The effect of endotoxin on hepatocyte nuclear factor 1 nuclear protein binding: potential implications on CYP2E1 expression in the rat.

The purpose of this study was to determine if changes in nuclear protein binding of hepatocyte nuclear factor 1 (HNF-1) occur after lipopolysaccharide (LPS) administration. In addition, the time-course of alterations in CYP2E1 regulation were evaluated. Rats were injected with 2.0 mg LPS and euthanized over a 72-h period. Nuclear protein binding to a consensus HNF-1 oligonucleotide was assessed by the electrophoretic mobility shift assay. CYP2E1 activity was analysed using chlorzoxazone as a substrate (60H-CLZ), and CYP2E1 protein concentration was determined by enzyme-linked immunosorbent assay. Endotoxin treatment resulted in decreased nuclear protein binding to an HNF-1 element as early as 1 h after treatment and returned to control levels by 72 h. This reduced binding persisted for 24 h and returned to control values 48 h after LPS administration. In addition, the reduction in binding was primarily attributable to a HNF-1alpha immunoreactive protein. The observed reduction in HNF-1 binding was followed in the time-course by decreases in CYP2E1 activity and protein content with maximal decreases to 50 and 67% of control, respectively, at 48 h after LPS administration. Endotoxin is a potent inducer of the acute phase response (APR). The APR stimulation by endotoxin administration reduced HNF-1alpha binding and decreased the expression of CYP2E1 in the rat liver. The time-course of alterations in HNF-1 and CYP2E1 lend support to the possibility that HNF-1alpha may play a role in the down-regulation of genes that require HNF-1alpha for their constitutive expression. These data serve as an important precedent for future studies evaluating the direct association of decreased HNF-1alpha binding and reduced gene expression after LPS administration.

Tissue-specific alterations in the 6-hydroxylation of chlorzoxazone following traumatic brain injury in the rat.

Interest in the non-neuronal alterations following traumatic brain injury (TBI) has led to research evaluating hepatic metabolism following injury. Several models of injury demonstrate tissue-specific alterations in cytochrome P450 activity. This study examined tissue-specific alterations in cytochrome P450-mediated hydroxylation in the rat model of TBI. Male Sprague-Dawley rats received anesthesia alone, craniotomy, or craniotomy plus TBI. Rats were sacrificed at 24 and 48 h. Liver, kidney, and brain cortex microsomes were isolated. Total liver P450 content, 6-hydroxychlorzoxazone formation rate, and CYP2E1 protein were evaluated. In liver microsomes, spectral P450 was decreased to 86 +/- 5% (p < 0.05) of control at 24 h following injury, and 6-hydroxychlorzoxazone formation rate decreased to 74 +/- 18% of control (p < 0.05) at 48 h following injury. In kidney microsomes, 6-hydroxychlorzoxazone formation rate was increased to 154% of control (p < 0.01) 24 h following injury. 6-Hydroxychlorzoxazone formation rate was unaffected by TBI in brain cortical microsomes. The CYP2E1 inhibitor, 4-methylpyrazole, inhibited the formation of 6-hydroxychlorzoxazone in brain, kidney, and liver microsomes. These data demonstrate that tissue-specific alterations in 6-hydroxychlorzoxazone formation rate occur following TBI.

The effect of endotoxin administration on the pharmacokinetics of chlorzoxazone in humans.

OBJECTIVE: Inflammation induced by Escherichia coli lipopolysaccharide alters the clearance of several hepatically eliminated drugs. Extensive rat liver research has shown CYP2E1 down-regulation after lipopolysaccharide administration. To further investigate this phenomenon in humans, lipopolysaccharide was administered to healthy male volunteers and chlorzoxazone was used as a CYP2E1 probe drug. METHODS: Twelve healthy men were given 500 mg oral chlorzoxazone after two daily lipopolysaccharide doses (20 endotoxin units/kg/day) and again after administration of saline solution in this balanced crossover study. Serum and urine chlorzoxazone and 6-hydroxychlorzoxazone were quantified, as well as cytokine and C-reactive protein levels. RESULTS: Lipopolysaccharide produced the expected induction of the acute-phase response shown by elevations in tumor necrosis factor, interleukin-6, C-reactive protein, and temperature. Lipopolysaccharide treatment failed to produce a significant change in the chlorzoxazone oral clearance (4.4 +/- 0.9 mL/min/kg for lipopolysaccharide versus 4.2 +/- 1.4 mL/min/kg for control) or the 6-hydroxychlorzoxazone formation clearance (2.8 +/- 0.65 mL/min/kg for lipopolysaccharide versus 2.5 +/- 0.9 mL/min/kg for control). The high intersubject variabilities in oral clearance and formation clearance were not accounted for by changes in protein binding, cytokine, or C-reactive protein values. In contrast, a significant increase in the 6-hydroxychlorzoxazone glucuronide renal clearance was observed (7.5 +/- 1.37 mL/min/kg for lipopolysaccharide versus 6.1 +/- 1.7 mL/min/kg for control). CONCLUSIONS: This study showed that the inflammatory response to lipopolysaccharide (20 endotoxin units/kg/day for 2 days) in humans does not consistently alter chlorzoxazone hepatic metabolism. However, the significant increase in renal clearance of the glucuronidated metabolite suggests that renal tubular secretion may be increased in humans with acute endotoxemia.

