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Treatment of rats with ethylbenzene (EB) modulates the hepatic expression of many P450s, with those induced after a single intraperitoneal hydrocarbon injection differing from those induced after more prolonged (3 day) administration. The goals of the current studies are (1) to characterize the induction response after prolonged hydrocarbon exposure, (2) to explain why the elevation of these P450s is attenuated after continued treatment, and (3) to determine how P450 2B protein remains elevated without an elevation of P450 2B1/2 RNA. P450 2C11 protein was decreased after a single EB injection and remained depressed throughout the treatment period. P450 2C11 RNA was only decreased with prolonged, but not acute treatment. P450 2E1 was induced after a single EB injection; however, the initial induction was attenuated with more prolonged treatment. P450 2B1 and P450 2B2 RNAs exhibited a similar response, being elevated after acute administration, but returned to control levels with prolonged EB administration. Interestingly, P450 2B protein levels remained elevated despite the decrease in P450 2B1 and P450 2B2 RNA to control levels. We then tested the possibility that the multiphasic induction pattern of P450 2E1 and P450 2B1/2 RNA was due to differences in the pharmacokinetics of EB. The disappearance of EB with time was measured in rats that were either (1) untreated, (2) pretreated with EB for 1 day, or (3) pretreated with EB for 3 days. These results demonstrated that prior hydrocarbon exposure caused an increase in EB clearance, which decreased the overall levels of EB in the body. Consequently, EB levels were sufficiently diminished to decrease EB's effectiveness as an inducer leading to the decrease in P450 2E1 protein and P450 2B1 and P450 2B2 RNA after continued EB administration. A further consequence of the decreased overall EB concentration is that the hydrocarbon was capable of producing only a transient elevation of P450 2B1 RNA levels. This transient elevation appears to be sufficient to maintain elevated P450 2B protein.

Free heme binds to heme oxygenase as a prosthetic group and substrate in the conversion of heme to biliverdin, carbon monoxide, and free iron. Current methods for quantifying heme oxygenase-1 (HO-1) involve reconstitution of the enzyme with heme, followed by a hydroxyapatite column to remove the excess heme. As a result of the hydroxyapatite chromatography, there are significant losses of purified protein. We have developed a method which allows accurate quantitation of HO-1 using a heme titration and elimination of the final hydroxyapatite column, increasing the amount of purified protein.

This report summarizes a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics at Experimental Biology held April 20-24 in Boston, MA. Presentations discussed the status of cytochrome P450 (P450) knowledge, emphasizing advances and challenges in relating structure with function and in applying this information to drug design. First, at least one structure of most major human drug-metabolizing P450 enzymes is known. However, the flexibility of these active sites can limit the predictive value of one structure for other ligands. A second limitation is our coarse-grain understanding of P450 interactions with membranes, other P450 enzymes, NADPH–cytochrome P450 reductase, and cytochrome b5. Recent work has examined differential P450 interactions with reductase in mixed P450 systems and P450:P450 complexes in reconstituted systems and cells, suggesting another level of functional control. In addition, protein nuclear magnetic resonance is a new approach to probe these protein/protein interactions, identifying interacting b5 and P450 surfaces, showing that b5 and reductase binding are mutually exclusive, and demonstrating ligand modulation of CYP17A1/b5 interactions. One desired outcome is the application of such information to control drug metabolism and/or design selective P450 inhibitors. A final presentation highlighted development of a CYP3A4 inhibitor that slows clearance of human immunodeficiency virus drugs otherwise rapidly metabolized by CYP3A4. Although understanding P450 structure/function relationships is an ongoing challenge, translational advances will benefit from continued integration of existing and new biophysical approaches.

Cytochrome P450 (P450) function requires the interaction of P450 and NADPH-cytochrome P450 reductase (CPR) in membranes, and is frequently studied using reconstituted systems composed solely of phosphatidylcholine. There is increasing evidence that other endoplasmic reticulum (ER) lipids can affect P450 structure, activity, and interactions with CPR. Some of these lipid effects have been attributed to the formation of organized liquid-ordered (l(o)) domains. The goal of this study was to determine if l(o) domains were formed in P450 reconstituted systems mimicking the ER membrane. CYP1A2, when incorporated in "ER-like" lipid vesicles, displayed detergent insolubility after treatment with Brij 98 and centrifugation in a sucrose gradient. Lipid probes were employed to identify domain formation in both ER-like vesicles and model membranes known to form l(o) domains. Changes in fluorescence resonance energy transfer (FRET) using an established donor/acceptor FRET pair in both ER-like and model l(o)-forming systems demonstrated the coexistence of l(o)- and liquid-disordered domains as a function of cholesterol and sphingomyelin content. Similarly, 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan), a probe that reports on membrane organization, showed that cholesterol and sphingomyelin increased membrane order. Finally, brominated-phosphatidylcholine allowed for monitoring of the location of both CPR and CYP1A2 within the l(o) regions of ER-like systems. Taken together, the results demonstrate that ER-like vesicles generate microdomains, and both CYP1A2 and CPR predominantly localize into l(o) membrane regions. Probe fluorescent responses suggest that lipid microdomains form in these vesicles whether or not enzymes are included in the reconstituted systems. Thus, it does not appear that the proteins are critical for stabilizing l(o) domains.

