F. Peter Guengerich

Mapping the Genetic Architecture of Gene Expression in Human Liver

Genetic variants that are associated with common human diseases do not lead directly to disease, but instead act on intermediate, molecular phenotypes that in turn induce changes in higher-order disease traits. Therefore, identifying the molecular phenotypes that vary in response to changes in DNA and that also associate with changes in disease traits has the potential to provide the functional information required to not only identify and validate the susceptibility genes that are directly affected by changes in DNA, but also to understand the molecular networks in which such genes operate and how changes in these networks lead to changes in disease traits. Toward that end, we profiled more than 39,000 transcripts and we genotyped 782,476 unique single nucleotide polymorphisms (SNPs) in more than 400 human liver samples to characterize the genetic architecture of gene expression in the human liver, a metabolically active tissue that is important in a number of common human diseases, including obesity, diabetes, and atherosclerosis. This genome-wide association study of gene expression resulted in the detection of more than 6,000 associations between SNP genotypes and liver gene expression traits, where many of the corresponding genes identified have already been implicated in a number of human diseases. The utility of these data for elucidating the causes of common human diseases is demonstrated by integrating them with genotypic and expression data from other human and mouse populations. This provides much-needed functional support for the candidate susceptibility genes being identified at a growing number of genetic loci that have been identified as key drivers of disease from genome-wide association studies of disease. By using an integrative genomics approach, we highlight how the gene RPS26 and not ERBB3 is supported by our data as the most likely susceptibility gene for a novel type 1 diabetes locus recently identified in a large-scale, genome-wide association study. We also identify SORT1 and CELSR2 as candidate susceptibility genes for a locus recently associated with coronary artery disease and plasma low-density lipoprotein cholesterol levels in the process.

Human cytochrome P-450 enzymes

Cytochrome P-450 (P-450) enzymes have been studied extensively in experimental animal models and much is known regarding their structures, regulation, and mechanisms of catalysis. In recent years investigations have been extended to the human P-450s. There are more than 30 different characterized human P-450s in the superfamily, and collectively they are probably the most significant enzymes involved in the metabolism of drugs, carcinogens, and steroids. The levels of many of the P-450s and their catalytic activities can vary considerably because of polymorphism, induction, and inhibition. The catalytic specificity of the P-450s can range from being very non-discriminatory to very exacting, and clinical consequences of drugs and steroids can be related to variations in P-450 levels. Defects in the rate-limiting P-450 reactions in steroidogenesis (due to genetic deficiencies) have been shown to be debilitating and even fatal.

Dealkylation of pentoxyresorufin: A rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat

The O-dealkylation of pentoxyresorufin (7-pentoxyphenoxazone) by rat liver microsomes was examined. The reaction appeared highly specific for certain phenobarbital inducible forms of cytochrome P-450 and was increased 95- to 140-fold by animal pretreatment with phenobarbital (75 mg/kg/day, four ip injections) and approximately 50-fold by Aroclor 1254 (500 mg/kg, one ip injection) while animal pretreatment with 3-methylcholanthrene (50 mg/kg/day, three ip injections) resulted in less than a 2-fold increase over the rate detected in control microsomes. It was observed that this activity, in microsomes for Aroclor-pretreated rats, was dependent on O2 and was inhibited by metyrapone and SKF 525-A, indicative of cytochrome(s) P-450 mediation in the reaction. When antibodies directed against purified cytochrome(s) P-450s were employed to inhibit the pentoxyresorufin O-dealkylation reaction, antibodies to P-450PB-B greatly inhibited the reaction (greater than 90%), while antibodies to P-450PB-C or P-450PB/PCN-E had minimal effects. Assay of hepatic microsomes from rats which were pretreated with varying doses of phenobarbital (0.9-75 mg/kg/day, four ip injections) indicated that while aminopyrine-N-demethylase activity was induced only 2-fold at the maximum dose (75 mg/kg/day), pentoxyresorufin O-dealkylase activity was induced approximately 140-fold at this dose and approximately 4-fold by a dose of phenobarbital as low as 0.9 mg/kg.

Cytochrome P450s and other enzymes in drug metabolism and toxicity

The cytochrome P450 (P450) enzymes are the major catalysts involved in the metabolism of drugs. bioavailability and toxicity are 2 of the most common barriers in drug development today, and P450 and the conjugation enzymes can influence these effects. The toxicity of drugs can be considered in 5 contexts: on-target toxicity, hypersensitivity and immunological reactions, off-target pharmacology, bioactivation to reactive intermediates, and idiosyncratic drug reactions. the chemistry of bioactivation is reasonably well understood, but the mechanisms underlying biological responses are not. In the article we consider what fraction of drug toxicity actually involves metabolism, and we examine how species and human interindividual variations affect pharmacokinetics and toxicity.

Human liver microsomal steroid metabolism: Identification of the major microsomal steroid hormone 6β-hydroxylase cytochrome P-450 enzyme

