Daniel W. Nebert

The in vivo and in vitro induction of aryl hydrocarbon hydroxylase in mammalian cells of different species, tissues, strains, and developmental and hormonal states.

Abstract Aryl hydrocarbon hydroxylase from rat liver metabolizes a variety of polycyclic hydrocarbons. This membrane-bound enzyme system is present in various tissues of the monkey, hamster, and rat. In vivo , the prior administration of a polycyclic hydrocarbon induces the hydroxylase to higher levels in the liver, lung, gastrointestinal tract, and kidney of each of these mammals. The enzyme is also present in various tissues of six strains of mice; the hepatic enzyme is inducible in the Swiss, strains C-57, C3H, and A, but not in strains AKR/N or DBA, of the mouse. Thus, there are genetic differences in the regulation of enzyme induction as well as in the control levels of enzyme. The enzyme system is also induced transplacentally: treatment of the pregnant hamster or rat, with either a polycyclic hydrocarbon or phenobarbital, induces the enzyme system in specific fetal tissues as well as in the placenta. The extent of enzyme induction is greatest in adult, less in neonatal tissues, and least in fetal tissues. Induction of hydroxylase activity by 3-methylcholanthrene occurs in the liver, lung, and kidney of the adrenalectomized or hypophysectomized male rat, and the hepatic enzyme is induced by phenobarbital in the adrenalectomized male rat. The magnitude of aryl hydrocarbon hydroxylase induction varies greatly with the tissue and species—from no induction to more than 100-fold increases in enzyme activity. The enzyme system is also present and inducible in fetal cell cultures derived from whole hamster, mouse, rat, and chick. Further, hydroxylase activity is inducible in cell cultures derived from hamster fetal liver, lung, small intestine, and limbs, and in mouse 3T3 culture, an established cell line.

The Ah Locus and the Metabolism of Chemical Carcinogens and Other Foreign Compounds

Publisher Summary Drug-metabolizing enzyme systems, which are localized principally in the liver, are generally divided into two groups—namely, phase I and phase II. One of the most interesting phase I enzyme systems include a group of enzymes collectively known as the “cytochrome P-450-mediated monooxygenases.” These membrane-bound enzyme systems effectively metabolize polycyclic aromatic hydrocarbons, mutagens, and numerous aromatic amines. The Ah locus in the mouse controls the induction of cytochrome P 1 -450, eleven associated monooxygenase activities, and UDP glucuronyltransferase activity. These enzyme systems potentiate and detoxify chemical carcinogens, environmental pollutants, drugs, and other chemicals, along with numerous endogenous substrates. Numerous conditions in the mouse, which include cancer, drug toxicity, and birth defects, are directly associated with the Ah locus— considered to be a single gene or a small number of genes. This chapter describes the background information on the characteristics of the P-450-mediated monooxygenases. It reviews the complexity of the genetic expression in the mouse and the large number of enzyme activities, the induction of which appears to be under common genetic regulation. It also examines evidence for an association of the Ah locus with conditions in the mouse such as, tumorigenesis and toxicity, evidence for an association of the Ah locus with in vitro test systems, and evidence for the existence of an Ah locus in the human.

Endogenous Functions of the Aryl Hydrocarbon Receptor (AHR): Intersection of Cytochrome P450 1 (CYP1)-metabolized Eicosanoids and AHR Biology

The AHR,2 a ligand-activated transcription factor, was first identified indirectly in the 1970s; the mouse and human genes were cloned in the early 1990s. Molecular mechanisms and biological consequences of AHR-mediated regulation of mammalian cytochrome P450 enzymes by foreign chemicals (e.g. polycyclic aromatic hydrocarbons and dioxins) have been studied extensively. Binding of such ligands to AHR leads to transcriptional activation of CYP1A1, CYP1A2, and CYP1B1; in turn, these enzymes catalyze oxidative detoxication or activation of most ligands. From the start, AHR endogenous ligands and functions were postulated; although this was initially controversial, the robust phenotype of Ahr–/– knock-out mice provided clear evidence of physiological roles (and endogenous ligands) for AHR. AHR has numerous important endogenous functions: during conception and embryonic and fetal development; in the immune, cardiovascular, neural, and reproductive systems; and in hepatocytes, skin cells, and adipocytes. These myriad AHR-mediated processes mirror the vast universe of action of the eicosanoids, lipid mediators known to undergo cytochrome P450-dependent oxidation. We propose that many endogenous and exogenous cellular stimuli lead to (i) AHR-dependent CYP1-dependent eicosanoid synthesis and degradation and (ii) AHR-dependent CYP1-independent (eicosanoid-dependent and -independent) responses. These two pathways can be delineated from one another in genetic models: the former is absent in the recently characterized Cyp1a1/1a2/1b1–/– triple-knock-out mouse, and the latter is absent in the Ahr–/– knock-out mouse. Identification of specific eicosanoids whose synthesis or degradation is carried out by each CYP1 enzyme should allow for identification of physiological endogenous AHR ligands. (For “History and Background,” see supplemental material (Box 1).)