1. Exposure to simple aromatic hydrocarbons has been shown to induce P450-dependent activities and the expression of particular P450 isozymes in a manner related to the molecular structure of the inducing hydrocarbon. In an attempt to identify the structural relationship controlling P450 induction, the effect of hydrocarbon treatment on the RNA levels for specific P450 isozymes was examined. 2. Rats were treated with daily injections of hydrocarbons (benzene, toluene, ethylbenzene, n-propylbenzene, m- and p-xylene) for 3 days, and the effects on specific RNA levels were examined by Northern blot hybridization. 3. Although P4502B1 mRNA was not elevated after hydrocarbon treatment, a significant elevation in 2B2 mRNA was observed after exposure to the larger aromatic hydrocarbons, ethylbenzene and m-xylene. It is interesting to note that despite the substantial elevation of P4502B protein levels, only a small elevation of P4502B1 and 2B2 RNA was observed. 4. P4502C11 mRNA was only suppressed by ethylbenzene administration, despite the depression of 2C11 protein levels by several hydrocarbons. 5. P4501A1 mRNA was not detectable and 2E1 mRNA was not changed by any aromatic hydrocarbon treatment investigated in this study. 6. The data indicate that the levels of mRNA species for a number of P450 isozymes are differentially regulated by exposure to hydrocarbons, and that small changes in hydrocarbon size or isomeric structure can influence the levels of these mRNA species.

The goal of these studies was to demonstrate that one P450 isozyme can influence the function of another isozyme when combined in a reconstituted system. Benzphetamine and 7-pentoxyresorufin were both shown to be preferred substrates for P450 2B4 (LM2) as compared to P450 1A2 (LM4). However, these substrates exhibited different characteristics when examined in reconstituted systems containing reductase and both P450 isozymes. Whereas benzphetamine demethylation showed a small increase in catalytic activity when both P450 1A2 and 2B4 were present over the activities obtained in simple reconstituted systems, 7-pentoxyresorufin O-dealkylation (PROD) was dramatically inhibited when both isozymes were present. These results indicate that the functional interactions between P450s in complex reconstituted systems are dependent on the substrate present. Inhibition of PROD was also dependent on reductase levels, with the inhibitory effect being more pronounced at subsaturating reductase. Finally, these protein-protein interactions were shown to be dependent on the reductase concentration in the reconstituted system rather the P450 concentration, supporting the view that P450 1A2 is inhibiting the reaction by competing with P450 2B4 for reductase molecules.

Rats were treated with a single intraperitoneal injection of ethylbenzene in corn oil and the effects on cytochrome P450 3A-dependent activities, immunoreactive protein levels and RNA levels were examined. Ethylbenzene increased both P450 3A-dependent 2β-hydroxylation of testosterone and immunoreactive protein levels. These levels were maximally induced by 24 hr and diminished thereafter. Despite the increases in P450 3A protein, neither P450 3A1 nor P450 3A2 mRNA levels were altered by treatment with the hydrocarbon. These results clearly demonstrate that this P450 isozyme can be induced by either translational activation or stabilization of P450 3A protein and are suggestive of an elevation of P450 3A2 levels.

The goal of this study was to characterize the effects of CYP1A2·CYP2B4 complex formation on the rates and efficiency of toluene metabolism by comparing the results from simple reconstituted systems containing P450 reductase (CPR) and a single P450 to those using a mixed system containing CPR and both P450s. In the mixed system, the rates of formation of CYP2B4-specific benzyl alcohol and p-cresol were inhibited, whereas that of CYP1A2-specific o-cresol was increased, results consistent with the formation of a CYP1A2·CYP2B4 complex in which the CYP1A2 moiety has a higher affinity for CPR binding. Comparison of the rates of NADPH oxidation and production of hydrogen peroxide and excess water by the simple and mixed systems indicated that excess water formed at a much lower rate in the mixed system. The commensurate increase in the rate of CYP1A2-specific product formation suggested the P450·P450 interaction increased the rate of the putative rate-limiting step of CYP1A2 catalysis, abstraction of a hydrogen radical from the substrate. Cumene hydroperoxide-supported metabolism was measured to determine whether the effects of the P450·P450 interaction required the presence of CPR. Peroxidative metabolism was not affected by the interaction of the two P450s, even with CPR present. However, CPR did stimulate peroxidative metabolism by the simple system containing CYP1A2. These results suggest the major functional effects of the P450·P450 interaction are mediated by changes in the relative abilities of the P450s to receive electrons from CPR. Furthermore, CPR may play an effector role by causing a conformational change in CYP1A2 that makes its metabolism more efficient.

