atom to water according to the following overall equation: RH + 0
Summary Cytochrome P450IIE1 is involved in the metabolic activation of many xenobiotics involved with human toxicity. In particular, cellular concentrations of P4501IE1 are significantly induced by the most widely abused drug in our society today, alcohol. As a result, the synthesis and degradation of this form of P450 has significant health consequences. The regulation of the steady-state concentration of P450IIE1 is an extremely complex process. The enzyme is regulated by transcriptional activation, mRNA stabilization, increased mRNA translatability and decreased protein degradation. The principal mechanism which controls the induction process depends on the chemical nature of the inducer, the age, and the nutritional and hormonal status of the animal. There also appear to be significant sex differences in the expression of P450IIE1. It is entirely possible that the regulation of the enzyme concentration under any given set of conditions will involve all of the mechanisms to different extents. Introduction Man is constantly exposed to a wide variety of xenobiotics which often have serious toxicological consequences. For many of the potential toxic substances to which we are exposed, either through voluntary action (i.e. smoking) or as a result of environmental exposure (i.e. passive smoke and air pollution), toxicity often requires the metabolic activation of the chemical. While there are a number of enzyme systems present in mammalian tissue which catalyze the oxidation of xenobiotics(l), one of the most important and widely studied of these is the cytochrome P450-dependent mixed function oxidase system. This system (hereafter referred to as P450) is present in highest concentrations in the endoplasmic reticulum of the liver although it has also been identified in numerous extrahepatic tissues, including, kidney, lung, placenta, skin and brain('-6). The P450 system consists of a FMN/FAD containing flavoprotein, NADPH cytochrome P450 oxidoreductase, which transfers reducing equivalents from NADPH to the terminal oxidase, P450. P450 catalyzes the addition of one atom of oxygen to the substrate while reducing the other
+ NADPH + R O H + H20 + NADP+
However, NADPH oxidation and oxygen consumption are not completely coupled to substrate hydroxylation. As a result, oxygen is also reduced to H 2 0 2 and/or 2 H 2 0 . In some instances, cytochrome b5 also has an important role in the metabolic process('32). The P450s represent a superfamily of enzymes. This superfamily has been subdivided into families and subfamilies based on global amino acid sequence similarities between different P450 forms. Ten families have been identified in mammals, six of which include forms of P450 involved in ~teroidogenesis(~-~). Due to species-specific evolution of these enzymed4),it is often difficult to identify those that share an immediate common ancestor (orthologue) in other species. Individual P450 forms can metabolize a large number of structurally diverse compounds. In some cases, a single substrate is metabolized by a single P450 form while other substrates can be oxidized, to varying degrees, by multiple P450 forms. The total P450 constituency within a given tissue determines whether a chemical. will undergo bioactivation or detoxification('.2). Cytochrome P45011E1 - An Overview The focus of the present review is the regulation of the P450IIE gene family. The first member of this family, P450IIE1, was originally purified from ethanol-treated rabbits and has subsequently been identified in all species examined including hamster, rats, mice, and human^(^.^,^). P450IIE1 is the only member of this family in rats, mice, and humans as determined by Southern blot analysis(3). A second highly related form, P450IIE2, differing by only 16 of 492 amino acids, is present in rabbits(g). P450IIE1 is of considerable interest because of its role in the metabolic activation of a variety of chemicals which have important toxicological effects in h u m a d ) . In addition, it catalyzes the first step in a proposed glucogenic pathway for acetone(''). This has important implications for the presence of the enzyme in the brain, where ketones are extensively utilized("). Table 1 summarizes several representative substrates, the products formed, and the toxicity associated with substrate metabolism. The list is not Table 1. Representative chemicals metabolized by P45011EI Substrate
Benzene/Phenol N-Nitrosodimethy lamine Acetaminophen CC14* Ethanol
Phenol/Hydroquinone Methyl carbonium ion
Leukemia Liver tumours
Benzoquinone imine Trichloromethyl radical Acetaldehyde
Liver toxicity Liver toxicity Liver damage due to protein binding
*The metabolism of CC4 occurs via a reductive dehalogenation and has been shown to be catalyzed by P450IIEl('*)).
exhaustive and is intended to represent some of the various classes of chemicals which can be oxidized by the enzyme. As can be seen, the list includes components present in cigarette smoke (alkylnitrosamines and benzene), industrial solvents (CC4 and benzene), and a commonly used drug (acetaminophen).
