Evidence supporting the interaction of CYP2B4 and CYP1A2 in microsomal preparations.

Recent studies have demonstrated that the catalytic behavior of one cytochrome P450 (P450) enzyme can be influenced by the presence of a second P450. This effect has been observed using reconstituted systems containing reductase, CYP2B4, and CYP1A2, primarily at subsaturating reductase. Addition of 1A2 caused a 75% inhibition of CYP2B4-dependent 7-pentoxyresorufin-O-dealkylation (PROD). Conversely, CYP2B4-dependent benzphetamine (bzp) demethylation did not exhibit this response after CYP1A2 addition. Addition of CYP2B4 to a reconstituted system containing reductase and CYP1A2 caused synergism of CYP1A2-dependent 7-ethoxyresorufin-O-dealkylation (EROD). This behavior was consistent with the formation of heteromeric CYP1A2-CYP2B4 complexes with altered catalytic properties. Although such responses have been documented in reconstituted systems, they have not been demonstrated in microsomal preparations. The goal of the present study was to determine whether such interactions were observed in rabbit liver microsomes. In an effort to detect such changes, we took advantage of the differential effect of CYP1A2 on CYP2B4-selective PROD and bzp metabolism. Rabbits were treated with phenobarbital (PB), beta-naphthoflavone (betaNF), and both PB + betaNF-conditions that enrich microsomes with CYP2B4, CYP1A2, or both enzymes, respectively. Benzphetamine demethylation activity was equivalently elevated in both the PB and the PB + betaNF groups, consistent with the induction of CYP2B4 in both groups. In contrast, PROD activity in the PB + betaNF group was less than 25% of that found in the PB-treated rabbits. These results demonstrate that the interactions observed in reconstituted systems are not an artifact of reconstitution but are observed under the more natural conditions of the microsomal membrane.

Effect of hypophysectomy and growth hormone replacement on the modulation of p450 expression after treatment with the aromatic hydrocarbon ethylbenzene.

Pituitary status has a significant effect on the expression of several cytochrome P450 enzymes. The goal of this study was to examine the role of pituitary input on the modulation of CYP2C11 and CYP2B after treatment with the aromatic hydrocarbon ethylbenzene (EB). Intact, hypophysectomized (HX), and HX rats supplemented with pulsatile growth hormone (GH) were treated with corn oil or EB and the effects on hepatic P450 expression were determined. Hypophysectomy caused a 50% decrease in CYP2C11 protein in untreated rats, whereas GH supplementation returned protein to control levels. EB administration also decreased CYP2C11 protein in intact rats; however, this decrease was not observed after EB treatment in HX or HX + GH groups. CYP2C11-dependent testosterone 2alpha-hydroxylation followed a similar pattern as CYP2C11 protein, except that the activity was only partially restored by GH replacement. CYP2B levels were also substantially influenced by hypophysectomy. Intact rats exhibited a 100-fold increase in CYP2B1 mRNA, reaching a maximum 12 h after EB administration. A much smaller response (ca. 20-fold) was observed in HX rats, reaching a maximum 24 h after EB treatment. This effect was not reversed by GH supplementation. The half-life for EB was significantly increased from 8 h in intact rats to 14 h in HX rats, suggesting higher plasma EB concentrations after EB administration to HX rats. These results indicate that CYP2C11 and CYP2B become less responsive to EB-dependent modulation in HX rats, a response that cannot be explained simply by absence of GH or by altered EB pharmacokinetics in HX animals.

Ethylbenzene induces microsomal oxygen free radical generation: antibody-directed characterization of the responsible cytochrome P450 enzymes.