Cytochrome P450 is part of an electron transport chain found in the endoplasmic reticulum (ER), with its catalytic function requiring interactions with NADPH-cytochrome P450 reductase (CPR). The goals of this study were to examine how the P450 system proteins are organized in the membrane and to determine whether they are distributed in detergent-resistant lipid microdomains (DRM). Isolated liver microsomes from untreated rabbits were treated with 1% Brij 98, and DRMs were isolated via sucrose gradient centrifugation. Lipid analysis showed that DRM fractions were enriched in cholesterol and sphingomyelin, similar to that found with plasma membrane DRMs. Approximately 73% of CYP1A2 and 68% of CPR resided in DRM fractions, compared with only 33% of total ER proteins. These DRMs were found to be cholesterol-dependent: CPR and CYP1A2 migrated to the more dense regions of the sucrose gradient after cholesterol depletion. CYP1A2 function was studied in three purified lipid vesicles consisting of 1) phosphatidylcholine (V-PC), 2) lipids with a composition similar to ER lipids (V-ER), and 3) lipids with a composition similar to the DRM fractions (V-DRM). Each system showed similar substrate binding characteristics. However, when the association between CPR and CYP1A2 was measured, V-ER and V-DRM liposomes produced lower apparent Km values compared with V-PC without any significant change in Vmax. These findings suggest that CYP1A2 and CPR reside in ER-DRMs and that the unique lipid components of these domains enhance CYP1A2 substrate metabolism through greater efficiency in CPR-CYP1A2 binding.

The subject of hydrocarbon inhibition of cytochrome P450-dependent reactions as well as data on other enzyme-catalyzed reactions from the literature was examined to determine the relationship between the "hydrophobicity" of the hydrocarbons and their ability to act as inhibitors. The compounds used in these studies (benzene, toluene, ethylbenzene, n -propylbenzene, and n -butylbenzene) behave as competitive inhibitors, with the affinity increasing as the size of the inhibiting hydrocarbon increases. A similarity was seen in the size dependence for both hydrocarbon inhibition of cytochrome P450-dependent activities (−0.6 to −0.7 kcal/mol/methylene group) and transfer of these compounds between aqueous and organic phases (−0.68 kcal/mol/methylene group), suggesting that the active site of cytochrome P450, in some ways, is comparable to an organic solvent in its ability to accommodate hydrophobic compounds. A more detailed examination of this process was initiated to separate the "hydrophobic effect" into its two component processes: (i) hydration of the hydrocarbon ligand and (ii) transfer of the unhydrated hydrocarbon onto the enzyme active site. In other words, do larger hydrocarbons bind more avidly to the active site because they are drawn more effectively into that site (pull), or is the size-dependent increase in hydrocarbon binding the result of the larger compounds being more efficiently expelled from the aqueous medium (push)? The results indicate that the predominant force involved in binding is the ability of the active site of cytochrome P450 and an impressive number of other enzymes to draw the hydrocarbon from the aqueous medium. The hydration of the hydrocarbon is much less dependent on the size of the hydrocarbon, indicating that dehydration or partial dehydration of the hydrocarbon molecule (upon leaving the solution and combining with the enzyme) contributes to the overall binding process to a much lesser extent; hydrophobic binding in the most widely used sense (entropy driven) is not the primary driving force that is responsible for the observed size dependence effects. It is pointed out that not all types of binding would be expected to follow the law which describes the size dependence for simple hydrocarbons because of heat-entropy relationships. The different temperature dependence of these heat-entropy relationships further complicates the analogy between enzyme-ligand binding and ligand partitioning between aqueous and organic phases. The maximum contribution that can be attributed to entropy driven hydrophobic binding (in the most widely used sense) is −0.1 to −0.2 kcal/mol/methylene group for the aromatic hydrocarbons examined here.

Male and female Holtzman rats were exposed to ethylbenzene, and the effect on liver microsomal activities was studied. Hydrocarbon- and sex-dependent effects on P450-dependent metabolism of drugs and aromatic hydrocarbons were investigated. Hydrocarbon treatment produced two patterns of induction in cytochrome P450-dependent activities: (1) induction common to both sexes; and (2) induction exclusively in females. Benzphetamine N-demethylation, 7-ethoxycoumarin O-deethylation, p-nitroanisole O-demethylation and aromatic hydroxylation of toluene were induced in both sexes after rats were exposed to ethylbenzene. The rate of benzphetamine N-demethylation increased 4-fold in females and nearly doubled in males. The increase in O-deethylation of 7-ethoxy-coumarin was 3-fold in females and doubled in males, while p-nitroanisole O-demethylation increased 4-fold in both sexes after exposure to ethylbenzene. Ethylbenzene had its greatest effect upon the formation of aromatic hydroxylated metabolites of toluene. Ethylbenzene exposure increased the rate of o-cresol formation by 4- and 9-fold in female and male rats, respectively. The formation rate of p-cresol was undetectable in either sex prior to hydrocarbon exposure; however, after the rats were given ethylbenzene, rates increased to 0.4 nmol/min/mg protein in females and to 0.9 nmol/min/mg protein in the males. Ethylbenzene exposure selectively induced aminopyrine demethylation, aniline hydroxylation, N,N-dimethylnitrosamine N-demethylation (DMNA) and aliphatic hydroxylation of toluene in females. Rates for aminopyrine, aniline, and DMNA were increased 50% over controls, while formation of benzyl alcohol from toluene was enhanced to 260% of control. Western immunoblotting indicated that ethylbenzene treatment induced cytochrome P450 2B1/2B2 to a greater extent in male rats and cytochrome P450 2E1 only in females. Ethylbenzene exposure did not affect significantly the level of cytochrome P450 1A1.