Sulfobetaine derivatives of bile acids: Nondenaturing surfactants for membrane biochemistry

The syntheses of four new sulfobetaine derivatives of bile salts are presented, along with a general set of criteria for useful detergents in membrane biochemistry. Physical properties including the critical micelle concentration, aggregation number, partial specific volume, critical micellar temperature, uv-vis spectrum, and circular dichroism spectrum are examined for the new compounds. To examine the interaction of this class of compounds with macromolecules, one of these (CHAPS) was further studied. Circular dichroism spectra of apolipoprotein C-III2 were measured in the presence of varying concentrations of CHAPS to determine the effect of this compound on secondary structure. Gel-exclusion chromatography and sedimentation equilibrium studies of cytochrome P-450 in the presence of CHAPS were also performed to establish the ability of this detergent to disaggregate cytochrome P-450 to a monomeric/dimeric state.

Multiple forms of inducible drug-metabolizing enzymes: A reasonable mechanism by which any organism can cope with adversity

SummaryAll organisms possess a number of genetically regulated mechanisms in order to cope with rapid adverse changes in the environment. The two systems which appear to respond to a seemingly endless array of chemical specificities are the immune response and the induction of drug-metabolizing enzymes. Similarities and differences between the immunoglobulin and the cytochrome P-450-mediated monooxygenase systems are described. DNA insertion sequences, plasmid “transposons,” maize “controlling elements,” gene duplication, intervening sequences, and high-frequency intergenic recombination are all discussed as possible methods by which organisms can “adapt” quickly to a new selective pressure. If the regulation of P-450 induction resembles in any way the other methods by which pro- and eukaryotes cope genetically with numerous forms of environmental adversity, therefore, it is very likely that mammalian tissues contain hundreds, if not thousands, of inducible forms of P-450.

The Ah regulatory gene product. Survey of nineteen polycyclic aromatic compounds' and fifteen benzo[a]pyrene metabolites' capacity to bind to the cytosolic receptor

The capacity of 19 polycyclic aromatic compounds and 15 benzo[a]pyrene metabolites to displace [1,6-3H]2,3,7,8-tetrachlorodibenzo-p-dioxine ([3H]TCDD) from the mouse liver cytosolic Ah receptor was examined. We compared our data with various parameters taken from previously published results: the capacity of seven polycyclic hydrocarbons to induce aryl hydrocarbon hydroxylase (AHH) activity in human cell cultures, the capacity of 10 polycyclic hydrocarbons to induce azo dye N-demethylase activity in rat liver, the capacity of 6 polycyclic hydrocarbons to shorten zoxazolamine paralysis times in the intact rat, and the capacity of 15 benzo[a]pyrene metabolites to induce AHH activity in rat hepatoma H-4-II-E cultures. An excellent correlation is seen between the capacity to displace the radioligand from the Ah receptor and the capacity to induce these monooxygenase activities. differences in the rate of cellular uptake and formation of alkali-extractable metabolites of dibenzo[a,h]anthracene, 3-methylcholanthrene, and benzo[a]anthracene in Hepa-1 mouse hepatoma cell cultures do not account for differences in the capacity of these three polycyclic hydrocarbons to displace [3H]TCDD from the Ah receptor.