Induction of P45011E1 P450IIE1 is readily inducible (induction being broadly defined, without mechanistic implications, as any increase in the cellular concentration of the enzyme) by the administration of a variety of structurally diverse chemicals as well as by changes in the hormonal and metabolic status of the animal(33698). The induction of this particular gene family is quite distinct from other P450 gene families. Considerable evidence has accumulated which indicates that several P450 genes are induced via transcriptional activation of the For example, the transcriptional activation of P450IA1 by aromatic hydrocarbons such as benzo(a)pyrene is well In contrast, the induction of P450IIE1 does not involve transcriptional activation of the gene except at birth. Depending on the inducer or treatment, it appears that the enzyme concentration can be regulated by a combination of mRNA stabilization, increased translation of existing mRNA, and inhibition of protein degradation. Table 2 summarizes various treatments and conditions and the general mechanism that has been implicated for the induction of the enzyme in each case. In general, all species respond in a similar manner to various treatments or hormonal changes although there are some unique differences. For example, rabbit P450IIE1 is extensively induced by imidazole treatment while induction of the rat enzyme has been reported to be refractory to this treatment(13’14). The reason for this difference is not
Table 2. Representative conditions or chemicals which induce P45OIIEl Chemical or Condition Birth Ethanol Pyridine Acetone Pyrazole Fasting Diabetes Hypophysectomy
Effect Observed transcriptional activation of gene no significant increase in mRNA, increased translatable mRNA no significant increase in mRNA, increase in translatable mRNA no significant increase in mRNA, increase largely due to stabilization of protein no significant increase in mRNA, increase largely due to stabilization of protein increase in mRNA (stabilization?), protein stability due to ketones? increase in mRNA due to mRNA stabilization, protein stability due to ketones? increase in mRNA (stabilization?)
understood. It is possible that there is polymorphism in human P450IIE1 expression and, if so, this could have important toxicological implication^('^). Clearly documented cases of ethanol exposure or drug treatment regimes which can induce P450IIE1 (i.e. isoniazid) have resulted in an increase in P450IIE1 in human liver sarnples(l6). Since expression of the enzyme can affect chemical metabolism and thus toxicity, it is important that a noninvasive method for estimating P450IIE1 in humans be established. This will require the use of a nontoxic drug, whereby the serum or urine concentration of a metabolite which specifically reflects the activity of P450IIE1 can be easily monitored. The metabolism of halogenated anesthetics such as enflurane or halothane may prove to be useful in this regard(17).
Regulation of P45011E1 at the Level of Transcription P450IIE1 was not detected in neonatal rat However, significant levels of P450IIE1 mRNA were detected within a few hours of birth. The increase in the mRNA was coincident with transcriptional activation of the P450IIE1 gene(”). The mRNA levels continued to increase and were maximal at 6 days after birth. The onset of transcription was accompanied by the demethylation of cytosine residues upstream from the transcription start site in the P450IIE1 gene one day after birth. Additional demeth lations were detected at 1 and 10 weeks after birth(’ ). As described in more detail below, exogenous chemicals induce P450IIE1 primarily by stabilization of existing protein. Since the enzyme was not detectable in the fetal liver, the transplacental transfer of many of the chemicals which induce P450IIE1 in adult liver may not be effective inducers in the fetus. However, one report has documented a small transplacental induction of immunodetectable P450IIE1 and its mRNA when the mother was treated with acetone during the last two days of pregnancy(”). While the increase was very small, the induced fetal form may potentiate the toxicity of other chemicals which may also be present. This could be significant in cases where the mother is smoking, drinking, and/or being exposed to other xenobiotics. In view of the importance of alcohol in teratogenesis, the induction of P450IIE1 by transplacental ethanol and other chemicals should be investigated. In an interesting preliminary report, Casazza et al. (20) found that pregnant rats exhibited lower acetone hydroxylase activity than nonpregnant controls and that there was a progressive loss of P450IIE1 during pregnancy. The mothers’ hepatic P450IIE1 was induced slightly by acetone, but the level never reached that of the untreated control. These results indicate that there may be some factor(s) present during pregnancy that limit the expression of P450IIE1. Whether the loss of
P450IIE1 during pregnancy is the result of increased degradation of protein or mRNA or of limited expression of the constitutive level of mRNA remains to be determined.
Regulation of P45011E1 by rnRNA Stabilization The transcriptional activation of the P450IIE1 gene at birth is in sharp contrast to the regulatory mechanisms employed in the adult, where mRNA and protein stabilization are the most important factors in the regulation of the enzyme level. The vast majority of chemical inducers of P450IIE1 lead to an increase in the level of P450IIE1 apoprotein as determined by immunoblot analysis with no or only a small increase in mRNA. However, there are certain treatments where P450IIE1 mRNA levels are significantly elevated compared to control animals. In almost all cases, elevated mRNA is observed in situations where hormonal changes are involved. The best documented case for mRNA stabilization occurs in alloxan or streptozotocin-induced diabetes(21). There have been numerous reports of the induction of P450IIE1 in chemically induced diabetes and in spontaneously diabetic rats(22-24).The induction of P450IIE1 by the diabetic state is variable; increases in the apoprotein as high as 6- to 8-fold were reported with a corresponding 10-fold increase in the mRNA levels(21).The increase in both the enzyme concentration and the mRNA levels is reversed by insulin administration. Donahue and Morgan(25) recently reported that the increase in P450IIE1 in streptozotocin-induced diabetic rats is also reversed by the administration of vanadate; the effect of vanadate on the mRNA levels was not reported. Initial suggestions were made that the induction of P450IIE1 by the diabetic state was due to an increase in the ketone body levels in the circulation(22). Similar suggestions were also made for the induction observed during fasting. However, fasting conditions do not give high enou h ketone levels to account for the induction observed(4i).