Substrate has recently been shown to affect (a) the high spin content of cytochrome P450 (b) the rate of first electron transfer when LM2 (P450 2B4) and reductase were in a preformed complex, and (c) the rate of functional complex formation between NADPH-cytochrome P450 reductase and cytochrome P450 LM2. When comparing the effect of substrate on each of these parameters, the strongest correlation was demonstrated between the rate of first electron transfer through the preformed complex and the rate of functional complex formation (W.L. Backes and C.S. Eyer, 1989, J. Biol. Chem. 264, 6252-6259). The relationship among high spin content, reduction rate, and the rate of functional complex formation was examined using a number of different cytochrome P450 isozymes. The goal of this study was to determine if the previously established relationship between reduction rate and the rate of reductase-P450 complex formation was a feature only of LM2, or a general characteristic of the cytochrome P450 system. Substrate addition caused an increase in first electron transfer for each of the isozymes examined, with high spin content being increased with cytochromes P450 2B1 (PBRLM5) and P450 2B2 (PBRLM6). Substrate addition to cytochrome P450 2C6 (PBRLM4) resulted in a small decrease in high spin content. P450 2B1 and P450 2B2 showed a positive correlation between substrate-mediated stimulation of reduction and high spin content, whereas P450 2C6 showed a negative correlation between these variables. Substrate also increased the rate of reductase-P450 association for each of the isozymes examined. When compared to the degree of stimulation of reduction through a preformed complex, a strong positive correlation was obtained with each isozyme examined. These results demonstrate that the increase in both the rate of functional reductase-P450 complex formation and the rate of first electron transfer is not simply a property of LM2, but appears to be a general characteristic of many cytochrome P450 isozymes.

Heme oxygenase-1 (HO-1) catalyzes the oxidative degradation of heme to biliverdin, carbon monoxide, and free iron in a reaction requiring the interaction of HO-1 with NADPH-cytochrome P450 reductase (CPR). HO-1 is bound to the endoplasmic reticulum by 23 C-terminal amino acids; however, a soluble HO-1 (sHO-1) lacking this membrane spanning region has been extensively studied. The goal of this project was to characterize the effect of the C-terminal hydrophobic domain on formation of the HO-1/CPR complex. Full-length HO-1 was shown to exhibit higher reaction rates than sHO-1, particularly at subsaturating CPR, indicating that the C-terminal region influences HO-1 binding to CPR. The increased activity of HO-1 was attributable to a time-dependent formation of a low K(m) HO-1/CPR complex that was not seen with sHO1. Gel filtration analysis confirmed the formation of multiple high molecular weight complexes in the presence and absence of the synthetic lipid dilauroylphosphatidylcholine (DLPC). However, the largest complex appeared following a 2 h incubation of HO-1 and CPR in DLPC, suggesting that the C-terminal region was required for the high-affinity HO-1/CPR complex formation and membrane incorporation. These data demonstrate that the C-terminal region of HO-1 influenced complex formation and ultimately its affinity for CPR.

In a previous study from our laboratory (Drug Metab Dispos 34: 660-666, 2006), we found several limitations with published methods (cholate gel filtration and cholate dialysis) for the incorporation of cytochromes P450 and P450 reductase into phospholipid vesicles. We found that a significant proportion of reductase was not incorporated in the vesicles when the amount of reductase was equal to or greater than that of CYP2B4 in the systems reconstituted with phosphatidylcholine. Furthermore, implementation of these methods compromised the ability of the CYP2B4 to form a ferrous carbon monoxy complex. In the current study, a comparison of results using the detergent-dialysis method with five similar detergents having the "bile salt" ring structure showed that glycocholate results in the greatest incorporation of reductase and the least loss in the ferrous carbon monoxy CYP2B4 complex. The method is further improved by using Bio-Beads SM-2 to remove detergent instead of the lengthy dialysis procedure or size exclusion chromatography that significantly dilutes the protein and lipid concentrations of the preparation. The method is shown to be applicable over a range of lipid/CYP2B4 ratios, and by using assay methods for total lipid, reductase, and CYP2B4, this improved reconstitution method resulted in increased incorporation efficiencies while minimizing the protein degradation inherent with these procedures.

Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes.

