Wayne L. Backes

Origin and health impacts of emissions of toxic by-products and fine particles from combustion and thermal treatment of hazardous wastes and materials.

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.

Formation of P450 · P450 complexes and their effect on P450 function

Cytochromes P450 (P450) are membrane-bound enzymes that catalyze the monooxygenation of a diverse array of xenobiotic and endogenous compounds. The P450s responsible for foreign compound metabolism generally are localized in the endoplasmic reticulum of the liver, lung and small intestine. P450 enzymes do not act alone but require an interaction with other electron transfer proteins such as NADPH-cytochrome P450 reductase (CPR) and cytochrome b(5). Because P450s are localized in the endoplasmic reticulum with these and other ER-resident proteins, there is a potential for protein-protein interactions to influence P450 function. There has been increasing evidence that P450 enzymes form complexes in the ER, with compelling support that formation of P450 · P450 complexes can significantly influence their function. Our goal is to review the research supporting the formation of P450 · P450 complexes, their specificity, and how drug metabolism may be affected. This review describes the potential mechanisms by which P450s may interact, and provides evidence to support each of the possible mechanisms. Additionally, evidence for the formation of both heteromeric and homomeric P450 complexes are reviewed. Finally, direct physical evidence for P450 complex formation in solution and in membranes is summarized, and questions directing the future research of functional P450 interactions are discussed with respect to their potential impact on drug metabolism.

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.

Functional Interactions between Cytochromes P450 1A2 and 2B4 Require Both Enzymes to Reside in the Same Phospholipid Vesicle EVIDENCE FOR PHYSICAL COMPLEX FORMATION

Previous studies have shown that the combined presence of two cytochrome P450 enzymes (P450s) can affect the function of both enzymes, results that are consistent with the formation of heteromeric P450·P450 complexes. The goal of this study was to provide direct evidence for a physical interaction between P450 1A2 (CYP1A2) and P450 2B4 (CYP2B4), by determining if the interactions required both enzymes to reside in the same lipid vesicles. When NADPH-cytochrome P450 reductase (CPR) and a single P450 were incorporated into separate vesicles, extremely slow reduction rates were observed, demonstrating that the enzymes were anchored in the vesicles. Next, several reconstituted systems were prepared: 1) CPR·CYP1A2, 2) CPR·CYP2B4, 3) a mixture of CPR·CYP1A2 vesicles with CPR·CYP2B4 vesicles, and 4) CPR·CYP1A2·CYP2B4 in the same vesicles (ternary system). When in the ternary system, CYP2B4-mediated metabolism was significantly inhibited, and CYP1A2 activities were stimulated by the presence of the alternate P450. In contrast, P450s in separate vesicles were unable to interact. These data demonstrate that P450s must be in the same vesicles to alter metabolism. Additional evidence for a physical interaction among CPR, CYP1A2, and CYP2B4 was provided by cross-linking with bis(sulfosuccinimidyl) suberate. The results showed that after cross-linking, antibody to CYP1A2 was able to co-immunoprecipitate CYP2B4 but only when both proteins were in the same phospholipid vesicles. These results clearly demonstrate that the alterations in P450 function require both P450s to be present in the same vesicles and support a mechanism whereby P450s form a physical complex in the membrane.

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 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.

Organization of NADPH-cytochrome P450 reductase and CYP1A2 in the endoplasmic reticulum--microdomain localization affects monooxygenase function.

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.