Growth hormone has been shown to regulate the concentration of a number of rat P450 Growth hormone levels are depressed in diabetic animals and this may have an important role in P450IIE1 induction. However, Thummel and Schenkman(24)found that while both male and female diabetic rats have elevated levels of P450IIE1, the diabetic state has no effect on the circulating growth hormone concentration in females. These investigators also reported that growth hormone administration to males failed to reverse the effect of diabetes(24).These results suggest that there is probably more than one mechanism important in the induction of P450IIE1 in diabetic rats. One involves the stabilization of the mRNA while others may operate through increased mRNA translation or a decrease in protein degradation.
Waxman et a1.(26) reported that P450IIE1 was a female predominant enzyme in the rat (1.5- to 2.0-fold greater in females than in males), and the difference was attributed to the male growth hormone secretion pattern which suppresses the expression of P45011E1(26).Hypophysectomy of adult rats resulted in increased P450IIE1 which was reversed by growth hormone. However, P450IIE1 mRNA levels were not reported and the difference could be due to increased mRNA levels in females, or the activation of proteolysis in males by growth hormone. In contrast, Yamazoe et u Z . ( ~ ~ )reported that in rats P450IIE1 was male predominant. The levels of P450IIE1 in untreated rats are low and these differences may reflect the error in determination of the enzyme at low levels. Thummel and S ~ h e n k m a n (reported ~~) that there was no significant difference between the constitutive level of P450IIE1 in male and female rats. Yamazoe et a1.(27) reported similar effects of hypophysectomy on P450IIE1 levels. Hypophysectomy increased the P450IIE1 level in both sexes and the increase was reversed by growth hormone. The change in the P450IIE1 level was paralleled by changes in the mRNA level. The effect of growth hormone was receptor mediated and appeared to have a direct effect on the liver(27). There is a large sex difference in the renal expression of P450IIE1 in mice(28). This difference has been related to sex-specific renal toxicities observed in this reported a 17-fold increase in species. Hong et both P450IIE1 mRNA and P450IIEl-dependent activities in the kidneys of female C3H/HeJ mice treated with testosterone. This increased the female P450IIE1 level to that of untreated males. Testosterone had no effect on the expression of P450IIE1 mRNA in the liver. The increase in the level of mRNA was not further evaluated and may be due to either increased transcriptional activity or mRNA stabilization. The effect of testosterone was recently confirmed by Henderson et These authors also established that the sex difference was not a reflection of growth hormone levels by using the mouse strain ‘little’. The homozygote (litllit) has only 5 1 0 % of the normal circulating levels of growth hormone. The administration of growth hormone did not alter the profile of P450 forms present in renal microsomes from the little strain or untreated mice. Castration feminized male kidneys, an effect which was reversed by testosterone treatment. P450IIE was not the only gene family affected by the androgen; all gene families detected in the kidney were regulated similarly(29).The testosterone-dependent renal P450IIE1 induction was attributed to hormone-responsive elements in the kidney. However, data demonstrating increased transcriptional activation of the gene was not reported and the increased mRNA levels may well represent increased P450IIE1 mRNA by stabilization. The effect of growth hormone on mouse liver P450IIE1 expression has not been reported.
Regulation of P45011E1 by Increased Translation of mRNA There have been only a few reports where the translation of P450IIE1 mRNA has been examined. Kubota et u Z . ( ~ ' ) isolated poly A+ RNA from ethanoland pyrazole-treated hamsters. They found that there was an increase in translatable P450IIE1 mRNA with both RNA preparations, although not to the same extent with each inducer. Similar re ulation of P450IIE1 was reported by Kim and Novak( 3, for pyridine induction of the enzyme. The administration of a single intraperitoneal dose of pyridine induced the level of P450IIE1 about 4-fold after 24 h; this induction was completely blocked by the administration of cycloheximide. Actinomycin D was without effect. However, as a result of the difficulty in interpreting the effect of cycloheximide in studies on whole animals, these results need to be confirmed in more rigorously controlled conditions. Northern blot analysis showed no increase in the mRNA for the enzyme. Synthesis of new P450IIE1 was inferred from an increase in incorporation of radioactive leucine into a protein with a molecular weight corresponding to P450IIE1 as determined by SDS gels of microsomal samples. This indirect comparison does not rigorously demonstrate incorporation into P450IIE1 and conclusions from such experiments should be viewed with caution. Immunoisolation will be required to confirm the incorporation of radioactivity into the enzyme. In contrast, Song et u Z . ( ~ ~ reported ) that when rats were pretreated with acetone for 10 days or untreated prior to pulse labeling, there was no change in the rate of P450IIE1 synthesis. In these studies, P450IIE1 was immunoisolated and the radiolabel in P450IIE1 was directly determined. The specific radioactivity of P45011E1 from acetone-treated rats was lower than that obtained from untreated animals. The difference was consistent with about 4-fold greater concentration of P450IIE1 in the acetone-treated rats(34). However, immunoquantitation for the level of total P450IIE1 was not presented for the samples which were used for immunoisolation.