2-Ethynylnaphthalene (2EN) is a mechanism-based inhibitor of CYP2B4 with two components to the inhibition, (1) enzyme inactivation, which requires covalent binding of the 2EN metabolite, and (2) reversible inhibition by 2EN itself. Both inhibitory components were examined using several different CYP2B4 substrates. Preincubation of CYP2B4 with 2EN led to a time-dependent inactivation of each of the CYP2B4-dependent activities examined; however, the ability of 2EN to reversibly inhibit CYP2B4 depended on the substrate employed, which is inconsistent with classical inhibition patterns. The degree 2EN’s reversible inhibition was shown not to correlate with the substrate affinity for the active site, but with parameters related to the molecular size of the substrate. The results are consistent with 2EN and the smaller substrates simultaneously fitting in the CYP2B4 active site, leading to very little inhibition. Larger substrates exhibited greater degrees of inhibition because of their inability to co-bind with inhibitor in the active site.

High-temperature, controlled incineration and thermal treatment of contaminated soils, sediments, and wastes at Superfund sites are often preferred methods of remediation of contaminated sites under the Comprehensive Environmental Response, Compensation, and Liability Act of 1980 and related legislation. Although these methods may be executed safely, formation of toxic combustion or reaction by-products is still a cause of concern. Emissions of polycyclic aromatic hydrocarbons (PAHs) ; chlorinated hydrocarbons (CHCs) , including polychlorinated dibenzo-p-dioxins and dibenzofurans ; and toxic metals (e.g., chromium VI) have historically been the focus of combustion and health effects research. However, fine particulate matter (PM) and ultrafine PM, which have been documented to be related to cardiovascular disease, pulmonary disease, and cancer, have more recently become the focus of research. Fine PM and ultrafine PM are effective delivery agents for PAHs, CHCs, and toxic metals. In addition, it has recently been realized that brominated hydrocarbons (including brominated/chlorinated dioxins) , redox-active metals, and redox-active persistent free radicals are also associated with PM emissions from combustion and thermal processes. In this article, we discuss the origin of each of these classes of pollutants, the nature of their association with combustion-generated PM, and the mechanisms of their known and potential health impacts.

Elevated mortality rates of lung cancer in the Mississippi River corridor in Louisiana have been clearly documented for the past half-century and rank among the highest in the nation. A population-based case-control study of lung cancer termed Lower Mississippi River Interagency Cancer Study was conducted in southern Louisiana. Lung tumor specimens were collected, isolated by laser capture microdissection, subjected to PCR to amplify KRAS, and sequenced to confirm mutation status and specificity. Of the 116 lung tumors analyzed to date, 32 (27.6%) contained mutations in either codon 12 or 13 of KRAS. This frequency is comparable to that reported in the literature; however, the mutation spectrum was strikingly different. Of the 32 mutations observed, 21 (65.6%) resulted in the inappropriate insertion of cysteine, 6 (18.8%) resulted in the insertion of serine, 3 (9.4%) resulted in the insertion of valine, and 1 (3.1%) each resulted in the insertion of aspartate and alanine. These data indicate that an abnormally high proportion of cysteine (P = 0.010) and serine (P = 0.002) mutations was observed in our sample group versus lung cancers reported in the literature. KRAS mutations were more common in African Americans with an odds ratio of 2.4 (P = 0.048), as were serine mutations, although the latter did not reach statistical significance (odds ratio, 2.6; P = 0.373). No association was found between the observed mutation spectrum and known lung cancer risk factors.

The purpose of this study is to characterize the interactions among P450 1A2, P450 2B4, and P450 reductase in mixed reconstituted systems. Previously, our laboratory demonstrated that in the presence of certain substrates, 1A2 can influence the catalytic characteristics of 2B4 [Cawley et al. (1995) Biochemistry 34, 1244-1247]. The goal of the current study is to distinguish between two models to explain these interactions: one model where substrate increases the affinity of one P450 enzyme for the reductase, and another model where substrate increases the affinity of one P450 for the reductase through the formation of a 1A2-2B4 complex. According to this model, the 1A2 moiety of 1A2-2B4 forms a high-affinity complex with reductase. Reductase, 1A2, and 2B4 were reconstituted with dilauroylphosphatidylcholine, and the effect of reductase concentration on 7-pentoxyresorufin-O-dealkylation was examined with 2B4-reductase and 1A2-reductase binary systems, and in ternary systems containing different 2B4:1A2 ratios. At subsaturating [reductase], there was a dramatic inhibition of the 2B4-dependent activity in the ternary system as compared with the binary systems. These results are consistent with the formation of a ternary (reductase-1A2-2B4) complex where the reductase is bound specifically to 1A2. At higher reductase concentrations where the reductase-binding sites on 1A2 become saturated, the results are consistent with the formation of a quaternary complex in which reductase binds to both P450 enzymes (reductase-1A2-2B4-reductase). Analogous experiments using the 1A2-preferred substrate 7-ethoxyresorufin showed a stimulation of 7-ethoxyresorufin-O-deethylation in the mixed reconstituted system, demonstrating that the high-affinity 2B4-1A2-reductase complex was functionally active and not merely an inhibitory complex.

