Perspective of the Induction of Liver Microsomal Cytochrome P450s by Chemical Compounds.

The concept of hepatic induction of drug-metabolizing cytochrome P450s (P450s) by xenobiotics, including therapeutic drugs, was proposed in the early 1960s. A polycyclic aromatic hydrocarbon and phenobarbital have been the two major inducers used to investigate this induction mechanism. Currently, the mechanisms mediated by aryl hydrocarbon receptor and constitutive androstane receptor are well-established. In addition to mammals, insects and fungi also express P450s and induce them following exposure to insecticides. These inductions may have environmental consequences. Finding the molecular mechanism regulating these inductions will be of major interest in the future. SIGNIFICANCE STATEMENT: This paper summarizes present and future of investigations into induction of drug-metabolizing enzymes.

Future perception in P450 research.

Cytochrome P450 (P450) of eukaryotes and prokaryotes diversified remarkably during the long course of their evolution. Since the diversification of P450 was the consequence of the adaptation of various living organisms to diverse environmental changes, the catalytic activities and physiological functions of the P450s of different taxa can be significantly different. It is likely that many P450s with novel physiological functions will be found in future when we further identify and examine the P450s of various eukaryotes and prokaryotes. The whole-genome sequences of a few thousand species are now available. The genome data will be helpful for the search of novel P450s in the eukaryotic and prokaryotic organisms that have not been studied so far. Discovery of new P450s with unique catalytic activities will expand the scope of biochemical and biophysical research on P450.

Evolutionary origin of mitochondrial cytochrome P450.

Different molecular species of cytochrome P450 (P450) are distributed between endoplasmic reticulum (microsomes) and mitochondria in animal cells. Plants and fungi have many microsomal P450s, but no mitochondrial P450 has so far been reported. To elucidate the evolutionary origin of mitochondrial P450s in animal cells, available evidence is examined, and the virtual absence of mitochondrial P450 in plants and fungi is confirmed. It is also suggested that a microsomal P450 is the ancestor of animal mitochondrial P450s. It is likely that the endoplasmic reticulum-targeting sequence at the amino-terminus of a microsomal P450 was converted to a mitochondria-targeting sequence possibly by point mutations of a few amino acid residues or by an exon-shuffling/moving event shortly after animal lineage diverged from plants and fungi in the course of evolution of eukaryotes. It is suggested that the microsome-type P450 first imported into mitochondria utilized the existing ferredoxin in the matrix to receive electrons from NADPH, retained its oxygenase activity in the mitochondria, and gradually diversified to several P450s with different substrate specificities in the course of the evolution of animals.

Contribution of cytochrome P450 to the diversification of eukaryotic organisms.

Emergence of eukaryotic cells in the ancient world of prokaryotic life was dependent on P450 as the synthesis of sterols, an essential constituent of the plasma membrane, required a P450-catalyzed reaction. As the ancestral monocellular eukaryotic organisms evolved into multicellular eukaryotes, and then diversified to plants, fungi, and animals with different body organizations and metabolic activities, many novel compounds were created in order to meet the requirements for increasing complex metabolic activities of a wide variety of eukaryotic organisms. Many new P450s, created by gene duplication and mutation, contributed to the synthesis of those novel compounds in animals, plants, and fungi, and supported the diversification of the eukaryotes. Many secondary metabolites of plants, which protect the plants from the predation by herbivorous animals, were also synthesized by P450-catalyzed reactions. The herbivorous animals detoxified the noxious foreign compounds in the plants by P450. This "chemical warfare" between animals and plants is particularly evident in plants-insects interaction, and contributed to the coevolution and diversification of both plants and insects. The interaction between flowering plants and insect pollinators, which contributed to their coevolution, also depends on various plant compounds synthesized by P450-catalyzed reactions. P450 has made highly important contributions to the evolution and diversification of eukaryotic organisms.

Recollection of the early years of the research on cytochrome P450.

Since the publication of the first paper on "cytochrome P450" in 1962, the biochemical research on this novel hemoprotein expanded rapidly in the 1960s and the 1970s as its principal roles in various important metabolic processes including steroid hormone biosynthesis in the steroidogenic organs and drug metabolism in the liver were elucidated. Establishment of the purification procedures of microsomal and mitochondrial P450s in the middle of the 1970s together with the introduction of molecular biological techniques accelerated the remarkable expansion of the research on P450 in the following years. This review paper summarizes the important developments in the research on P450 in the early years, for about two decades from the beginning, together with my personal recollections.

