Abstract
Cytochrome P450 (P450) enzymes are involved in the metabolism of carcinogens, as well as drugs, steroids, vitamins, and other classes of chemicals. P450s also oxidize ethanol, in particular P450 2E1. P450 2E1 oxidizes ethanol to acetaldehyde and then to acetic acid, roles also played by alcohol and aldehyde dehydrogenases. The role of P450 2E1 in cancer is complex in that P450 2E1 is also induced by ethanol, P450 2E1 is involved in the bioactivation and detoxication of a number of chemical carcinogens, and ethanol is an inhibitor of P450 2E1. Contrary to some literature, P450 2E1 expression and induction itself does not cause global oxidative stress in vivo, as demonstrated in studies using isoniazid treatment and gene deletion studies with rats and mice. However, a major fraction of P450 2E1 is localized in liver mitochondria instead of the endoplasmic reticulum, and studies with site-directed rat P450 2E1 mutants and natural human P450 2E1 N-terminal variants have shown that P450 2E1 localized in mitochondria is catalytically active and more proficient in producing reactive oxygen species and damage. The role of the mitochondrial oxidative stress in ethanol toxicity is still under investigation, as is the mechanism of altered electron transport to P450s that localize inside mitochondria instead of their typical endoplasmic reticulum environment.
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Keywords
- Ethanol
- Oxidation by cytochrome P450, in ethanol oxidation
- Mitochondria, and cytochrome P450
- Endoplasmic reticulum, and cytochrome P450
- Cytochrome P450 2E1
- P450 2E1
- Enzyme kinetics
- Reactive oxygen species
- Mitochondrial toxicity
- Chemical carcinogens
2.1 Introduction to Cytochrome P450 (P450)
The field of P450 research stemmed from work in the areas of the metabolism of steroids, drugs, and carcinogens [18, 29]. The human genome contains 57 P450 (CYP) genes, of which almost all are expressed in some tissue [29]. The P450s are classified into families and subfamilies on the basis of sequence similarity [52]. However, sequence similarity may or may not be a guide to function with P450s [23, 29].
Another way to classify P450s is by the types of substrates they use (Table 2.1) [29]. Even this approach has caveats in that some P450s could be placed in multiple categories in Table 2.1 (e.g., P450s 1B1 and 27A1) [29]. Another point to make is that several human P450s have yet to have functions identified, i.e. those in the column labeled “unknown.” These are often termed the “orphans [30]. However, even when P450s are classified in the “xenobiotics” category and do happen to be able to oxidize a substrate found in the body (e.g. P450 3A4), it is not clear that this reaction has a significant physiological role [29]. In general, the mouse orthologues of this column of P450s can be deleted without profound physiological effects [26].
Collectively the P450s catalyze about 75% of all reactions involved in the metabolism of drugs [81] (some drugs are not readily metabolized, however, and biologicals are not included in this analysis). In the previous analyses [80, 81], the 3A and 2C Subfamily P450s were those most involved in drug metabolism (i.e., 3A4, 2C9, 2C19). This pattern still holds for P450s [64]. Further, when one considers oxidation-reduction reactions with all chemicals (for the human enzymes), P450 catalyze 95% of the reactions. Again, the 3A and 2C Subfamily P450s are most prominent [64].
The localization of individual P450s has been reviewed elsewhere [29]. Those P450s involved in critical physiological functions are often found only in a few relevant tissues, e.g. many of the steroidogenic P450s are found only in the adrenals, gonads, etc. Those involved in bile acid processing are generally localized in the liver (e.g. P450 7A1). In general, the xenobiotic-processing P450s are localized principally in the liver, and some are never found outside of the liver, e.g. 1A2, 7A1 [29]. However, some of these are also found at lower levels in many other tissues. Some caution should be used in reviewing the literature, however, in that much of the work is at the mRNA level, not protein.
One of the reasons for the interest in the P450s is their role in the metabolism of toxicants and carcinogens. In general, this process of oxidation (and sometimes reduction) aids in the removal of chemicals from the body and may, along with transporters, be viewed as a mechanism for the body to use a limited set of genes to eliminate natural products that could prove harmful if allowed to accumulate in cells (e.g. alkaloids, terpenes, flavonoids). In general, the oxidation and reduction reactions catalyzed by P450s detoxicate chemicals. However, these same reactions also sometimes create electrophilic species that can react with cellular macromolecules and initiate biological damage, general with DNA or proteins. DNA damage can be misreplicated and fits into the somatic mutation theory of cancer [10]. The relevance of metabolism to chemical carcinogenesis was established largely by the late James and Elizabeth Miller in animal studies [50, 51]. This same concept applies to the toxicity of drugs, with protein damage generally being considered an initial event [24, 37].