Regulation of P45011E1 by Inhibition of Protein Degradation As previously indicated, P450IIE1 is induced by treatment of animals with a variety of structurally diverse compounds, including ethanol, acetone, propanol, imidazole, pyrazole, trichloroethylene, isoniazid, 4-methylpyrazole, and benzene(138313). The enzyme is unequally distributed across the hepatic acinus, with the highest concentration in zone 3 near the terminal hepatic vein(33734).After induction, the form distribution is the same with higher concentrations in each region. For all chemical inducers examined in adult animals, there is no significant increase in the hepatic concentration of P450IIE1 mRNA(35). These results, with the exceptions noted above in hormonal imbal-
ance, have led to the suggestion that P450ICE1 is induced primarily by post-transcriptional mechanisms. The chemicals do not cause a generalized depression of transcriptional activity or an increase in mRNA degradation since acetone also induced P450IIB 1 mRNA in the rat, although not to the same extent as is seen with the well characterized inducer of this form, phenobarbital(36), Support for a mechanism involving a decrease in protein degradation as a result of protein stabilization is provided by a number of investigations. Early experiments suggested that P450IIE1 had a very rapid turnover; induced activities returned to control levels within 12 to 24 h after termination of t r e a t m e ~ ~ t ( ~ ~ - ~ ~ ) . Song et u Z . ( ~ ~ )directly measured the degradation of P450IIE1 in untreated and acetone-treated rats by pulse labeling with Hi4C03. They reported a biphasic degradation of P450IIE1 in untreated rats, with estimated half-lives of about 7 and 37h. From the limited data presented, it is not possible to determine the proportion of the total P45011E1 that is degraded in each phase, but the data su6qst that there may be two populations of P450IIE1 . Whether the kinetic expression of two populations is due to a heterogeneous distribution within the endoplasmic reticulum, the presence of both apo- and holoenzyme, or possibly the unequal distribution across the liver acinus where degradation may occur at different rates remains to be determined. However, in the presence of acetone, which was continuously administered to the rats before, during, and after the pulse of H14C03, there was no rapid phase of degradation. Only a slow phase of degradation was observed with a rate about the same as the slow phase observed with untreated rats, suggesting that the compound stabilized the protein and inhibited de radation. In an interesting preliminary report, Ito et u Z . ~ ~ ' ) , reported a monophasic loss of P450IIE1 pulse labelled with [35S]rnethionine in both untreated and ethanol-treated rats with no change in the apparent half-life of the protein, suggesting that ethanol induction may not involve decreased degradation of P450IIE1. Additional support for the stabilization of P450IIE1 was provided by studies of Eliasson et a1.(41)investigating the effect of ligands on the loss of preinduced enzyme when hepatocytes were placed in primary culture, a system which does not express P450 forms identically to the intact liver(42). In the absence of additional ligands, the induced P450IIE1 was decreased by about 70 % in 24 h and was completely lost in 72 h. The level of mRNA for P450IIE1 was negligible after 24 h in culture. In contrast, when the cells were cultured in the presence of various ligands for the enzyme, including ethanol, imidazole, 2-propanol, and dimethylsulfoxide, the loss of enzyme was not as great; after three days in culture, about 50% of the original P450IIE1 that was present at the time of plating still remained. The level of mRNA was still reduced to near zero after one day in culture. The amount of enzyme