1. The aim was to determine if the ethylbenzene (EB)-mediated expression of CYP2B and CYP2C11 involved a hormonally controlled component. 2. The hypophysectomized (HX) and intact rats were treated with EB for 1 or 2 days, and the effects on specific CYP levels measured. 3. Differences were observed in the inducibility of CYP2B by EB in the HX rat when compared with intact controls. Whereas significant elevations of CYP2B-dependent activities and protein levels were observed after both 1 and 2 days of EB injection in intact controls, CYP2B levels were significantly elevated in the HX rat only after 2 days of hydrocarbon treatment. 4. Both CYP2C11-dependent activities and protein levels were decreased after EB administration to the intact rat. In contrast, CYP2C11 levels were unaffected by EB in the HX rat at any of the time points indicated. 5. CYP2C11 protein levels were unaffected by treatment with EB for 24 h in cultured hepatocytes, also supporting the hypothesis that hormones are involved in CYP2C11 expression. 6. This study indicates that pituitary input influences the EB-mediated changes in both CYP2B and CYP2C11. CYP2C11 is affected by EB administration in a manner similar to other xenobiotics such as phenobarbital. On the other hand, the smaller induction of CYP2B1/2 in response to EB differs from that observed with phenobarbital where HX augmented the response of the inducer.

Ethylbenzene (EB) treatment to male Holtzman rats was shown to alter the expression of cytochrome P-450s 1A1, 2B, 2C11, 2E1, and 3A, with several isozymes exhibiting complex multiphasic induction patterns when treated for 1 and 3 days with the alkylbenzene. Male rats were treated with daily i.p. injections of EB for either one or three days, and the effects on P-450 dependent activities, P-450 immunoreactive protein levels and their corresponding mRNA levels were measured. Although levels of P-450 2B, 2C11, 2E1, and 3A were all modulated by EB treatment, each exhibited different temporal characteristics. P-450 2B1/2B2 were induced after a single EB exposure and continued to be elevated after EB treatment for 3 days. However, P-450 2B1 and 2B2 mRNA levels were elevated about 50-fold after a single injection, and returned to control values after continued EB administration. P-450 2C11 expression was decreased to about 45% of controls after either single or repeated EB exposure with corresponding changes being observed in the levels of 2C11 mRNA. P-450 2E1 was induced by EB according to a complex multistep induction pattern. Both P-450 2E1 protein and RNA levels were increased 2-4-fold after a single EB treatment but returned to control values after continued administration. P-450 3A-dependent testosterone 2beta-hydroxylation and P-450 3A immunoreactive protein levels were both increased about 3-fold after a single EB treatment, whereas levels were only elevated 2-fold after EB treatment for 3 days. In contrast, P-450 3A2 mRNA was unaffected by a single EB injection but was increased 3.5-fold with repeated administration. Changes in P-450 3A1/2 were similar to those observed with P-450 3A2, whereas changes in P-450 3A1/23 and 3A23 mRNAs were not detectable. These data indicate that while EB can influence the expression of several P-450 isozymes, the hydrocarbon appears to alter P-450 expression by acting at different regulatory steps.

The goal of the present study was to examine the time course for changes in P450 expression and hydrocarbon metabolism after acute treatment with the simple aromatic hydrocarbon ethylbenzene (EB) and to correlate these alterations with the changes observed in alkylbenzene metabolism. Male Holtzman rats were treated with a single intraperitoneal injection of EB, and the effects on specific P450-dependent activities, immunoreactive P450 isozyme levels, and RNA levels were measured at various times after injection. Toluene was used as the test alkylbenzene for examination of the EB-mediated changes on in vitro hydrocarbon metabolism. In untreated rats, toluene was metabolized almost entirely by aliphatic hydroxylation (to benzyl alcohol); however, in EB-treated rats, significant quantities of benzyl alcohol, o-cresol, and p-cresol were produced. Interestingly, 5-10 h after EB treatment, there was a 40% decrease in benzyl alcohol production. By 24 h, rates of benzyl alcohol formation returned to control levels, whereas there was a 7-fold increase in o-cresol and a greater that 50-fold increase in p-cresol production. The changes in the disposition of toluene were then correlated with changes in particular P450 isozymes. Several P450 isozymes were induced after EB administration. P450 2B1/2-dependent testosterone 16 beta-hydroxylation and P450 2B1/2-immunoreactive protein were elevated 30-fold after EB administration, reaching maxima by 24 h and remaining elevated 48 h after exposure. Changes in P450 2B1 and 2B2 RNA preceded those of the proteins. Similar results were observed with P450 1A1. P450 2E1 RNA levels were elevated after a single EB injection. However, the elevation in P450 2E1-dependent activities and immunoreactive protein levels preceded the changes in RNA, suggesting that multiple steps are affected by EB exposure. In contrast to the increases in some isozymes, P450 2C11 protein was rapidly suppressed (within the first 2-10 h) after hydrocarbon exposure, suggestive of a destabilization of the protein. When comparing the changes in P450 isozymes to alterations in toluene metabolism, the immediate suppression in aliphatic hydroxylation of toluene (in the first 5-10 h) was consistent with the decrease in P450 2C11. Subsequent to this effect, P450 2B1/2 and 2E1 were induced, which elevated production of this metabolite to control levels. The increase in the aromatic hydroxylation of toluene to both o, and p-cresol was consistent with the induction of P450s 2B1/2, 2E1, and 1A1.