Small aromatic hydrocarbons cause changes in oxidative metabolism by modulating the levels of cytochrome P450 enzymes, with the changes in these enzymes being responsible for qualitative changes in aromatic hydrocarbon metabolism. The goal of this study was to determine if exposure to the small alkylbenzene ethylbenzene (EB) leads to an increase in hepatic free radical production. Male F344 rats were treated with ip injections of EB (10 mmol/kg) and compared to corn oil controls. Hepatic free radical production was examined by measuring the conversion of 2',7'-dichlorofluorescin diacetate (DCFH-DA) to its fluorescent product 2',7'-dichlorofluorescein (DCF). A significant elevation of fluorescent DCF production was observed after treatment with EB, despite the lack of effect on overall cytochrome P450 levels. This process was shown to be inhibitable by metyrapone, an inhibitor of P450. DCF production was also inhibited by catalase, suggesting that hydrogen peroxide (H(2)O(2)) is one of the reactive oxygen intermediates involved in EB-mediated reactive oxygen species (ROS) formation. Interestingly, superoxide dismutase (SOD) did not inhibit DCF production in corn oil-treated rats but was an effective inhibitor in the EB-treated groups. In an effort to determine if the increase in ROS production was related to changes in specific P450 enzymes, DCF production was measured in the presence of anti-CYP2B, anti-CYP2C11, anti-CYP2E1, and anti-CYP3A2 inhibitory antibodies. Anti-CYP2B antibodies inhibited DCF production in EB-treated, but not corn oil groups, which is consistent with the low constitutive levels of this enzyme and its induction by EB. The data also demonstrate that CYP2B contributes to ROS production. Anti-CYP2C11 did not influence DCF production in either group. ROS formation in corn oil-treated rats as well as in ethylbenzene-treated rats was also inhibited with antibodies to anti-CYP2E1 and anti-CYP3A2. These results suggest that CYP2C11 does not appear to influence free radical production and that the increase in free radical production in EB treated rats is consistent with the EB-mediated elevation of CYP2B, CYP 2E1, and CYP3A2. Such alterations in free radical generation in response to hydrocarbon treatment may contribute to the toxicity of these compounds.

Changes in the expression of cytochrome P450s 2B1, 2B2, 2E1, and 2C11 in response to daily aromatic hydrocarbon treatment.

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.

Interactions among P450 enzymes when combined in reconstituted systems: formation of a 2B4-1A2 complex with a high affinity for NADPH-cytochrome P450 reductase.

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.

Pituitary component of the aromatic hydrocarbon-mediated expression of CYP2B and CYP2C11.

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 modulates the expression of different cytochrome P-450 isozymes by discrete multistep processes.

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.

Time course for the modulation of hepatic cytochrome P450 after administration of ethylbenzene and its correlation with toluene metabolism.

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.

Substrate-dependent competition of different P450 isozymes for limiting NADPH-cytochrome P450 reductase.

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.

Relationship between hydrocarbon structure and induction of P450: effect on RNA levels.

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.

Induction of P450 3A by ethylbenzene without altering RNA levels.

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 2 beta-hydroxylation of testosterone and immunoreactive protein levels. These levels were maximally induced by 24 hr and diminished thereafter. Despite the increase 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.

Temporal changes in P-450 2E1 expression with continued ethylbenzene exposure.

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.

Relationship between hydrocarbon structure and induction of P450: effects on protein levels and enzyme activities.

1. Treatment of male rat with the small aromatic hydrocarbons, benzene, toluene, ethylbenzene, n-propylbenzene, m-xylene, and p-xylene increased several P450-dependent activities, with ethylbenzene, m-xylene, and n-propylbenzene producing the greatest response. Hydrocarbon treatment differentially affected toluene metabolism, producing a response dependent on the metabolite monitored. In untreated rats, benzyl alcohol was the major hydroxylation product of toluene metabolism, comprising > 99% of the total metabolites formed. Hydrocarbon treatment increased the overall rate of toluene metabolism by dramatically increasing the amount of aromatic hydroxylation. Ethylbenzene, n-propylbenzene and m-xylene were the most effective inducers of aromatic hydroxylation of toluene. In contrast, production of the major toluene metabolite benzyl alcohol was increased only after treatment with m-xylene. 2. P450 2B1/2B2 levels were induced by each of the hydrocarbons examined, with the magnitude of induction increasing with increasing hydrocarbon size. P450 1A1 was also induced after hydrocarbon exposure; however, the degree of induction was smaller than that observed for P450 2B1/2B2. P450 2C11 levels were suppressed after treatment with benzene, ethylbenzene and n-propylbenzene. 3. Taken together these results display two induction patterns. The first generally corresponds to changes in the P450 2B subfamily, where activities (e.g. the aromatic hydroxylations of toluene) were most effectively induced by ethylbenzene, n-propylbenzene and m-xylene. In the second, induction was observed only after m-xylene treatment, a pattern that was found when the metabolism of the substrate was catalysed by both the P450 2B subfamily and P450 2C11. Hydrocarbons that both induced P450 2B1/2B2 and suppressed P450 2C11 (such as ethylbenzene and n-propylbenzene) showed little change in activities catalysed by both isozymes (e.g. aliphatic hydroxylation of toluene, and aniline hydroxylation); however, m-xylene treatment led to elevated P450 2B1/2B2 levels without significantly suppressing P450 2C11. m-Xylene produced significant increases in activities efficiently catalysed by both isozymes. Therefore, the unique induction pattern observed after m-xylene treatment can be accounted for by induction of P450 2B1/2B2 without concomitant suppression of P450 2C11.