that was protected from degradation was dependent on the extent of induction and was variable. About 150pmol P450IIE1 per mg microsomal protein was not degraded in hepatocytes from ethanol-treated rats while 61pmol P450IIE1 per mg protein was not degraded in hepatocytes from 2-propanol-treated rats. In all cases about 50 % of the induced enzyme remained after 3 days. It should be emphasized that the enzyme that was measured was determined immunochemically and represents the total cytochrome present, both apo and holoenzyme. It has been shown that there is a discrepancy between the immunochemically determined enzyme and the catalytically competent en~ y m e ( ~ ~ It’ ~would ~ ’ ~ be ~ ) of . interest to determine whether the difference observed in hepatocytes in culture was due to differences between the holoenzyme and apoenzyme present after induction. For each ligand tested, the effectiveness of protection was related to the spectral binding constant of the ligand for the purified enzyme, which suggests that ligation to the enzyme was important for stabili~atiod~l). Imidazole was a very effective protective agent although others have reported that the in vivo administration of imidazole is not an effective inducer in the rat(14).Eliasson et al. (41) reported that imidazole induced P450IIE1 about 2-fold, which is in sharp contrast to other reports(14). The ability of imidazole to protect P450IIE1 in hepatocyte cultures suggests that the ineffectiveness of the ligand in vivo may represent a pharmacokinetic problem. A question remains as to how the ligands protect the enzyme from degradation. Very little is known about the mechanism of degradation of P450 in the endoplasmic reticulum membrane(43). The presence of ligand may maintain the enzyme in a stable conformation which prevents a degradative pathway, illustrated schematically in Fig. 1. This stabilization may prevent the labilization of an intermediate conformation of the enzyme. The mechanism of labilization is not established but may involve heme oxidation and/or protein modification, as has been suggested for rat P 4 5 0 ~ ( ~or~ ) , direct oxidation of critical amino acid residues by H202 or other species produced by the uncoupled oxidation of NADPH(43).Recognition of the labilized form of the enzyme may proceed by many mechanism^(^^-^*). These include but are not limited to phosphorylation, ubiquitination and subsequent degradation by the ubiquitin-dependent proteosome in the cytosol, exposure of a PEST sequence which is present in the heme-binding region in most P450 forms sequenced, or specific degradation by other roteases in the endoplasmic reticulum or cytos01(~ 48). In all cases, the labilization of the enzyme may not be reversible and may result in a commitment to the degradative pathways. In order to begin to understand ligand-dependent stabilization, the mechanism of selective degradation of P450IIE1 must be understood. After a single dose of ethanol, maximal catalytic and immunochemical detectable P450IIE1 was present about 24h after the
Ligand/substrate binding (acetone and pyrazole) P45011E 1 intermediate conformation
, Uncoupled NADPH oxidation? Protein/heme modification?
P45011E 1 labilized conformation
P45011E 1 degradation
Lysosomal autophagocytosis ? Microsomal protease? Cytosolic protease?
Fig. 1. Hypothetical scheme for the degradation of cytochrome P450IIEl. The stabilized conformation is obtained in the presence of substrates and/or ligands. The intermediate conformation represents the unligated enzyme which partitions between the stabilized and labilized conformation. The labilized conformation is targeted for degradation.
dose(3634).However, while there was a rapid loss of P450IIEl-dependent p-nitrophenol hydroxylase activity (it returned to control levels within 24 h), the immunochemical detectable enzyme was still elevated 96 h after the initial dose(44).These results suggest that the degradation of the apoprotein does not immediately follow the loss of catalytic activity which is assumed to represent inactivation of P450IIE1. Other factors in addition to the loss of catalytic activity are important for the eventual degradation of the enzyme. Ronis and Ingelman-Sundberg(u) detected both P450IIB1 and P450IIE1 in the lysosomal compartment in the presence of leupeptin and thus suggested that the enzyme was degraded by an autophagosomal/autolysosomal pathway. The loss of P450IIB1 did not parallel the loss of P450IIE1 and NADPH cytochrome P450 reductase was not present in the lysosomal compartment. This suggests that a mechanism such as microa~tophagy(~~) exists to selectively target a portion of the endoplasmic reticulum containing P450IIE1 for lysosomal degradation. Eliasson et aZ.(49)recently reported that in primary hepatocyte cultures glucagon or 8-bromoadenosine 3’,5’-cyclic monophosphate mediated the phosphorylation of P450IIE1, and that the phosphorylation was correlated with an enhanced degradation of the 30 % of the P450IIE1 that was not normally lost when the cells were placed in culture. When the hepatocytes were cultured in the presence of ligand, phosphorylation was
