Paul F. Hollenberg, PhD

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, 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. More than 30 different forms of cytochromes P450 have been found in human tissues. Each form 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. For these studies we have been using purified P450 2B1 and P450 2B4, the major forms of P450 induced by phenobarbital in rats and rabbits, respectively. We are also studying P450 2E1, a form which is thought to play a major role in the metabolic activation of many low molecular weight carcinogens and other toxic agents.

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 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 a-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, and 3A4.

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 each inactivator for protection against different carcinogens in cells expressing various P450s. 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 aimed at understanding the relationship between the structure and function of P450 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 and 2B2, which differ by only 14 amino acid residues and yet exhibit marked differences in their specificities for some substrates and on P450 2E1. 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.

A second area of interest is concerned with the metabolism of dialkylnitrosamine carcinogens such as N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) and N-nitrosodipropylamine (NDPA). These compounds are of great interest and concern because of widespread human exposure, high carcinogenic potency, and their demonstrated importance in human carcinogenesis. Nitrosamines are readily metabolized by various forms of cytochrome P450 which play a critical role both in their metabolic activation and their detoxication.

We are currently studying the metabolism of a series of dialkylnitrosamines ranging from C1 (dimethyl) to C4 (dibutyl). Areas under investigation include: determination of the metabolic products formed from each nitrosamine; identification of the forms of P450 responsible for the metabolism of each nitrosamine leading to the formation of the various products; elucidation of the role(s) of the various metabolites in cancer induction; and investigations of the effects of modifying agents such as phenobarbital, ethyl alcohol and pyridine on the metabolic activation and detoxication of these nitrosamines.

Cytochrome P450 2E1, which is elevated in diabetic animals and induced by ethyl alcohol, acetone, and isoniazid, plays a key role in the metabolic activation of low molecular weight nitrosamines such as NDMA and NDEA. We have cloned the gene for P452 2E1, inserted it into an appropriate expression vector, transfected the plasmid into human cells in vitro, and stably expressed this P450 in human cells in culture. Studies aimed at characterizing nitrosamine carcinogen metabolism, cytotoxicity, and DNA modification in human cells containing 2E1 are under investigation. These studies will be expanded to include other forms of P450 such as P450s 3A4 and 2B6 which are important for human metabolism of xenobiotics and other toxic chemicals.

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 rat P450 2B1 and rabbit 2E1. Rabbit 2E1 is also inactivated by tert-butylisothiocyanate. We have characterized all three inactivations in detail. We are currently used the radio-labeled isothiocyanates as probes for the structures of the active sites of these P450s. We are also investigating the ability of these compounds to protect human cells from nitrosamine-induced toxicity. 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 and exploring the effects of BG on drug bioavailability.