Recollections of my friendship with Klaus Ruckpaul.

Klaus Ruckpaul was the leader of cytochrome P450 research in the "East" Germany when the world was politically divided into "East" and "West". Under strong political pressure during the "Cold War", the communication between the scientists in the "East" countries with those in the "West" countries was badly restricted. He wanted to improve the situation, and organized an international gathering of the biochemists studying cytochrome P450. The first meeting was held in 1976, and it developed later into a big international conference named "International Conference on Cytochrome P450, Biochemistry and Biophysics". He and his colleagues also contributed greatly to the elucidation of the mechanism of P450-catalyzed reactions. I respect him for his great contribution to the advancement of biochemical study on cytochrome P450, and feel happy that I have enjoyed a long friendship with him.

Structural diversity of cytochrome P450 enzyme system.

Cytochrome P450 enzyme system consists of P450 and its NAD(P)H-linked reductase or reducing system, and catalyses monooxygenation reactions. The most prevalent type in eukaryotic organisms is 'microsomes type', which consists of membrane-bound P450 and NADPH-P450 reductase. The second type is 'mitochondria type', in which P450 is bound to the inner membrane while the reducing system consisting of an NADPH-linked flavoprotein and a ferredoxin-type iron-sulphur protein is soluble in the matrix space. The third type is 'bacteria type', in which both P450 and the reducing system are soluble in the cytoplasm. In addition to these three types, several forms of P450-reductase fusion proteins have been found in prokaryotic organisms. On the other hand, some P450s catalyse the re-arrangement of the oxygen atoms in the substrate molecules that does not require the supply of reducing equivalents for the reaction. A peculiar P450, P450nor, receives electrons directly from NADH for the reduction of nitric oxide.

Mitochondrial P450s.

Cytochrome P450 was first found in the microsomes from animal tissues, and then the presence of P450 in mitochondria was reported for the steroidogenic organs, adrenal gland and gonads. Three forms of mitochondrial P450 (11A, 11B1, and 11B2) were purified from these organs and their functions in steroid hormone biosynthesis were confirmed. Later studies showed the presence of several other forms of P450 (24A, 27A, 27B, and 27C) in the mitochondria of various non-steroidogenic organs including liver and kidney. These mitochondrial P450s were found to participate in the biosynthesis of bile acids from cholesterol in the liver, and the metabolic activation of Vitamin D3 to its active form, 1,25-dihydroxyvitamin D3, in the liver and the kidney. In contrast to the "drug-metabolizing" P450s in microsomes, most mitochondrial P450s show high specificity to their endogenous substrates, and have negligible activity towards xenobiotic compounds. In contrast to these established roles of mitochondrial P450s in the metabolism of endogenous substrates, the metabolism of xenobiotic chemicals by P450-catalyzed reactions in mitochondria has long been a subject of controversy. It is now known that all P450s in eukaryotic organisms are coded by nuclear genes, and the nascent peptides of various forms of P450 synthesized by cytoplasmic ribosomes are targeted to either endoplasmic reticulum (ER) or mitochondria depending on the ER-targeting sequence or the mitochondria-targeting sequence present in their amino-terminal portion. However, the presence of some microsome-type P450s in the mitochondria from various animal tissues including liver and brain has been reported. Possible mechanisms of intracellular sorting of some microsome-type P450s to mitochondria have been proposed, although physiological significance of the contribution of P450s in mitochondria to the metabolism of xenobiotic chemicals in animal tissues is still elusive.

Heme-thiolate proteins.

Cytochrome P450 was the first hemoprotein found to have a thiolate anion as the axial ligand of the heme. Several other heme-thiolate proteins, including nitric oxide synthase, were later found in animals, plants, and microorganisms. Both cytochrome P450 and nitric oxide synthase, two major members of the heme-thiolate protein family, catalyze monooxygenase reactions, but the physiological functions of other heme-thiolate proteins are apparently highly diverse. Chloroperoxidase of a mold, Caldaryomyces fumago, catalyzes a haloperoxidase reaction. CooA of a bacterium, Rhodospirillum rubrum, and heme-regulated eIF2alpha kinase of animals function as the sensors for carbon monoxide and nitric oxide, respectively, to elicit biological responses to these gases. The role of heme in the enzymatic activity of cystathionine beta-synthase is still unknown. It is likely that more heme-thiolate proteins with diversified functions will be found in various organisms in the future.