamide by P450 3A4 may represent a bioactivation pathway. We have also demonstrated that the expression of anandamide-metabolizing P450s 4F13 and 3A1 in mouse microglial cells is upregulated following activation of the microglial cells in vitro with the cytokine interferon-gamma. This elevation in the protein expression correlates with the increased ability of the microglial cells to convert anandamide to 5,6-EET-EA.
Current studies are concerned with developing more sensitive approaches that could be used for the separation and detection of the HETE-EAs, EET-EAs, and other potential metabolites of anandamide so that these can be used to determine their presence and levels in biological samples. Because of the wide ranging effects of anandamide in many organ systems, itís metabolism by P450s from various tissues such as the brain and the spleen will be examined in detail. It is hoped that a better understanding of the metabolic routes and biological activities of the endocannabinoid metabolites will lead to the development of new drugs that will be useful in the treatment of inflammation, cancer, neurodegeneration, and chronic pain, as well as other diseases involving the endocannabinoid system.