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.

Distribution of nitric oxide synthase in rat dorsal vagal complex and effects of microinjection of nitric oxide compounds upon gastric motor function.

Nitric oxide (NO) has received attention as a vagal nonadrenergic-noncholinergic (NANC) mediator of gastrointestinal relaxation. The dorsal vagal complex (DVC) is the primary hindbrain site of vagal control of the gastrointestinal tract, and yet the subnuclear distribution of NO and its physiological effects have not been analyzed in this nucleus. Therefore, this study estimates the relative number of NO synthase (NOS)-containing neurons in subnuclear regions of the DVC, identifies NOS-containing vagal abdominal preganglionic neurons in the dorsal motor nucleus of the vagus, and defines a role of NO in the DVC in control of gastric motor function. The location of NADPH-diaphorase-positive staining (a marker of NOS activity) and NOS immunoreactivity overlap in the DVC. In the dorsal motor nucleus of the vagus there are positively stained cells caudal to the obex and at its most rostral extent, but not at the intermediate level. Intraperitoneal fluorogold combined with NADPH-diaphorase activity labels approximately 5% and 15% of fluorogold-immunoreactive cells in the caudal and rostral dorsal motor nucleus of the vagus, respectively. Thus, a portion of NOS-containing neurons are preganglionic vagal neurons projecting to the abdominal viscera. In the nucleus tractus solitarius, the majority of NADPH-diaphorase-positive cells are within the centralis, medial, and ventral/ventrolateral subnuclei. Fiber/terminal staining is present in the subnucleus centralis, subnucleus gelatinosus, subpostremal zone, and the medial nucleus tractus solitarius. The presence of NOS terminal staining implicates NO in afferent control of gastric function in the DVC (e.g., vago-vagal circuits in subnucleus gelatinosus). To determine a role of NO in the DVC, NO-related agents were microinjected into the DVC in alpha-chloralose-anesthetized rats while recording indices of gastric motor function. L-Arginine, microinjected into the DVC, significantly decreases intragastric pressure (-2.2 +/- 0.4 cm2, N = 12), and this effect is abolished by vagotomy. Microinjection of an NOS inhibitor, NG-nitro-L-arginine methyl ester, increases intragastric pressure (1.9 +/- 0.7 cm2, N = 10), with the greatest effect in the DVC rostral to the obex. Overall, it was concluded that tonic release of NO in the DVC mediates gastric relaxation, at least in anesthetized animals, and NOS-containing preganglionic neurons in the dorsal motor nucleus of the vagus may be "command" NANC neurons which control a variety of gastrointestinal functions.

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.

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.

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.

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.