The goal of this study was to examine the effect of duration of ethylbenzene exposure on cytochrome P-450-dependent activities. Male rats were treated with ethylbenzene by intraperitoneal injection for either 1 or 3 days, and microsomal preparations were examined for changes in the microsomal proteins and activities as well as the expression of specific P-450 isozymes. Two general patterns of induction were evident when different P-450-dependent activities were examined. (i) Cytochrome P-450 2B-dependent activities (e.g., p-nitroanisole demethylation, benzphetamine demethylation, and aromatic toluene hydroxylations) were induced both after 1 and 3 days of ethylbenzene exposure. (ii) Cytochrome P-450 2E1-dependent activities (e.g., N,N-dimethylnitrosamine demethylation and aniline hydroxylation) were induced after treatment with ethylbenzene for one day; however, after 3 days of ethylbenzene treatment these activities returned to control levels. Changes in these activities were consistent with changes in the levels of specific P-450 isozymes as determined by immunoblotting. Cytochrome P-450 2B levels were increased and P-450 2C11 levels were suppressed at both 1 and 3 days of ethylbenzene exposure. A temporal response in P-450 2E1 expression was observed, with P-450 2E1 levels increasing after a single ethylbenzene injection and returning to controls after administration of the hydrocarbon for 3 days. Rats were also subjected to a pair-feeding regimen to determine whether these effects were related to altered dietary status in ethylbenzene-treated rats. Neither P-450-dependent activities nor immunoreactive protein levels were altered in pair-fed rats. These results demonstrate that prolonging the duration of hydrocarbon exposure can produce differential effects on the expression of P-450 2E1, with levels being elevated after acute hydrocarbon administration, but not after more prolonged hydrocarbon exposure.

In 1950, a report was published describing the isolation of a protein involved in the reduction of cytochrome c in animal tissue (Horecker 1950). The search for this enzyme was prompted by the previous discovery of an NADPH-cytochrome c reductase in yeast (Haas et al. 1940). The yeast enzyme was shown to use NADPH as the source of reducing equivalents, and contained flavin mononucleotide (FMN) as a prosthetic group. This newly discovered enzyme was isolated from an acetone powder of pig liver after trypsin treatment. It had a molecular weight of 68000, used NADPH as its source of reducing equivalents, and was reported to contain the prosthetic group flavine adenine dinucleotide (FAD). The flavin was shown to be a necessary cofactor, since its removal from the enzyme led to the elimination of its cytochrome c reductase activity. Addition of either FMN or FAD to the apoenzyme led to its reactivation. The reductase was subsequently found to be located in the microsomal fraction (Phillips and Langdon 1962; Williams and Kamin 1962). These earlier findings provided the setting for raising a number of questions concerning reductase function: Since cytochrome c is located in the mitochondria, what is the physiological substrate for NADPH-cytochrome c reductase? How many flavins are actually present, and how are they involved in the electron transfer process? The reductase is now commonly called NADPH-cytochrome P450 reductase, referring to the endogenous electron acceptor, cytochrome P450.

The effect of substrate on LM2 reduction was examined using a reconstituted system containing dilauroylphosphatidylcholine, NADPH-cytochrome P-450 reductase, and cytochrome P-450 LM2 in a 160:1.5:1 molar ratio. In general, most substrates increased the rate constants of both the first and second phases of reduction as well as the fraction of LM2 reduced in the first phase. The correlation between the high spin content of the cytochrome and each of these kinetic parameters was weaker than expected if spin state controlled LM2 reduction. Further, substrate was shown to exert a rapid effect on both the high spin content and stimulation of reduction indicating that the low spin to high spin shift cannot be responsible for the slow phase of reduction for this particular isoform. Cytochrome P-450 reduction was also examined in both phospholipid-containing and soluble systems where the LM2 and reductase were not present as a preformed complex. In these systems the reactions were substantially slower than with the standard reconstituted system. Addition of substrate enhanced the rate of reduction, indicating that the rate of association between LM2 and the reductase was increased by substrate addition. The strong correlation between the rate of LM2 reduction in a preformed complex and the logarithm of the rate of LM2 and reductase association implicates the rate of functional complex formation as the factor controlling the slow phase of reduction.