Cytochrome P-450-dependent steroid hormone metabolism was studied in isolated human liver microsomal fractions. 6 beta hydroxylation was shown to be the major route of NADPH-dependent oxidative metabolism (greater than or equal to 75% of total hydroxylated metabolites) with each of three steroid substrates, testosterone, androstenedione, and progesterone. With testosterone, 2 beta and 15 beta hydroxylation also occurred, proceeding at approximately 10% and 3-4% the rate of microsomal 6 beta hydroxylation, respectively, in each of the liver samples examined. Rates for the three steroid 6 beta-hydroxylase activities were highly correlated with each other (r = 0.95-0.97 for 25 individual microsomal preparations), suggesting that a single human liver P-450 enzyme is the principal microsomal 6 beta-hydroxylase catalyst with all three steroid substrates. Steroid 6 beta-hydroxylase rates correlated well with the specific content of human P-450NF (r = 0.69-0.83) and with its associated nifedipine oxidase activity (r = 0.80), but not with the rates for debrisoquine 4-hydroxylase, phenacetin O-deethylase, or S-mephenytoin 4-hydroxylase activities or the specific contents of their respective associated P-450 forms in these same liver microsomes (r less than 0.2). These correlative observations were supported by the selective inhibition of human liver microsomal 6 beta hydroxylation by antibody raised to either human P-450NF or a rat homolog, P-450 PB-2a. Anti-P-450NF also inhibited human microsomal testosterone 2 beta and 15 beta hydroxylation in parallel to the 6 beta-hydroxylation reaction. This antibody also inhibited rat P-450 2a-dependent steroid hormone 6 beta hydroxylation in uninduced adult male rat liver microsomes but not the steroid 2 alpha, 16 alpha, or 7 alpha hydroxylation reactions catalyzed by other rat P-450 forms. Finally, steroid 6 beta hydroxylation catalyzed by either human or rat liver microsomes was selectively inhibited by NADPH-dependent complexation of the macrolide antibiotic triacetyloleandomycin, a reaction that is characteristic of members of the P-450NF gene subfamily (P-450 IIIA subfamily). These observations establish that P-450NF or a closely related enzyme is the major catalyst of steroid hormone 6 beta hydroxylation in human liver microsomes, and furthermore suggest that steroid 6 beta hydroxylation may provide a useful, noninvasive monitor for the monooxygenase activity of this hepatic P-450 form.

Enzymatic activation of chemicals to toxic metabolites

A variety of enzymes function in the oxygenation, oxidation-reduction, conjugation, and hydrolysis of drugs and other foreign chemicals. Often these enzymes detoxicate chemicals to prevent detrimental effects. In this review we will, however, concentrate on cases in which metabolism activates chemicals to reactive species which cause cellular damage. Particular attention will be given to mixed-function oxidases, which carry out a variety of oxygenations, as well as other reactions. (We will focus on cellular toxicity as opposed to initiation of tumorigenesis in this review.) In many cases, considerable circumstantial evidence exists linking these enzymes to enhanced toxicity of chemicals, although causal relationships have seldom been demonstrated. Further, in very few cases is the explicit cause of toxicity known. Modification of critical protein residues is suspected, although oxidative stress may also be involved in some cases. We discuss general aspects of mechanisms of toxic action, briefly list all cases in which metabolism is suspected to play a role in enhancing toxicity, and review a few examples in detail where substantial chemical and enzymatic information is available. The latter instances would involve knowledge of the enzymes involved, chemical evidence on the structures of the reactive metabolites, identification of adducts, and some inference into the biological processes which are effected to elicit toxicity. We consider, in this regard, vinyl halides (which have been a focus in our own laboratory), acetaminophen, pyrrolizidine alkaloids, and fluoroxene.

COMPARISONS OF CATALYTIC SELECTIVITY OF CYTOCHROME P450 SUBFAMILY ENZYMES FROM DIFFERENT SPECIES

Historically there has been considerable interest in comparing patterns of biotransformation of xenobiotic chemicals in experimental animal models and humans, e.g. in areas such as drug metabolism and chemical carcinogenesis. With the availability of more basic knowledge it has become possible to attribute the oxidation of selected chemicals to individual cytochrome P450 (P450) enzymes in animals and humans. Further, these P450s can be characterized by their classification into distinct subfamilies, which are defined as having > 59% amino acid sequence identity. Questions arise about how similar these enzymes are with regard to structure and function. More practically, how much can be predicted about reaction specificity and catalysis? In order to address these issues, we need to consider not only the relatedness of P450s from different species but also (i) functional similarity within P450 subfamilies and (ii) the effects of small changes imposed by site-directed mutagenesis. Relationships in the P450 1A, 2A, 2B, 2C, 2D, 2E, 3A, and 17A subfamilies are briefly reviewed. Overall functional similarity is generally seen in subfamily enzymes but many examples exist of important changes in catalysis due to very small differences, even a single conservative amino acid substitution. Some general conclusions are presented about predictability within various P450 subfamilies.

Genetic polymorphism of S-mephenytoin hydroxylation

This review describes the pharmacological, biochemical and clinical studies that have led to a functional characterization of the mephenytoin polymorphism. In addition, attempts to understand the genetic basis of the metabolic defect at the biochemical and molecular levels are also described

Metabolic activation of carcinogens

Most chemical carcinogens are not active in themselves but require bioactivation to electrophiles that bind covalently to DNA and often act by producing mutations. In recent years it has been realized that mutations can be important at many stages of carcinogenesis. A variety of different enzymes are involved in bioactivation reactions, which include oxidation, reduction, thiol conjugation, acetyl transfer, sulfur transfer, methyl transfer, glucuronosyl transfer, and epoxide hydrolysis. These processes often occur in concert with a single carcinogen. Humans vary considerably in activities of these enzymes and this variation may contribute to differences in risk.

Activation of procarcinogens by human cytochrome P450 enzymes

Enzymatic transformation of most chemical carcinogens is requisite to the formation of electrophiles that cause genotoxicity, and the cytochrome P450 (P450) enzymes are the most prominent enzymes involved in such activation reactions. During the past 15 years the human P450 enzymes have been extensively characterized. Considerable evidence exists that the variation in activity of these enzymes can have important consequences in the actions of drugs. Other studies have been concerned with the activation of procarcinogens by human P450s. Assignments of roles of particular P450s in the metabolism of chemical carcinogens are discussed, along with the current state of evidence for relationships of particular P450s with human cancer.