inhibited as was loss of the enzyme, leading to the suggestion that phosphorylation targeted the enzyme for degradation. However, the significance of this increase in the rate of degradation after two days in culture when about 70% of the P450IIE1 is already degraded by undefined mechanisms remains to be established. Parker et al. (50) reported that the in vivo phosphorylation of rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was correlated with the loss of the 97-kDa 32P band from microsomes. This loss was decreased by inhibitors of lysosomal function. In addition, an in vitro system with the calcium-dependent protease calpain-2 produced greater levels of hydrolysis of the 97-kDa protein when it was pho~phorylated(~~). If phosphorylation does enhance degradation of P450IIE1 in a similar manner, then during fasting when glucagon and CAMP levels are increased, it would be expected that a rapid loss of enzyme should occur in the liver before enough substrate (i.e. ketone body) is formed to protect the enzyme. Many forms of P450 are phosphorylated by both cyclic AMP-dependent protein kinases and protein kinase C(51). It remains to be established whether phosphorylation is selective for P450IIE1 and, if so, how this occurs. The regulation of the expression of P450IIE1 is extremely complex and involves both transcriptional and post-transcriptional mechanisms. In adult animals and presumably in man, the control of degradation of the enzyme is one of the principal components in the regulatory pathway and remains one of the least understood. Acknowledgements The work performed in the authors' laboratory was supported by the National Institute on Alcohol Abuse and Alcoholism (AA07219). Due to space limitations, it was necessary to limit the citations for this review. We have made every effort to refer to the most recent publications in the area. Readers are referred to the extensive recent reviews in the area for more complete citations('-6). References 1 GUENGERICH, F. P. (1990). Enzymatic oxidation of xenobiotic chemicals. CRC Crir. Rev. Biochem. Mol. Biol. 25, 97-153. 2 RYAN,D. E. AND LEVIN, W. (1990). Purification and characterization of
hepatic microsomal cytochrome P-450. Pharmac. Ther. 45, 153-239. 3 GONZALEZ, F. J. (1989). The molecular biology of cytochrome P450s. Pharmacol. Rev. 40, 243-288. 4 GONZALEZ, F. J . AND NEBERT,D . W. (1990). Evolution of the P450-gene superfamily - Animal plant warfare, molecular drive and human genetic differences in drug oxidation. Trends Gener. 6, 182-186. 5 GONZALEZ, F. J. (1990). Molecular genetics of the P-450 superfamily. Pharm. Ther. 45, 1-38. 6 OKEY,A. B. (1990). Enzyme induction in the cytochrome P-450 system. Pharm. Ther. 45, 241-298. 7 KOOP, D. R., MORGAN,E. T., TARR,G. E. AND COON,M. J . (1982). Purification and characterization of a unique isozyme of cytochrome P-450 from liver microsomes of ethanol-treated rabbits. J. Biol. Chem. 257, 8472-8480. 8 KOOP,D. R. AND COON,M. J. (1986). Ethanol oxidation and toxicity: Role of alcohol P-450 oxygenase. Alcohol. Clin. Exp. Res. 10, 44-49s. 9 KHANI, s. c . , PORTER, T. D., FUJITA, V. s. AND COON, M. J . (1988).
Organization and differential expression of two highly similar genes in the rabbit alcohol-inducible cytochrome P-450 subfamily. J . Biol. Chem. 263, 7170-7175. 10 KOOP, D. R . AND CASAZZA, J. P. (1985). Identification of ethanol inducible P-450 isozyme 3a as the acetone and acetol monooxygenase of rabbit microsomes. J . Biol. Chem. 260, 13607-13612, 11 HANSSON, T., TINDBERG, N., INGELMAN-SUNDBERG, M. AND KOHLER, C. (1990). Regional distribution of ethanol-inducible cytochrome P450 IIEl in the rat central nervous system. Neuroscience 34, 451-463. 12 JOHANSSON, I . AND INGELMAN-SUNDBERG, M. (1985). Carbon tetrdchlorideinduced lipid peroxidation dependent o n an ethanol-inducible form of rabbit liver microsomal cytochrome P-450. FEBS Lett. 183, 265-269. 