The reduction kinetics of cytochrome P-450 are known to be biphasic, with a rapid initial phase and a slower subsequent phase, both of which appear linear in semilogarithmic plots. The present report demonstrates that these biphasic reduction kinetics can be described in terms of a preequilibrium between high- and low-spin ferric states. Computer simulations are used which express the rate and extent of the fast phase or burst as being due to the initial proportion of high-spin cytochrome P-450. According to this simplified sequential model, the slow phase of reduction is controlled by the rate of formation of high-spin cytochrome P-450. The substrate-induced alterations in the reduction kinetics are likewise consistent with the model, which indicates that type I compounds exert their effect by virtue of a decrease in the rate constant controlling the shift from high-spin to low-spin ferric cytochrome. The model is further supported by the influence of temperature on the spin equilibrium and reduction kinetics. Potential influences of other intermediate steps in the reduction and assumptions in the hypothesis are described.

Studies have been undertaken to explain the observed variation of the apparent association constant for water-insoluble substrates, which were diluted in common organic solvents, as a direct function of the solvent/solute ratio. By the use of suitable equations, the solvents methanol, ethanol, propanol, and acetone are shown to interact with hydrocarbon substrates in a competitive manner in PB-treated male rats, with the solvent producing a type I spectral component. Such solvents are shown to elicit, in addition to the type I component, a modified type II component. In untreated rats, ethanol does not produce a type I component, and also does not affect the apparent association constant for the hydrocarbon substrates when used as a solvent for those substrates. All perturbations of the enzyme which cause a change in the apparent association constant of the substrate also cause a quantitatively similar change in the apparent association of the solvent for the enzyme. A sex difference, with respect to competitive solvent binding, is also observed. Cytochrome P-450 from untreated male rats is apparently unable to bind small polar solvent substrates at the hydrocarbon binding site, whereas untreated female rats possess such an ability. In PB-treated rats, solvent binding is found to be sex-dependent. With respect to PB induction in female rats, the binding affinity for ethanol in the PB-treated animals is significantly larger than that observed in untreated females.

Cytochrome P-450 LM2 reduction was measured at a series of NADPH concentrations in the absence of substrate and in the presence of 1 mM benzphetamine. In the absence of substrate reduction could be described as a biphasic process with 55% of the reaction occurring in the first phase (at 20 microM NADPH). When benzphetamine was present, the fraction of the reaction occurring in the first phase was increased to 91%. When examined either in the absence or presence of benzphetamine, the rate constant and fraction of LM2 reduced in the fast phase were decreased as the NADPH concentration was decreased. In each case the fraction of LM2 reduced in the second phase was not substantially altered over the NADPH concentrations examined. To explain the effect of NADPH concentration on the initial rate of LM2 reduction, the effect of NADPH on the reduction of NADPH-cytochrome P-450 reductase was examined. Due to the presence of two flavins within each reductase molecule, there would be nine possible oxidation-reduction states of the reductase which may be present at a given NADPH concentration. Based on the redox potentials for the flavin half-reactions and for NADPH oxidation, the relative concentrations of each of the reductase subspecies could be determined. Rate constants were assigned for the reduction of LM2 by the various reductase subspecies, and the theoretical initial rates of LM2 reduction at various NADPH concentrations were compared with values obtained experimentally. The experimental data are consistent with a model where, under the conditions of this assay, the fully reduced reductase is the form primarily responsible for the reduction of LM2.

Phenobarbital pretreatment of male rats induced four microsomal cytochrome P-450 enzymes, at least one of which has previously not been reported. Three of the induced forms of the cytochrome possess i

The substrates hexobarbital and ethylbenzene have been shown to compete for the spectral binding site of phenobarbital-induced rat hepatic microsomal cytochrome p-450. The two substrates produce different delta Absmax values, and the presence of one substrate does not affect the delta Absmax of the other substrate and vice versa. The respective binding constants for the two substrates are similarly unaffected. The conclusion drawn from these observations is that, over the concentration ranges studied, there is no change in the availability of the enzyme as a result of substrate addition; the difference in delta Absmax apparently being due to varying abilities of different substrates to bring about a spin shift in the enzyme. Evidence is presented to indicate that differences between enzymes from untreated male rats and phenobarbital-treated male rats are attributable to differences in the enzyme itself and not to changes in the nature of the membrane brought about by phenobarbital administration, at least insofar as heat entropy compensation is concerned. The enthalpy-entropy compensation observed in the binding of a homologous series of barbiturates to the microsomal membrane as determined from the membrane concentration dependence of their binding constants is shown to agree surprisingly well with the direct determination performed by Sitar and Mannering.

When hydrophobic compounds were added to a solution of protoferriheme, a reverse type I spectral change was produced when observed by difference spectroscopy. The spectrum has a peak at 422 nm and a trough at 387 nm, and the characteristics were dependent on the pH of the sample. An association constant for the complex could be determined and was also found to be pH sensitive, with the association constant dropping to zero at values below pH 7.0 and above pH 8.5. The determination of the δAbsmax for the ethylbenzene-hemin complex at various hemin concentrations indications monomeric heme to be the species responsible for binding the hydrocarbon with the concomitant generation of the reverse type I spectral change.