The effect of pregnenolone 16alpha-carbonitrile on the pharmacokinetics and metabolism of dapsone in rats.

The purpose of this study was to evaluate the effect of pregnenolone 16 alpha-carbonitrile (PCN) on the interconversion pharmacokinetics and metabolism of dapsone. To determine microsomal CYP3A activity and protein, eight rats (4 PCN, 4 corn oil) received a 1 mg kg(-1) intravenous bolus dose of dapsone, followed by blood and urine sampling. The formation clearance of dapsone hydroxylamine (CLf DDS-NOH) was calculated from the obtained samples. Interconversion pharmacokinetics estimates were obtained after 10 rats (5 PCN, 5 control) received 1 mg kg(-1) dapsone or 1.17 mg kg(-1) monoacetyldapsone, with a 24-h wash-out. Results from the interconversion analysis demonstrated that PCN significantly increased systemic clearance (CLs) of dapsone, but not its interconversion. The in-vivo/in-vitro correlation study demonstrated that PCN significantly increased CLs of dapsone (8.55 to 16.39mLmin(-1); P<0.01) and CLf DDS-NOH (0.13 to 0.18mLmin(-1); P<0.01). PCN treatment produced a 69% increase in CYP3A protein, and increased 6beta- and 2beta-hydroxytestosterone formation rates. Significant correlations were found between CLf DDS-NOH and either 6beta- (r2 = 0.925), 2beta-hydroxytestosterone (r2 = 0.92), or CYP3A1/2 protein (r2= 0.60). We conclude that PCN treatment produces significant increases in CLs (dapsone) and CLf (DDS-NOH) in rats. These changes were not due to changes in the reversible metabolism of dapsone. These results suggest that the formation clearance of dapsone hydroxylamine reflects alterations in CYP3A activity, despite the fact that it accounted for a small part of the systemic clearance of dapsone.

Effect of leptin on cytochrome P-450, conjugation, and antioxidant enzymes in the ob/ob mouse.

Leptin is a hormone that is secreted by adipocytes and regulates body weight through its effect on satiety and energy metabolism. The ob/ob mouse is deficient in this protein and is characterized by obesity and other metabolic disorders. This study investigated the alterations of several hepatic cytochrome P-450 (CYP), conjugation, and antioxidant enzymes in lean and ob/ob mice and the role leptin plays in the modulation of these enzymes. Lean and ob/ob male mice were injected with leptin (100 microg) or PBS for 15 days. Liver microsomes from ob/ob mice, when compared with lean controls, displayed significantly reduced chlorzoxazone 6-hydroxylation activity (27%); however, 7alpha- and 16alpha- testosterone hydroxylation and pentoxyresorufin O-dealkylation activities were significantly higher (47%, 22%, and 39%, respectively). Leptin administration corrected alterations seen with all P-450 activities. Dealkylation of ethoxyresorufin and omega-hydroxylation of lauric acid activities from ob/ob and lean mice were not statistically different; however, leptin exposure significantly increased ethoxyresorufin activity in lean mice (14%) and decreased the activity in ob/ob mice (36%). UDP-glucuronosyl-transferase and glutathione S-transferase activities were not altered. The antioxidant enzymes, catalase (11%) and glutathione peroxidase (26%), as well as glutathione reductase (17%), were lower in the ob/ob mice and leptin treatment corrected these alterations. The results of this study demonstrate alterations in constitutive expression of CYP2B, CYP2E, CYP2A, catalase, glutathione peroxidase, and glutathione reductase in ob/ob mice that were restored to lean control values following leptin treatment. Additionally, CYP3A activity was increased following leptin treatment in ob/ob mice. The mechanism for the observed alterations may be due to direct leptin effects or via indirect alterations in insulin, corticosterone, and/or growth hormone.

Hepatic cytochrome P-450 expression in tumor necrosis factor-alpha receptor (p55/p75) knockout mice after endotoxin administration.

Hepatic cytochromes P-450 (CYP) are well characterized drug and xenobiotic metabolizing enzymes that are extensively regulated by genetic and environmental factors. Inflammatory mediators, including interleukins (ILs), interferons (IFNs), and tumor necrosis factor-alpha (TNF-alpha), have been shown to down-regulate several CYP isoforms; however, elucidation of the inflammatory mediators that are responsible for specific CYP down-regulation is difficult. The purpose of this experiment was to evaluate the role endogenous TNF-alpha plays in the regulation of liver CYP expression after endotoxin administration. Mice deficient in the p55 and p75 TNF receptors and wild-type mice were given Gram-negative bacterial lipopolysaccharide (LPS) and killed 24 h after administration. CYP analysis indicates that LPS decreases CYP1A, CYP2B, CYP3A, and CYP4A independently of TNF-alpha. CYP2D9 and CYP2E1 activities show differential responses to LPS between wild-type and TNF p55/p75 receptor knockout mice, indicating the down-regulation of CYP2D9 and CYP2E1 is differentially modulated by TNF-alpha expression. Furthermore, TNF-alpha appears to affect the constitutive expression of CYP2D9 and CYP2E1. To date, this is the first evidence suggesting that a proinflammatory cytokine is involved in the constitutive regulation of drug-metabolizing enzymes.