13 KOOP,D. R., CRUMP, B. L., NORDBLOM, G . D. AND COON,M. J. (1985). Immunochemical evidence for induction of the alcohol-oxidizing cytochrome P450 of rabbit liver microsomes by diverse agents: ethanol, imidazole, trichloroethylene, acetone, pyrazole, and isoniazid. Proc. Nut1 Acad. Sci. USA 82, 4065-4069. 14 SONG,B.-J., GELBOIN, H. V., PARK,S . - S . , YANG,C. S. AND GONZALEZ, F. J. (1986). Complementary DNA and protein sequences of ethanol-inducible rat and human cytochrome P-450s: Transcriptional and post-transcriptional regulation of the rat enzyme. J . B i d . Chem. 261, 16689-16697. 15 PERROT,N., NALPAS,B., YANG, C. S . AND BEAUNE,P. H. (1989). Modulation of cytochrome P450 isozymes in human liver by ethanol and drug intake. Eur. J. Clin. Invesrig. 19, 549-555. 16 WRICHTON, S. A,, THOMAS, P. E., MOLOWA, D. T., HANIU,M., SHIVELY, J. E . , MAINES, S . L., WATKINS, P. B., PARKER, G., MENDEZ-PICON, G., LEVIN, W. AND GUZELIAN, P. S . (1986). Characterization of ethanol-inducible human liver N-nitrosodimethylamine demethylase. Biochemistry 25, 6731-6735. 17 HOFFMAN, J., KONOPKA, K., BUCKHORN, c . , KOOP, D. R. AND WASKELL, L. (1989). Ethanol-inducible cytochrome P450 in rabbits metabolizes enflurane. Br. J . Anaesth. 63, 103-108. 18 UMENO, M., SONG, B. J., KOZAK,c . , GELBOIN, H. V. AND GONZALEZ, F. J. (1988). The rat P450IIE1 gene: Complete intron and exon sequence, chromosome mapping, and correlation of developmental expression with specific 5' cytosine demethylation. J . Biol. Chem. 263, 4956-4962. 19 HONG,J., PAN, J., DONG,Z . , NING,S . M. AND YANC,C. S . (1987). Regulation of N-nitrosodimethylamine demethylase in rat liver and kidney. Cancer Res. 47, 5948-5953. 20 CASAZZA, J. P., YUN,Y. P., VEECH,R. L. AND SONG,B.-J. (1990). Serum acetone and P450IIE1 levels in pregnant rats. FASEB J . 4, A2244. 21 SONG,B.-J., MATSUNAGA, T . , HARDWICK, J. P., PARK,S . S . , VEECH.R. L., YANG,C. S . , GELBOIN, H. V. AND GONZALEZ, F. J. (1987). Stabilization of cytochrome P450j messenger ribonucleic acid in the diabetic rat. Mol. Endocrinol. 1, 542-547. 22 DONG,Z., HONG,J . , MA, Q., LI, D., BULLOCK, J., GONZALEZ, F. J., PARK, S. S . , GELBOIN, H. V. A N D YANG,C. S . (1988). Mechanism of induction of cytochrome P-450ac (P-45Oj) in chemically induced and spontaneously diabetic rats. Arch. Biochem. Biophys. 263, 29-35. 23 BELLWARD, G. D., CHANG,T., RODRIGUES, B., MCNEILL,J. H., MAINES, S . , RYAN,D. E., LEVIN,W. AND THOMAS, P. E. (1988). Hepatic cytochrome P450j induction in the spontaneously diabetic BB rat. Mol. Pharmacol. 33, 140-143. 24 THUMMEI., K. E. AND SCHENKMAN, J . B. (1990). Effects of testosterone and growth hormone treatment on hepatic microsomal P450 expression in the diabetic rat. Mol. Pharmacol. 37, 119-129. 25 DONAHUE, B. S. AND MORGAN,E. T. (1990). Effects of vanadate on expression of hepatic cytochrome P450 in streptozotocin-diabetic expression rats. Drug Metab. Dispos. 18, 519-526. 26 WAXMAN, D. J . , MORRISSEY, J. J. AND LEBLANC, G. A. (1989). Femalepredominant rat hepatic P-450 forms j (IIE1) and 3 (IIA1) are under hormonal regulatory controls distinct from those of the sex-specific P-450 forms. Endocrinology 124, 2954-2966. 27 YAMAZOE, Y., MURAYAMA, N., SHIMADA, M., IMAOKA,S., FUNAE, Y. A N D KATO,R . (1989). Suppression of hepatic levels of an ethanol-inducible P450DM/j by growth hormone: Relationship between the increased level of P450DM/j and depletion of growth hormone in diabetes. Mol. Pharmacol. 36, 716-722. 28 HONG,J.-Y., PAN,J., NING,S . M. AND YANG,C. S . (1989). Molecular basis for the sex-related difference in renal N-nitrosodimethylamine demethylase in C3H/HeJ mice. Cancer Res. 49, 2973-2979. 29 HENDERSON, C. J., SCOTT,A. R., YANG,C. S . AND WOLF,R. C. (1990). Testosterone-mediated regulation of mouse renal cytochrome P-450 isoenzymes. Biochem. J. 266, 675-681. 30 KUBOTA, S., LASKER, J. M. AND LIEBER, C. S. (1988). Molecular regulation of ethanol-inducible cytochrome P450-IIEI in hamsters. Biochem. Biophys. Res. Commun. 150, 304-310. 31 KIM, S . G. AND NOVAK, R. F. (1990). Induction of rat hepatic P450IIE1 (CUP 2E1) by pyridine: Evidence for a role of protein synthesis in the absence of transcriptional activation. Biochem. Biophys. Res. Commun. 166, 1072- 1079. 32 SONG,B.-J., VEECH,R. L., PARK,S . S . , GELBOIN, H. V. AND GONZALEZ, F.