Update of human and mouse forkhead box (FOX) gene families

The forkhead box (FOX) proteins are transcription factors that play complex and important roles in processes from development and organogenesis to regulation of metabolism and the immune system. There are 50 FOX genes in the human genome and 44 in the mouse, divided into 19 subfamilies. All human FOX genes have close mouse orthologues, with one exception: the mouse has a single Foxd4, whereas the human gene has undergone a recent duplication to a total of seven (FOXD4 and FOXD4L1 → FOXD4L6). Evolutionarily ancient family members can be found as far back as the fungi and metazoans. The DNA-binding domain, the forkhead domain, is an example of the winged-helix domain, and is very well conserved across the FOX family and across species, with a few notable exceptions in which divergence has created new functionality. Mutations in FOX genes have been implicated in at least four familial human diseases, and differential expression may play a role in a number of other pathologies -- ranging from metabolic disorders to autoimmunity. Furthermore, FOX genes are differentially expressed in a large number of cancers; their role can be either as an oncogene or tumour suppressor, depending on the family member and cell type. Although some drugs that target FOX gene expression or activity, notably proteasome inhibitors, appear to work well, much more basic research is needed to unlock the complex interplay of upstream and downstream interactions with FOX family transcription factors.

The Ah locus, a multigene family necessary for survival in a chemically adverse environment: comparison with the immune system.

Publisher Summary This chapter discusses the drug-metabolizing enzyme systems, presents the Ah system, and provides data for multiple Ah-structural gene products. A cytosolic receptor is regarded as the major product of the Ah-regulatory genes. Sucrose-density gradient analysis following dextran–charcoal treatment is among the most reliable methods for characterizing an Ah receptor. The aryl hydrocarbon hydroxylase (AHH) fluorescent assay is simple and extremely sensitive. This assay, using benzo[α]pyrene as the substrate in vitro, is most commonly used as the biochemical marker for the Ah locus in laboratory animal and human studies. When males and females of four inbred strains were housed together and allowed to breed randomly for 46 to 48 months, the AHH inducibility by 3-methylcholanthrene of weanlings between 18 and 22 generations approximated the distribution found normally for out-bred or random-bred mouse strains. These data indicate the involvement of multiple Ah regulatory genes and a “natural selection” tendency of the induction process to “drift” toward lower intensity in populations having heterogeneous genetic input.

Cytochrome P450 gene expression and regulation

Abstract Cytochrome P 450 proteins are critical in the synthesis and degradation of steroids, fatty acids, prostaglandins, biogenic amines and pheromones and have probably existed since early days of evolution of eukaryotes. Daniel Nebert and Frank Gonzalez describe the different classes of P 450 and suggest that the use of mammalian (and perhaps even non-mammalian) P 450 genes will aid in the diagnosis and treatment of certain clinical disorders.

Bone marrow toxicity induced by oral benzo[a]pyrene: Protection resides at the level of the intestine and liver

The Ah locus encodes a cytosolic receptor that regulates the induction of certain drug-metabolizing enzymes by polycyclic aromatic hydrocarbons such as benzo[a]pyrene. Some inbred mouse strains such as C57BL/6N have the high-affinity Ah receptor (Ahb/Ahb), others such as DBA/2N, the poor-affinity receptor (Ahd/Ahd). Presence of the high-affinity receptor leads to greater cytochrome P1-450 induction by benzo[a]pyrene; in turn, enhanced benzo[a]pyrene metabolism can result in more toxic intermediates or greater detoxication, depending upon the test system studied. Benzo[a]pyrene in the growth medium, in direct contact with cultured myeloid cells, is more toxic to C57BL/6N than DBA/2N cultured cells. Oral benzo[a]pyrene induces P1-450 (measured by benzo[a]pyrene trans-7,8-dihydrodiol formation determined by high-performance liquid chromatography) in C57BL/6N but not DBA/2N intestine and liver. In the bone marrow of oral benzo[a]pyrene-treated C57BL/6N and DBA/2N mice, the magnitude of P1-450 induction is about the same. WB/ReJ (Ahd/Ahd), C57BL/6J (Ahb/Ahb), or (WB/ReJ)(C57BL/6J)F1 (Ahb/Ahd) marrow was transplanted into lethally irradiated (WB/ReJ)(C57BL/6J)F1 mice. DBA/2J (Ahd/Ahd) marrow was transplanted into lethally irradiated BALB/cByJ (Ahb/Ahb) mice and vice versa. Mice having the Ahd/Ahd intestine and liver died in less than 3 weeks of benzo[a]pyrene feeding (120 mg/kg/day), irrespective of the source of transfused marrow. All the data are consistent with pharmacokinetic differences in the tissue distribution of benzo[a]pyrene: mice having the high-affinity receptor, and therefore the P1-450 induction process in the intestine and liver, are protected from oral benzo[a]pyrene-induced myelotoxicity.