Almost all of the information on the roles of human P450s in the metabolism of carcinogens has been published in the past 26 years [72, 73]. One classification indicates that (human) P450s are involved in ~ 2/3 of the reactions in which chemical carcinogens are bioactivated (Fig. 2.1a) [63]. Six human P450s recount for ~ ¾ of these reactions, i.e. P450s 1A1, 1A2, 1B1, 2A6, 2E1, and 3A4 (Fig. 2.1b) [63]. However, P450s are also involved in detoxication of chemical carcinogens [63]. The complexity of these systems can be seen in the classic 1952 Richardson experiment, in which treatment of rats with one carcinogen induced P450s that detoxicate another carcinogen and then had a protective role [65]. We now know that a single P450 catalyze both activation and detoxication of a single carcinogenic chemical, e.g. P450 3A4 oxidizes the hepatocarcinogen aflatoxin B1 to the 8,9-exo epoxide (which reacts efficiently with DNA [36]) and to aflatoxin Q1 (3α-hydroxylation product), which does not appear to be a carcinogen [77]. This dichotomy appears to be due to the presence of multiple binding modes of the substrate (aflatoxin B1 ) in the enzyme active site.
2.2 P450 2E1
The discovery of P450 2E1 followed largely from work on a microsomal ethanol oxidizing system [47, 57]. P450 2E1 was characterized in rabbit [43] and rat liver [68]. The enzyme is found at highest abundance in liver but also in numerous other tissues of relevance to cancer [25] (Table 2.2).
P450 2E1 is also induced by ethanol [47]. The study of this process has been complicated, in that ethanol also inhibits the enzyme (as expected for a substrate) [82]. Regulation of the CYP2E1 gene is complex and includes both transcriptional and post-transcriptional aspects [25].
P450 2E1 substrates include ethanol, a number of drugs (especially anesthetics), N-alkylnitrosamines , vinyl monomers, and halogenated hydrocarbons (Table 2.3) [29, 31]. Several of these are of interest in that a number of the nitrosamines, vinyl monomers, and halogenated hydrocarbons are carcinogens [31]. Nitrosamines are present in tobacco, and the vinyl monomers and halogenated hydrocarbons are used on a large scale in industry. These substrates have the general property of small molecular size, consistent with the crystal structure of P450 2E1 [60, 61]. However, it should be pointed out that even fatty acids can be substrates [61]. Because of the induction and inhibition of P450 2E1 by ethanol, the multiples sites of expression of the enzyme (Table 2.2), and the relevance to humans drinking ethanol and exposed to carcinogens in Table 2.2, the co-carcinogenicity of ethanol has been considered in a number of studies with experimental animals [15, 48]. (Co-carcinogenesis studies involve simultaneous administration of chemicals, as opposed to initiation-promotion regimens, with distinct temporal relationships and mechanisms [58]).
2.3 Ethanol Oxidation by P450 2E1
A general view in the alcohol field has been that alcohol dehydrogenase is the most relevant enzyme at low concentrations of ethanol and that P450 (2E1) is more involved at higher concentrations, i.e. those that would be relevant with high consumption. The situation is also complicated in that catalase can also contribute to ethanol metabolism [14, 76].
Even though ethanol has only two carbons and one oxygen, its oxidation has several complexities (Fig. 2.2). The general view in the field is that there are no receptors as such for ethanol, and essentially all of its biological effects are related to acetaldehyde. In aqueous solution, acetaldehyde exists in a roughly equimolar equilibrium with its hydrate, a “gem diol” (Fig. 2.2a) [27, 28].
We were interested in kinetic hydrogen isotope effects (KIEs) for ethanol oxidation because of some reports on KIEs for nitrosamine oxidation by liver microsomes [41, 49]. Work with recombinant human P450 2E1 showed, not unexpectedly, that α-deuterated ethanol showed a 5-fold KIE, expressed in the Km but not kcat (Fig. 2.3) [11]. This was shown to be the result of “burst” kinetics, i.e. a rate-determining step following product formation [11, 32]. The kinetic analysis is not novel [79], and Km is a constant that obviously has nothing to do with Kd here (a general axiom in enzymology) [35, 56]. Km even contains kcat as a variable in this case [32]. That is, for the simplified expression
where S is the substrate and P the product . Then
with Km having units of molarity. k3 must be considerably larger than k5, the rate of a step following product formation [44]. The original expressions for Vmax and Km are then reduced to
\( {\mathrm{V}}_{\mathrm{max}}\cong {\mathrm{k}}_5{\left[\mathrm{E}\right]}_{\mathrm{T}} \)
Km even contains kcat as a variable in this case [32].