J. (1989). Induction of rat hepatic N-nitrosodimethylamine demethylase by acetone is due to protein stabilization. J. Biol. Chem. 264, 3568-3572. 33 INGELMAN-SUNDBERG, M., JOHANSSON, I . , PENTTILA, K. E., GLAUMANN, H. AND LINDROS, K. 0. (1988). Centrilobular expression of ethanol-inducible cytochrome P-450 (IIE1) in rat liver. Biochem. Biophys. Res. Commun. 157, 55-60. 34 TSUTSUMI, M., LASKER, J. M., SHIMIZU, M., ROSMAN, A. S. AND LIEBER. C. S. (1989). The intralobular distribution of ethanol-inducible P450IIE1 in rat and human liver. Hepatology 10, 437-446. 35 KHANI,S. C., ZAPHIROPOULOS, P. G., FUJITA,V. S., PORTER, T. D., KOOP, D. R. AND COON,M. J. (1987). cDNA and derived amino acid sequence of the ethanol-inducible rabbit liver cytochrome P-450 isozyme 3a (P-450ALC). Proc. Natl Acad. Sci. USA 84, 638-642. 36 JOHANSSON, I . , EKSTROM, G., SCHOLTE, B., PUZYCKI, D . , JORNVALL, H. AND INGELMAN-SUNDBERG, M. (1988). Ethanol-, fasting, and acetone-inducible cytochromes P-450 in rat liver: Regulation and characteristics of enzymes belonging to the IIB and IIE gene subfamilies. Biochemistry 27, 192551934, 37 MORGAN, E . T . , DEVINE,M. AND SKEIT, P. (1981). Changes in the rat hepatic mixed function oxidase system associated with chronic ethanol vapor inhalation. Biochem. Pharmacol. 30, 595-600. 38 PANTUCK, E. J., PANTUCK, C. B.,RYAN,D. E. AND CONNEY, A . H. (1985). Inhibition and stimulation of enflurane metabolism in the rat following a single dose or chronic administration of ethanol. Anesthesiology 62, 255-262. 39 HETU, C. AND JOLY,J.-G. (1985). Differences in the duration of the enhancement of liver mixed-function oxidase activities in ethanol-fed rats after withdrawal. Biochem. Pharmacol. 34, 1211-1216. 40 ITO, Y., TSUTSUMI, M., LIEBER,C. S. AND LASKER, J. M. (1990). Induction of rat liver P450IIE1 by ethanol involves increased enzyme synthesis. FASEB J. 4, A1972. 41 ELIASSON, E., JOHANSSON, I. AND INGELMAN-SUNDBERG, M. (1988). Liganddependent maintenance of ethanol-inducible cytochrome P-450 in primary hepatocyte cell cultures. Biochem. Biophys. Res. Commun. 150, 436-443. 42 SCHUETZ, E. G., LI, D., OMIECINSKI, C. J., MULLER-EBERHARD, U.. KLEINMAN, H. K . , ELSWICK, B. AND GUZELIAN, P. S . (1988). Regulation of gene
exoression in adult rat heoatocvtes cultured on a basement membrane matrix. J. Ctk. Physiol. 134, 30!-j23. ’ 43 WATKINS, P. B., BOND,J. S. AND GUZELIAN, P. S . (1987). Degradation of the hepatic cytochromes P-450. In Mammalian Cytochromes P-450, Volume IZ. (ed. F . P. Guengerich) pp. 173-193, CRC Press, Inc. Boca Raton, FL. 44 RONIS,M. J. J. AND INGELMAN-SUNDBERG, M. (1989). Acetone-dependent regulation of cytochrome P-45Oj (IIE1) and P-450b (1181) in rat livcr. Xenobiorica 19, 1161-1165. 45 CORREIA, M. A,, SUGIYAMA, K. AND YAO,K. (1989). Degradation of rat hepatic cytochrome P450p. Drug Metab. Rev. 20, 615-628. 46 RIVEIT,A. J. (1986). Regulation of intracellular protein turnover: Covalent modification as a mechanism of marking proteins for degradation. Current Topics Cell. Reg. 28, 291-337. 47 RECHSTEINER, M. (1987). Ubiquitin-mediated pathways for intracellular proteolysis. Annu. Rev. Cell Biol. 3, 1-30. 48 HARE,J. F. (1990). Mechanisms of membrane protein turnover. Biochem. Biophys. Acra 1031, 71-90. 49 ELIASSON,E., JOHANSSON, I. AND INGELMAN-SUNOBERG, M. (1990). Substrate-, hormone-, and CAMP-regulated cytochrome P450 degradation. Proc. Natl Acad. Sci. U S A 87, 322553229, 50 PARKER, R. A., MILLER,S. J . AND GIBSON,D. M. (1989). Phosphorylation of native 97-kDA 3-hydroxy-3-methylglutaryl-coenzymeA reductase from rat liver. Impact on activity and degradation of the enzyme. J. Biol. Chem. 264, 4877-4887. 51 EPSTEIN, P. M., CURTI,M., JANSSON, I., HUANG, C.-K. AND SCHENKMAN, J. B. (1989). Phosphorylation of cytochrome P450: Regulation by cytochrome b5. Arch. Biochem. Biophys. 271, 424-432.
Dennis R. KoopP and Daniel J. Tierney are at the Departments of Environmental Health Sciences and Pharmacology, Case Western Reserve University, School of Medicine, Cleveland, OH 44106, USA. ?To whom correspondence should be sent.
THE 1991 MIAMI BIO/TECHNOLOGY WINTER SYMPOSIA January 27-February 1, 1991 Miami Beach, Florida, USA “Advances in Gene Technology: The Molecular Biology of Human Genetic Diseases” Organized by
The University Biochemistry & Molecular Biology Foundation, Inc. and Nature Publishing C o m p a n y Topics Genetic disease genes, Mitochondria1 genes and human genetic disease, DNA sequencing technology, Physical mapping of human DNA, Some specific results from human genetic mapping, Methods for DNA transfer, Cancer related genes, New uses for viral vectors in human genetics, Diagnostic technology for human genetic diseases, Progress toward gene therapy. If you wish to receive a program, registration forms and poster session information, please write to: The Miami Bio/Technology Winter Symposia, P.O. Box 016129, Miami, FL 33101-6129, USA or call toll free, 1-800-MIA-GENE (1-800-642-4363) or fax us at 1-305-324-5665.