Aromatic hydrocarbon binding to cytochrome P450 and other enzyme binding sites: are hydrophobic compounds drawn into the active site or pushed from the aqueous phase?

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.

Ethylbenzene-mediated induction of cytochrome P450 isozymes in male and female rats.

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-ethoxycoumarin 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.

Relationship between the rate of reductase-cytochrome P450 complex formation and the rate of first electron transfer.

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.

Dual role of phospholipid in the reconstitution of cytochrome P-450 LM2-dependent activities.

The effect of dilauroylphosphatidylcholine (DLPC) concentration on cytochrome P-450 LM2 (LM2)-dependent reduction and monooxygenase activities was examined as a function of preincubation time. Purified NADPH-cytochrome P-450 reductase (reductase) and LM2 were reconstituted at different DLPC to LM2 ratios by preincubation of the proteins in the presence of DLPC for either 5 min or 2 hr at room temperature. After preincubation was complete, the samples were assayed for either monooxygenase activity or first-electron transfer activity. When preincubated for 5 min, overall monooxygenase activity was dependent on the [DLPC]:[LM2] ratio, beginning at a low level in the absence of phospholipid and increasing to a maximum at a 160:1 ratio. At [DLPC]:[LM2] ratios above 160:1, the rate was decreased to 80% of the maximum rate. When the samples were preincubated for 2 hr, again low monooxygenase activities were obtained in the absence of DLPC, which increased to a maximum at 160:1 [DLPC]:[LM2] ratio. Above this [DLPC]:[LM2]ratio, the rate was decreased to less than 50% of the maximum value. These changes in overall activities appear to be related to changes in the amount of functional reductase-LM2 complex formed. Similar results were found when LM2 reduction was examined. When preincubated for 5 min, LM2 reduction was shown to be diminished as the DLPC to LM2 ratio decreased below 160:1. The DLPC-dependent effect on reduction was primarily characterized by alterations in the fraction of LM2 reduced in the first phase, with the first-phase rate constant and the slow phase parameters being largely unaffected. Below a 16:1 ratio [( DLPC]:[LM2]), no phospholipid stimulation of LM2 reduction was observed. When the [DLPC]:[LM2] ratio was increased above a 160:1 ratio, only a small effect on the kinetic constants was observed, which was characterized by a 20% decrease in the fraction of LM2 reduced in the first phase. LM2 reduction was more sensitive to DLPC concentration after longer preincubations (2 hr), with a 50% decrease in the fraction of reduction in the first phase being observed at [DLPC]:[LM2] ratios above 160:1. The results are consistent with a dual role for phospholipid in the stimulation of LM2-dependent activities. First, DLPC facilitates the association of reductase and LM2 and, second, DLPC provides a matrix for the incorporation of LM2 and reductase. Facilitation of the protein association appears to be a relatively rapid process, occurring after a 5-min preincubation, whereas a 2-hr preincubation altered the protein interactions in a manner consistent with incorporation of the LM2 and reductase into the phospholipid.

Cytochrome P-450 LM2 reduction. Substrate effects on the rate of reductase-LM2 association.

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.