We also showed that (human) P450 2E1 can oxidize acetaldehyde to acetic acid [12], in support of reports by others with the rat enzyme [45, 75] (Figs. 2.2, 2.3b, c). This oxidation also displays burst kinetics [12] and a KIE in the Km parameter. Pulse-chase experiments showed that the conversion of radiolabeled ethanol into acetic acid was not attenuated by the addition of unlabeled acetaldehyde. However, the results cannot be explained by a high affinity of P450 2E1 for acetaldehyde. A scheme involving a slow conformational step after the formation of acetaldehyde (and acetic acid) has been proposed [12]. Some similar results were found in the oxidation of N,N-dimethyl- and N,N-diethyl nitrosamine by P450 2E1 [17].
2.4 Source of Oxidative Stress Due to P450 2E1 in Tissues
A role for P450 2E1 in generation of reactive oxygen species (ROS) is often assumed in much of the literature in the field. Much of this view is based on early studies with liver microsomes [22, 62] and studies in cell culture [16]. The ROS field is complicated in that the vast majority of the work is in vitro and many of the methods of detecting ROS are not validated, e.g. see [40]. In the field, a “gold standard” is F2-isoprostane production, which can be measured both in vitro and in vivo [38, 39].
Treatment of rats with typical P450 enzyme inducers did not produce enhanced levels of liver isoprostanes, except in the case of barbiturate-type induction [20]. In particular, treatment of rats or mice with isoniazid, an established inducer of P450 2E1 [68], did not increase isoprostane levels [20, 21]. Further, Cyp2e1−/− mice had very similar levels of liver, kidney, brain, and urinary isoprostanes [21]. Thus, P450 2E1 does not appear to increase global levels of ROS. Increased levels of ROS related to treatment of animals with ethanol are not linked only to induction of P450 2E1. Further, although the utilization of reducing equivalents (NADPH) by purified P450 2E1 is low, it is not inferior to several other (human) P450s we have worked with [74, 84].
As has been suggested in the literature [42, 83], ROS production in the presence of ethanol is probably not due to P450 2E1 induction per se but possibly to the generation (and reaction) of CH3CHO· radicals.
The ROS increases following treatment of rats and mice with barbiturates have been attributed to induction of nicotinamide N-methyl transferase and depletion of pyridine nucleotides to support ROS destruction [21].
2.5 Roles of Mitochondrial P450s
Of the 57 human P450s (Table 2.1), seven are bona fide mitochondrial enzymes (11A1, 11B1, 11B2, 24A1, 27A1, 27B1, 27C1). These P450s have distinct roles in the metabolism of sterols and fat-soluble vitamins and they utilize electrons supplied by NADPH-adrenodoxin reductase (ADR) and the iron-sulfur protein adrenodoxin (Adx) [29].
The other 50 P450 proteins, when expressed, are targeted to the endoplasmic reticulum (ER). However, fractions of these P450s can, in some cases, be found in mitochondria. This was first shown in rats, where P450 2B1 could be found in mitochondria and catalyze the oxidation of aflatoxin B1 [53]. Further studies established this phenomenon, i.e. partial localization in mitochondria and catalytic activity there [55, 71]. Studies from several groups showed that mitochondrial Adx (plus ADR) and bacterial ferredoxin (plus NADPH-ferredoxin reductase) could efficiently donate electrons to mitochondria-localized P450 1A1, 2E1, 2C8, 2B1, 2D6, 3A4, and 17A1 [5, 6, 9, 19, 59, 70], demonstrating that mitochondria-localized P450s are catalytically active. It was also shown that the N-terminal acidic domain of Adx interacted with mitochondria-localized P450 1A1 through charge-charge interaction [1]. These Adx-interacting domains are conserved in other P450 Family 1 members and also in P450s 2B1, 2B4, 2D6, 2E1, 3A4, 17A1, and 21A2 [5, 6, 9, 46, 59, 70].
One of the important factors in signaling proteins for endoplasmic reticulum (ER) localization is signal recognition particle (SRP) [69]. SRP interacts with hydrophobic residues in the N-terminal regions of proteins declined for ER localization. We reasoned that the introduction of charged residues in the N-terminal region of rat P450 2E1 might lead to decreased SRP recognition and increased mitochondrial import [7]. This hypothesis was tested, and positive results were obtained, particularly with the Mt++ derivative (Fig. 2.4). Mitochondria from cells transfected and expressing the Mt++ variant also showed increased mitochondrial ROS production, as judged by isoprostane measurements (Fig. 2.5). When the variants were expressed in yeast, the cell showed similar phenotypes when the yeast were grown in glucose medium regardless of the N-terminal sequence (Fig. 2.6). However, with the non-fermentable carbon source lactic acid in the media, the cells transfected with the Mt++ variant, localized to the mitochondria, showed selective toxicity. The phenotype was petite, indicating a loss of mitochondrial function.
