George F. Cawley

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.

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).

Acute Choline Supplementation In Vivo Enhances Acetylcholine Synthesis In Vitro when Neurotransmitter Release Is Increased by Potassium

Abstract: The main objective of these studies was to determine whether the acute administration of choline to rats provides supplemental precursor that can be used to support acetylcholine synthesis when the demand for choline is increased by increasing neurotransmitter release. For these experiments, hippocampal and striatal slices were prepared from rats that had received saline or an acute injection of choline. Slices were incubated in a choline‐free buffer containing 4.74–35 mM KC, and acetylcholine synthesis and release and choline production were measured. The initial tissue contents of acetylcholine and choline did not differ between experimental groups for either brain region. When hippocampal slices from the controls were incubated for 10 min with depolarizing concentrations of KCl, acetylcholine release increased and the tissue content decreased in a concentration‐dependent fashion; no net synthesis of acetylcholine occurred. In contrast, hippocampal slices from the choline‐injected animals maintained their tissue content in the presence of high concentrations of KCl, despite an increase in acetylcholine release that was similar in magnitude to that of the controls; positive net synthesis of acetylcholine resulted. Although the molar concentration of choline achieved in the incubation media at the end of the 10‐min period did not differ between groups, the mobilization of free choline from bound stores was significantly greater in hippocampal slices from the choline‐injected group than the controls. In addition, the synthesis of acetylcholine by hippocampal slices from the cholineinjected group was prevented by the presence of hemicholinium‐3 (1 μM) in the media. Acetylcholine synthesis and choline mobilization by striatal slices did not differ between groups. Results demonstrate that acute supplementation with choline provides precursor to support acetylcholine synthesis when the release of neurotransmitter is increased.

Effect of Homomeric P450•P450 Complexes on P450 Function

Previous studies have shown that the presence of one P450 enzyme can affect the function of another. The goal of the present study was to determine if P450 enzymes are capable of forming homomeric complexes that affect P450 function. To address this problem, the catalytic activities of several P450s were examined in reconstituted systems containing NADPH-POR (cytochrome P450 reductase) and a single P450. CYP2B4 (cytochrome P450 2B4)-, CYP2E1 (cytochrome P450 2E1)- and CYP1A2 (cytochrome P450 1A2)-mediated activities were measured as a function of POR concentration using reconstituted systems containing different concentrations of P450. Although CYP2B4-dependent activities could be explained by a simple Michaelis-Menten interaction between POR and CYP2B4, both CYP2E1 and CYP1A2 activities generally produced a sigmoidal response as a function of [POR]. Interestingly, the non-Michaelis behaviour of CYP1A2 could be converted into a simple mass-action response by increasing the ionic strength of the buffer. Next, physical interactions between CYP1A2 enzymes were demonstrated in reconstituted systems by chemical cross-linking and in cellular systems by BRET (bioluminescence resonance energy transfer). Cross-linking data were consistent with the kinetic responses in that both were similarly modulated by increasing the ionic strength of the surrounding solution. Taken together, these results show that CYP1A2 forms CYP1A2-CYP1A2 complexes that exhibit altered catalytic activity.

Temporal changes in P-4502E1 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.

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.

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.

Chronic choline supplementation attenuates the behavioral effects of pentobarbital

The behavioral and neurochemical effects of pentobarbital were investigated in rats maintained for 28-35 days on a standard choline-containing diet or on a diet containing 10 times the concentration of choline present in standard rodent chow. The supplemented dietary regimen increased the concentration of free choline in serum by 52%, but did not alter the steady-state concentrations of either choline or acetylcholine in brain. Choline supplementation attenuated both the sedative/hypnotic and hypothermic effects of pentobarbital through an action that could not be attributed to either an enhanced peripheral metabolism of pentobarbital or to an attenuation of the cholinergic effects of pentobarbital. Rather, results indicate that chronic supplementation with choline increases cerebral glucose metabolism and causes a behavioral hyperactivity, effects that may mediate the attenuation of the behavioral response of pentobarbital.

Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes

Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230°C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition.