In Situ X-ray Absorption Study of Surface Complexes: Selenium Oxyanions on agr-FeOOH.

A novel application of x-ray absorption spectroscopy has provided structural information for ions sorbed at oxide-water interfaces. As an example, in situ extended x-ray absorption fine structure (EXAFS) measurements of adsorbed selenate and selenite ions at ah alpha-FeOOH(goethite)-water interface have been performed; these measurements show that selenate forms a weakly bonded, outer-sphere complex and that selenite forms a strongly bonded, inner-sphere complex. The selenite ion is bonded directly to the goethite surface in a bidentate fashion with two iron atoms 3.38 angstroms from the selenium atom. Adsorbed selenate has no iron atom in the second coordination shell of selenium, which indicates retention of its hydration sphere upon sorption. This method provides direct structural information for adsorbed species at solid-liquid interfaces.

The metabolic profile of azimilide in man: in vivo and in vitro evaluations.

The metabolic fate of azimilide in man is unusual as it undergoes a cleavage in vivo resulting in the formation of two classes of structurally distinct metabolites. During a metabolite profiling study conducted in human volunteers to assess the contribution of all pathways to the clearance of (14)C-azimilide, greater than 82% of radioactivity was recovered in urine (49%-58%) and feces (33%). Urine, feces, and plasma were profiled for metabolites. A cleaved metabolite, 4-chloro-2-phenyl furoic acid was present at high concentration in plasma (metabolite/parent AUC ratio approx. 4), while other plasma metabolites, azimilide N-oxide (metabolite/parent AUC ratio 0.001), and a cleaved hydantoin metabolite (metabolite/parent AUC ratio = 0.3) were present at lower concentrations than azimilide. In urine, the cleaved metabolites were the major metabolites, (> 35% of the dose) along with phenols (as conjugates, 7%-8%), azimilide N-oxide (4%-10%), a butanoic acid metabolite (2%-3%), and desmethyl azimilide (2%). A limited investigation of fecal metabolites indicated that azimilide (3%-5%), desmethyl azimilide (1%-3%), and the butanoic acid metabolite (< 1%) were present. Contributing pathways for metabolism of azimilide, identified through in vitro and in-vivo studies, were CYPs 1A1 (est. 28%), 3A4/5 (est. 20%), 2D6 (< 1%), FMO (est. 14%), and cleavage (35%). Enzyme(s) involved in the cleavage of azimilide were not identified.

Dot-blot hybridization: quantitative analysis with direct beta counting.

The suitability of using direct beta counting (DBC) for quantitating radioactivity of the probe:target complex in dot-blot hybridization was evaluated using a Packard Matrix 96. A comparison of blots analyzed using autoradiography followed by densitometry scanning (film/densitometry) with those analyzed using direct beta counting revealed similar data trends with the two methods. However, direct beta counting quantitated the amount of radioactivity in the dot blots directly (without film exposure or additional sample preparation), which significantly reduced the time required to obtain results. Blots analyzed first with direct beta counting and then liquid scintillation counting exhibited similar data trends with both methods. Despite a decreased counting efficiency, analysis with direct beta counting has the following advantages compared with liquid scintillation counting: 1) no additional sample preparation is required (no vials or cocktail are used), 2) no sample destruction occurs due to analysis and 3) quantitative results are obtained more rapidly (since the radioactivity for all 96 samples in a dot blot is simultaneously determined in real time). Analysis with direct beta counting was also shown not to interfere with the successful reprobing of stripped dot blots with either unique sequence or total genomic probes. Overall, direct beta counting provides quick, quantitative results for dot blots while saving considerable time and effort.

Role of the aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control, and apoptosis.

The chronology and history of characterizing the aromatic hydrocarbon [Ah] battery is reviewed. This battery represents the Ah receptor (AHR)-mediated control of at least six, and probably many more, dioxin-inducible genes; two cytochrome P450 genes-P450 1A1 and 1A2 (Cypla1, Cypla2-and four non-P450 genes, have experimentally been documented to be members of this battery. Metabolism of endogenous and exogenous substrates by perhaps every P450 enzyme, but certainly CYP1A1 and CYP1A2 (which are located, in part, in the mitochondrion), have been shown to cause reactive oxygenated metabolite (ROM)-mediated oxidative stress. Oxidative stress activates genes via the electrophile response element (EPRE) DNA motif, whereas dioxin (acutely) activates genes via the AHR-mediated aromatic hydrocarbon response element (AHRE) DNA motif. In contrast to dioxin, AHR ligands that are readily metabolized to ROMs (e.g. benzo[a]pyrene, beta-naphthoflavone) activate genes via both AHREs and the EPRE. The importance of the AHR in cell cycle regulation and apoptosis has just begun to be realized. Current evidence suggests that the CYP1A1 and CYP1A2 enzymes might control the level of the putative endogenous ligand of the AHR, but that CYPA1/1A2 metabolism generates ROM-mediated oxidative stress which can be ameliorated by the four non-P450 EPRE-driven genes in the [Ah] battery. Oxidative stress is a major signal in precipitating apoptosis; however, the precise mechanism, or molecule, which determines the cell's decision between apoptosis and continuation with the cell cycle, remains to be elucidated. The total action of AHR and the [Ah] battery genes therefore represents a pivotal upstream event in the apoptosis cascade, providing an intricate balance between promoting and preventing ROM-mediated oxidative stress. These proposed endogenous functions of the AHR and [Ah] enzymes are, of course, in addition to the frequently described functions of "metabolic potentiation" and "detoxification" of various foreign chemicals.

Nitrobenzo[a]pyrene-induced DNA amplification in SV40-transformed Chinese hamster embryo cells.

Nitrobenzo[a]pyrenes (NBaPs) are ubiquitous environmental pollutants that produce mutations in Salmonella typhimurium and Chinese hamster ovary cells. In this study, 1-, 3-, and 6-NBaP induced amplification of SV40 DNA sequences in an SV40-transformed Chinese hamster embryo cell line which is sensitive to DNA amplification by various known carcinogens. Of the three isomers, 3-NBaP produced the highest level of gene amplification, which was 4.8 relative to untreated controls at a dose of 5 micrograms/ml. Considering the relationship between gene amplification and tumorigenesis, it seems prudent to carry out a more exhaustive analysis of the carcinogenic potential of these agents.

Characterization of nuclear protein binding (AP-1, GR, and STAT) in the genetically obese (fa/fa) Zucker rat.

There is evidence to suggest that obese populations have an increased susceptibility to various pathologic disorders. Both AP-1 and STAT nuclear binding proteins have been suggested to play a role in certain obesity-related diseases. The objective of our studies reported herein was to compare constitutive binding activity of nuclear proteins (AP-1, GR, and STAT), that may be relevant to obesity-related diseases in the obese (fa/fa) Zucker rat to lean (Fa/?) littermates. AP-1, GR, and STAT liver nuclear protein binding activity was analyzed using the electrophoretic mobility shift assay (EMSA). EMSA analysis of liver nuclear protein from obese and lean Zucker rats revealed high constitutive AP-1 binding activity in the obese animals. AP-1 binding activity in the obese rats was not further elevated by treatment with phenobarbital, a known inducer of AP-1 binding activity. No differences were observed in GR binding to a consensus GRE between obese and lean animals; however, STAT binding activity to a consensus GAS element was lower in liver tissue from obese Zucker rats. Our findings presented herein suggest that the fa/fa Zucker rat may be a suitable obese rodent model for studying the roles AP-1 and STAT may play in the pathologies of these diseases.

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.

Ethnic and genetic differences in metabolism genes and risk of toxicity and cancer.

Individual risk of toxicity or cancer can be affected by one's exposure to sufficiently high doses of particular environmental agents (or mixtures), combined with each person's underlying genetic predisposition. The development of unequivocal DNA tests for genetic susceptibility to toxicity and cancer and the identification of individuals at increased risk, would revolutionize the fields of public health and preventive medicine. A growing number of human genetic polymorphisms in drug-metabolizing enzymes (DMEs) and the receptors controlling DME expression, are being characterized, some of these have been shown to be correlated with risk of toxicity or cancer, whereas, others presently remain equivocal and require further study. 'Phase I' DMEs, many of which represent cytochromes P450, sometimes metabolically activate pro-carcinogens to genotoxic electrophilic intermediates and other times are involved in detoxification. 'Phase II' DMEs are sometimes activating, but usually they conjugate Phase I intermediates to water-soluble derivatives, to complete the detoxification cycle. Genetic differences in the regulation, expression and activity of genes coding for Phase I and Phase II DMEs and DME receptors that control DME activity levels, can be crucial factors in defining cancer susceptibility and the toxic or carcinogenic power of environmental chemicals. In this review, our current knowledge about polymorphisms in several of these genes is summarized.

Characterization and regulation of angiotensin II receptors in rat adipose tissue. Angiotensin receptors in adipose tissue.

Characterization and regulation of angiotensin II (AII) receptor binding sites was performed in rat membrane preparations from nonadipose (liver, lung) and adipose (interscapular (ISBAT) and periaortic (PA) brown adipose tissue; epididymal (EF) and retroperitoneal (RPF) white adipose tissue). In membrane preparations from brown and white adipose sources, [125I]AII saturation binding revealed a single, high affinity (Kd range of 0.3 -0.6 nM) binding site with a modest AII receptor density (Bmax range of 17-120 fmol/mg protein) comparable to rat lung (130 fmol/mg protein). White adipose tissue contained a greater number of AII receptor sites than brown adipose tissue. Competition displacement studies demonstrated the AT1 receptor is the only angiotensin receptor subtype localized in adipose tissue, with the rank order for competition of [125I]AII binding in all adipose tissues examined AIII > AII > losartan > angiotensin I (AI) > PD123319. The AT2 specific receptor antagonist, PD123319, was ineffective at displacing [125I]AII binding in all adipose tissues examined. Since components of the renin-angiotensin system are regulated in adipose tissue, we determined if the AII receptor is also regulated in the obese state. AII receptor binding characteristics were determined in liver, lung, ISBAT and EF membrane preparations from adult Zucker obese (fa/fa) and lean (Fa/?) rats. AII receptor density was decreased in liver from obese rats. In contrast, the affinity for [125I]AII binding was not altered in tissues from obese rats. In a separate group of obese and lean rats, regulation of the AII receptor by phenobarbital (PB) was examined. Administration of PB restored AII receptor density in liver from obese rats to levels obtained in lean rats. In summary, these results demonstrate the presence of AT1 receptor sites in brown and white adipose tissue. Moreover, AII receptor density is decreased in tissues from obese rats, with restoration of receptor density by administration of PB. Future studies will determine if PB regulates the AT1 receptor at the level of gene expression.

In vivo phenobarbital treatment increases protein binding to a putative AP-1 site in the CYP2B2 promoter.

Phenobarbital (PB) is a potent inducer of cytochrome P450 enzymes, particularly CYP2B1/2B2. Although the mechanism(s) of PB induction of CYP2B1/2B2 is not fully understood, current research is focusing on the role PB may play in altering the binding of nuclear proteins to critical DNA response elements in the 5'-flanking region of these genes. In this study, rat liver nuclear proteins were analyzed for DNA binding ability using both a general consensus and a CYP2B2 sequence-specific AP-1 oligonucleotide. We demonstrate that in vivo PB treatment enhances protein binding activity to the consensus AP-1 oligonucleotide. Likewise, a putative AP-1 site, identified at -1441 in the CYP2B2 5'-flanking region, also formed a sequence specific DNA/protein complex which was enhanced after PB exposure. These data may support a role of AP-1 in the PB induction mechanism of CYP2B1/2B2.

Loss of CYP2E1 and CYP1A2 activity as a function of acetaminophen dose: relation to toxicity.

The effect of acetaminophen (APAP) dose on the cytochrome P450s responsible for its bioactivation was examined in control mice and mice treated with acetone to induce CYP2E1, or beta-napthaflavone to induce CYP1A2. In non-induced mice, 150 mg/kg APAP caused minimal hepatotoxicity and loss of CYP2E1- but not CYP1A2-dependent activity. In contrast, 400 mg/kg APAP was hepatotoxic and diminished both CYP2E1 and CYP1A2 activities. In acetone-pretreated mice, the 150 and 400 mg/kg APAP doses caused similar depletion of CYP2E1 activity and similar levels of covalent binding of APAP to liver proteins. In beta-napthaflavone-pretreated mice, CYP1A2 activity was decreased only by the high dose of APAP, and covalent binding was > 2-fold higher at the high APAP dose. The data indicate CYP2E1 is important in the bioactivation of APAP at the low dose with little additional contribution at the high dose, whereas CYP1A2 contributes more to the bioactivation and toxicity APAP at high doses.

Dinitropyrene metabolism, DNA adduct formation, and DNA amplification in an SV40-transformed Chinese hamster embryo cell line.

The environmental pollutants 1,6-dinitropyrene (1,6-DNP) and 1,8-dinitropyrene (1,8-DNP) are strongly carcinogenic in a number of animal models. These DNPs are metabolized by nitroreduction to N-hydroxy arylamine derivatives that either directly or after acetylation bind to cellular DNA. In the experiments reported here, we examined whether DNA adduct formation by 1,6-DNP and 1,8-DNP was associated with amplification of specific DNA sequences, a process that may be causally related to tumorigenesis. CO60 cells, an SV40-transformed Chinese hamster embryo cell line, were incubated with 2.5 or 50 ng/mL [4,5,9,10(-3)H]1,6-DNP for 5 h. High-pressure liquid chromatographic analysis of organic extracts of the medium indicated the presence of 1-acetylamino-6-nitropyrene, suggesting that these cells are capable of nitroreduction and acetylation. 32P-Postlabeling analysis of DNA isolated from cells exposed to 1.0 or 2.5 ng/mL 1,6-DNP revealed dose-related formation of N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene. A similar adduct, presumably N-(deoxyguanosin-8-yl)-1-amino-8-nitropyrene, was detected after incubations with 1,8-DNP. DNA isolated from analogous experiments was slot-blotted onto nylon membranes and hybridized with 32P-labeled SV40, c-fos, or beta-actin DNA probes. beta-Actin was not amplified and c-fos was amplified only a small amount; however, there was dose-related amplification of SV40 sequences, whose levels were in some instances approximately 20 times that observed in solvent-treated controls. These data indicate that DNA adduct formation by 1,6-DNP and 1,8-DNP is associated with the amplification of certain DNA sequences, a response that may be related to the tumorigenic potential of these compounds.

Resonance Raman studies of pyrocatechase-inhibitor complexes.

The resonance Raman spectra of native pyrocatechase and its benzoate and phenolate complexes were investigated by using the available lines of an argon and a krypton laser. The data provide evidence for the presence of two distinct tyrosines coordinated to the active-site iron. The two tyrosines exhibit different upsilon CO values which show maximum resonance enhancements at different excitation wavelengths. Moreover, one tyrosine is more susceptible to changes in the active-site environment. Pyrocatechase is the only example thus far among iron-tyrosinate proteins where the tyrosines coordinating the iron are distinguishable.

Characterization of cytochrome P450 and glutathione S-transferase activity and expression in male and female ob/ob mice.

OBJECTIVE: To characterize the effect(s) of gender, age (glycemic status) and obese state, on hepatic biotransformation activities, expression of cytochrome P450 (CYP450) mRNAs and glutathione transferase activity in the ob/ob mouse. DESIGN: Male and female, ob/ob or ob/+ mice were killed at 3-4 months or 7-8 months of age. Hepatic microsomes, cytosol and RNA were prepared from each animal. ANIMALS: Male and female ob/ob and ob/+ mice, 3-4 or 7-8 months of age. MEASUREMENTS: CYP450 form-specific activities of CYP1A1/1A2, CYP3A and CYP2B were estimated by determining the 0-dealkylation of alkoxyresorufin substrates (ethoxy-EROD, benzoxy-BROD and pentoxy-resorufin, PROD, respectively). CYP2E1-dependent, 4-nitrophenol hydroxylase (PNP-OH) and CYP3A-dependent erythromycin N-demethylase (ERY-DM) were also measured in hepatic microsomes. CYP1A2, CYP2E1 and CYP3A protein in microsomal fractions was determined by ELISA. Glutathione transferase activity (GST) was determined in hepatic cytosol and CYP1A2 and CYP2E1 mRNA was estimated by Northern blot analysis. RESULTS: Female mice, regardless of glycemic status, showed an obesity enhanced level of CYP2E1-dependent PNP-OH activity and CYP2E1 protein as shown by ELISA. These increases were observed to be independent of the diabetic state, since 7-8 month-old mice had blood glucose levels identical to lean mice. The mRNA level of CYP2E1 in female mice also exhibited age-and obesity-influenced decreases in expression. No significant differences in CYP2E1 activity or expression were observed in male mice. CYP3A-dependent ERY-DM activity was significantly higher in young males, regardless of phenotype. CYP3A and CYP2B activities did not differ among any animals; however, CYP1A activity, while depressed in obese animals of both genders, was significantly different in old animals. Glutathione S-transferase activity was lower in obese male mice, whereas no difference was observed between lean and obese females CONCLUSION: This study supports earlier observations in man and rats that the obese state produces alterations in the expression of important oxidation and conjugation pathways. In addition, this report more thoroughly examines the role of gender and glycemic status on biotransformation activities in the ob/ob mouse as demonstrated by increased CYP2E1 protein and CYP2E1-dependent activity in obese females, decreased CYP1A2 protein and CYP1A2-dependent activity in obese animals, and obesity had no effect of glutathione transferase in female mice, in contrast with the previously reported obesity-dependent decrease of this activity in male mice.

Evidence for synergistic anion binding to iron in ovotransferrin complexes from resonance Raman and extended X-ray absorption fine structure analysis.

The 2,3-dihydroxybenzoate and thioglycolate complexes of iron(III)-ovotransferrin have been studied with resonance Raman and extended x-ray absorption fine structure spectroscopies, respectively, to obtain evidence for the coordination of the synergistic anion to the iron center. The dihydroxybenzoate complex exhibits resonance-enhanced Raman vibrations arising from both the endogenous tyrosinates and the added dihydroxybenzoate. A comparison of the extended x-ray absorption fine structure spectra of the carbonate and thioglycolate complexes shows a large feature at about 1.95 A assigned to Fe-(O,N) interactions. The latter complex exhibits an added feature at 2.32 A assigned to an Fe-S interaction. These experiments demonstrate that the Lewis base functions in the synergistic anions coordinate to the iron in ovotransferrin.

A large reservoir of sulfate and sulfonate resides within plasma cells from Ascidia ceratodes, revealed by X-ray absorption near-edge structure spectroscopy.

The study of sulfur within the plasma cells of Ascidia ceratodes [Carlson, R. M. K. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2217-2221; Frank, P., Carlson, R. M. K., & Hodgson, K. O. (1986) Inorg. Chem. 25, 470-478; Hedman, B., Frank, P., Penner-Hahn, J. E., Roe, A. L., Hodgson, K. O., Carlson, R. M. K., Brown, G., Cerino, J., Hettel, R., Troxel, T., Winick, H., & Yang, J. (1986) Nucl. Instrum. Methods Phys. Res., Sect. A 246, 797-800] has been extended with X-ray absorption near-edge structure (XANES) spectroscopy. An intense absorption feature at 2482.4 eV and a second feature at 2473.7 eV indicate a large endogenous sulfate concentration, as well as smaller though significant amounts of thiol or thioether sulfur, respectively. A strong shoulder was observed at 2481.7 eV on the low-energy side of the sulfate absorption edge, deriving from a novel type of sulfur having a slightly lower oxidation state than sulfate sulfur. The line width of the primary transition on the sulfur edge of a vanadium (III) sulfate solution was found to be broadened relative to that of sodium sulfate, possibly deriving from the formation of the VSO4+ complex ion [Britton, H. T. S., & Welford, G. (1940) J. Chem. Soc., 761-764; Duffy, J. A., & Macdonald, W. J. D. (1970) J. Chem. Soc., 977-980; Kimura, T., Morinaga, M., & Nakano, J. (1972) Nippon Kagaku Zaishi, 664-667]. Similar broadening appears to characterize the oxidized sulfur types in vanadocytes. A very good linear correlation between oxidation state and peak position (in electronvolts) was found for a series of related sulfur compounds. This correlation was used to determine a 5+ oxidation state for the additional sulfur type at 2481.7 eV. (ABSTRACT TRUNCATED AT 250 WORDS)

4-Hydroxyphenylpyruvate dioxygenase is an iron-tyrosinate protein.

A resonance Raman investigation into the blue chromophore of 4-hydroxyphenylpyruvate dioxygenase, a non-heme iron enzyme from Pseudomonas P. J. 874, reveals the presence of enhanced vibrations characteristic of tyrosinate coordination to the iron center. The excitation profiles for these features show that they are associated with the 595 nm absorption feature. EPR studies of this enzyme indicate the presence of a high-spin ferric center in a rhombic environment, as evidenced by a signal at g = 4.3 with the correct intensity for the measured iron content. This enzyme thus belongs to the emerging class of iron-tyrosinate proteins.

HepG2 cells: an in vitro model for P450-dependent metabolism of acetaminophen.

The human hepatoma cell line, HepG2, retains many cellular functions often lost by cells in culture. This research examined the constitutive bioactivation of acetaminophen and P450-dependent activity in microsomes from HepG2 cells and the effect of 0.1% acetone pretreatment on these activities. Low levels of acetaminophen bioactivation, P450 IIE1 activity, and P450 IA1-IA2 activity were demonstrated in non-induced HepG2 microsomes. Acetone increased acetaminophen bioactivation and IIE1-dependent metabolism but not P450 IA1-IA2-dependent activity. Thus, HepG2 cells may provide an in vitro model for assessing human xenobiotic metabolism of acetaminophen and other drugs.

The effect of high dose endotoxin on CYP3A2 expression in the rat.

PURPOSE: The purpose of our research was two-fold: 1) to further characterize the downregulation of CYP3A2 mRNA, protein, and activity during an acute phase response (APR); 2) most importantly, to relate the time-dependent activation of nuclear proteins to putative DNA binding sequences within the CYP3A2 5'-flanking region, with the loss in CYP3A2 expression. METHODS: Rats were injected (2.0 mg/animal, i.p.) with LPS and sacrificed at 1, 2, 4, 6, 8, 24, 48, and 72 hours. Hepatic nuclear protein was isolated and analyzed for binding activity to AP-1, NFkappaB, and NF-IL6 consensus sequences. Hepatic CYP3A2 mRNA levels were determined by solution hybridization and CYP3A2 protein, CYP3A2 activity, and total P450 were measured in hepatic microsomes. RESULTS: Computer analysis of the 5'-flanking region of CYP3A2 revealed the presence of 5 NF-IL6 and 4 AP-1 putative DNA binding sites. The strongest increase in AP-1 binding activity occurred between 6 and 24 hr, and the alteration in binding complexes to an NF-IL6 oligonucleotide occurred between 4 and 24 hr. Maximum loss in CYP3A2 mRNA occurred at 8 hr post-LPS injection and remained lowered at the 24 hr timepoint. CYP3A2 protein was significantly decreased at 24, 48, and 72 hours post-LPS treatment with corresponding decreases in CYP3A2 activity and total P450. CONCLUSIONS: The changes in NF-IL6 and AP-1 binding after LPS treatment, which appears to correlate with the changes in CYP3A2 mRNA, combined with the presence of putative NF-IL6 and AP-1 sites located in the CYP3A25'-flanking region, may indicate a potential role for NF-IL6 and AP-1 in CYP3A2 downregulation during an APR.