Consistent with the data obtained with COS and yeast cells expressing the Mt++ variant of P450 Δ2E1, hepatic mitochondria from rats fed ethanol showed a time-dependent increase in F2-isoprostane production, while hepatic microsomal membranes from these rats showed no significant increase [7]. The increased mitochondrial ROS production was accompanied by attenuated mitochondrial DNA contents, as well as cytochrome c oxidase and other electron transport complex activities, suggesting that mitochondrial P450 2E1 promotes alcohol-mediated mitochondrial dysfunction [1, 2, 13, 66, 70]. A recent study with livers of alcohol-fed mice suggested that mitochondrial P450 2E1 contributes significantly to the metabolic profiles of the liver tissue with several P450 2E1 substrates, including 4-nitrophenol, aniline, and styrene [34].
Studies with rat P450 2E1 [3, 67] showed that the N-terminal 160 amino acid region of the protein contained signals for targeting to both the ER and mitochondria. The positively charges residues of immediately upstream of the transmembrane domain of the protein functioned as a cryptic mitochondria targeting signal. Furthermore protein kinase A-mediated phosphorylation at Ser-129 enhanced mitochondrial translocation of the protein through enhanced affinity for binding to cytosolic HSP70 chaperone protein—which has been implicated in presenting mitochondrial passenger proteins to mitochondrial outer membrane translocators. Subsequent work [7] showed that the hydrophobicity of the N-terminal targeting domains of P450 Family 2 proteins determines their affinity for SRP binding and thus the level of ER vs. mitochondrial targeting [7]. P450 2E1—with a more hydrophobic N-terminus—was mostly targeted to the ER, and proteins with higher hydrophilicity were increasingly targeted to mitochondria.
Analysis of human liver samples from our collection showed varying degrees of mitochondrial localization, > 90% in some cases [8] (Fig. 2.7). DNA sequence analysis showed the presence of nucleotides coding for positively charged residues in the N-terminal region of P450 2E1 in these human samples. Liver samples with high mitochondrial P450 2E1 contents showed reduced cytochrome c oxidase activity and markedly depleted cytochrome c oxidase I, IVi1, and Vb subunits [8]. Consisted with observations with the rat P450 2E1, the N-terminal 160 amino acids of human P450 2E1 were sufficient to direct the organelle localization of P450s [8]. Accordingly, this region could be fused to a reporter protein (dihydrofolate reductase, DHFR ) to monitor membrane localization. Sequence analysis of the N-terminal coding regions cDNAs from human liver samples showed W23R and W30R substitutions and, as with rat P450 2E1 [7], the N-terminal sequence variants with the more basic residue substitutions (i.e., W23R/W30R) showed higher mitochondrial translocation and more mitochondrial ROS production (Fig. 2.8). In other assays, mitochondria from HepG2 cells transfected with the mitochondria-directed variants showed increased uncoupled respiratory rates and partial mitochondrial DNA depletion (Fig. 2.9). Thus, although the direct role of P450 2E1 in ROS production at the whole organ level remains suspect, the mitochondria-targeted P450 2E1 appears to augment alcohol toxicity by inducing mitochondrial respiratory dysfunction.
2.6 Future Questions
Collectively, the studies with mitochondria show a selective response of P450 2E1 localized there, in terms of ROS production. The mitochondrial P450 2E1 showed a similar catalytic activity in N,N-dimethylnitrosamine oxidation (as the microsomal P450 2E1) [7]. The results, taken collectively, argue for a selective role of mitochondrial P450 2E1 in ethanol toxicity. Exactly how this relates to ethanol-linked cancer is not clear. P450 2E1 may be generating more mitochondrial DNA adducts, and there is evidence for a higher level of DNA adducts in mitochondria with some carcinogens [4, 54, 78]. What is not clear is how genetic mutations in the mitochondrial DNA would yield tumors, in the context of current theories regarding roles of mutations in oncogenes and tumor suppressor genes.
Another basic question is how Adx interacts with mitochondrial P450 2E1 to deliver electrons. Several other human P450s have now been found to have significant fractions localized in mitochondria (i.e., 1B1, 2C8, 2D6) [5, 6, 9, 29, 70] and this is a similar question in these cases. One possibility is that electron transfer to the P450 with Adx occurs through a different domain, or with altered efficiency as with the normal redox partner NADPH-P450 reductase (an ER protein) and may determine the relative activities. Additionally, the mitochondria localized P450s 1A1, 2B1, and 2E1 show significant changes in their α-helical and β-sheet contents, suggesting altered folding. Many of these questions remain to be addressed in a more detailed manner.
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Peter Guengerich, F., Avadhani, N.G. (2018). Roles of Cytochrome P450 in Metabolism of Ethanol and Carcinogens. In: Vasiliou, V., Zakhari, S., Mishra, L., Seitz, H. (eds) Alcohol and Cancer. Advances in Experimental Medicine and Biology, vol 1032. Springer, Cham. https://doi.org/10.1007/978-3-319-98788-0_2
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