Inhibition of Cytochrome P450 1A2-Mediated Metabolism and Production of Reactive Oxygen Species by Heme Oxygenase-1 in Rat Liver Microsomes

Heme oxygenase-1 (HO-1) is induced in most cell types by many forms of environmental stress and is believed to play a protective role in cells exposed to oxidative stress. Metabolism by cytochromes P450 (P450) is highly inefficient as the oxidation of substrate is associated with the production of varying proportions of hydrogen peroxide and/or superoxide. This study tests the hypothesis that heme oxygenase-1 (HO-1) plays a protective role against oxidative stress by competing with P450 for binding to the common redox partner, the NADPH P450 reductase (CPR) and in the process, diminishing P450 metabolism and the associated production of reactive oxygen species (ROS). Liver microsomes were isolated from uninduced rats and rats that were treated with cadmium and/or β-napthoflavone (BNF) to induce HO-1 and/or CYP1A2. HO-1 induction was associated with slower rates of metabolism of the CYP1A2-specific substrate, 7-ethoxyresorufin. Furthermore, HO-1 induction also was associated with slower rates of hydrogen peroxide and hydroxyl radical production by microsomes from rats induced for CYP1A2. The inhibition associated with HO-1 induction was not dependent on the addition of heme to the microsomal incubations. The effects of HO-1 induction were less dramatic in the absence of substrate for CYP1A2, suggesting that the enzyme was more effective in inhibiting the CYP1A2-related activity than the CPR-related production of superoxide (that dismutates to form hydrogen peroxide).

Isolation and Comparison of Four Cytochrome P-450 Enzymes from Phenobarbital-Induced Rat Liver: Three Forms Possessing Identical NH2-Terminal Sequences

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 identical NH2-terminal amino acid sequence for the first 32 residues (PBRLM5, 6 and 7). This sequence is identical to that shown earlier for PB-4 and PB-5. Apparent values for minimum molecular weight on SDS-PAGE were 52,000 (PBRLM4), 53,000 (PBRLM5), 53,500 (PBRLM6) and 54,000 (PBRLM7). Isomeric metabolite patterns from testosterone and progesterone differed for each enzyme further indicating their unique natures. Studies reveal similarity of PBRLM4 to PB-1, of PBRLM5 to P-450b and PB-4, and PBRLM6 to P-450e and PB-5. PBRLM7, which does not correspond to any reported forms, metabolizes steroids poorly. It preferentially hydroxylates testosterone at the 16 beta-position. It is the largest and least active of the enzymes shown for all of the substrates tested. This study further provided a cautionary note against assuming that chromatographic pools, like a P-450 PB-B fraction, are homogeneous.

AN EVALUATION OF METHODS FOR THE RECONSTITUTION OF CYTOCHROMES P450 AND NADPH P450 REDUCTASE INTO LIPID VESICLES

Two methods (cholate dialysis and cholate gel filtration) used to incorporate cytochromes P450 (P450s) and reductase into unilamellar phospholipid vesicles were compared with a standard reconstituted system (SRS) in which the proteins were reconstituted with preformed liposomes. Both cholate dialysis and gel filtration methods were comparable in their ability to physically incorporate reductase and either CYP2B4 or CYP1A2 into phospholipid, as determined by the elution of enzymes in the void volume using size exclusion chromatography (mol. wt. cutoff –5,000,000). Incorporation of these proteins was more efficient with both cholate methods than when reductase and P450 were mixed with preformed vesicles (SRS). Using either cholate method, more than 85% of the P450 was physically incorporated into the phospholipid vesicles, whereas less than 40% of the P450 was physically incorporated into the phospholipid vesicles using the SRS. Catalytic activities of the vesicular preparations of reductase and either CYP1A2 or CYP2B4 also were significantly higher than those resulting from the SRS using dilaurylphosphatidylcholine. Although both cholate dialysis and gel filtration methods improved protein incorporation when compared with preincubation of proteins with preformed liposomes, reductase incorporation was dependent on the relative amount of reductase used. Reductase incorporation was complete at a 0.2:1 reductase/P450 ratio; however, the efficiency of incorporation decreased to less than 50% at equimolar reductase/P450. Interestingly, reductase incorporation was higher in the presence of CYP1A2 than with CYP2B4. Both cholate methods resulted in the loss of a proportion of spectrally detectable carbon monoxyferrous P450, resulting from incubation of the proteins with detergent.