ProMAT calibrator: A tool for reducing experimental bias in antibody microarrays.

Our research group has been developing enzyme-linked immunosorbent assays (ELISA) microarray technology for the rapid and quantitative evaluation of biomarker panels. Studies using antibody microarrays are susceptible to systematic bias from the various steps in the experimental process, and these biases can mask biologically significant differences. For this reason, we have developed a calibration system that can identify and reduce systematic bias due to processing factors. Specifically, we developed a sandwich ELISA for green fluorescent protein (GFP) that is included on each chip. The GFP antigen is spiked into each biological sample or standard mixture and the resulting signal is used for calibration between chips. We developed ProMAT Calibrator, an open-source bioinformatics tool, for the rapid visualization and interpretation of the calibrator data and, if desired, data normalization. We demonstrate that data normalization using this system markedly reduces bias from processing factors. Equally useful, this calibrator system can help reveal the source of the bias, thereby facilitating the elimination of the underlying problem. ProMAT Calibrator can be downloaded at http://www.pnl.gov/statistics/ProMAT .

The nuclear factor-kappa B pathway regulates cytochrome P450 3A4 protein stability.

We have previously observed that CYP3A4 protein levels are suppressed by inhibition of the proteasome in primary cultured hepatocytes. Because this result is opposite of what would be expected if CYP3A4 were degraded by the proteasome, it seemed likely that there might be another protein susceptible to proteasomal degradation that regulated CYP3A4 expression. In this study, we evaluated whether the nuclear factor-kappaB (NF-kappaB) pathway was involved in that process. Our model system used an adenovirus system to express CYP3A4 protein in HepG2 cells, which are derived from human cancer cells. Similar to results in primary hepatocytes, the inhibition of the proteasome with N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) suppresses CYP3A4 protein levels. We also found that MG132 treatment had a broad affect on the NF-kappaB pathway, including down-regulation of NF-kappaB DNA binding activity and IkappaB kinase (IKK)alpha levels and up-regulation of IKKbeta and inhibitory kappaB levels. Treatment of the HepG2 cells with several structurally distinct NF-kappaB inhibitors also suppressed CYP3A4 protein levels. When the HepG2 cells were treated with cycloheximide, a general inhibitor of protein synthesis, the loss of CYP3A4 protein was accelerated by cotreatment with either proteasome or NF-kappaB inhibitors. These results indicate that NF-kappaB activity regulated CYP3A4 protein stability, and they suggest that the NF-kappaB pathway was responsible for the decrease in CYP3A4 protein levels that resulted from the proteasomal inhibition.

Cytochrome P450 3A conjugation to ubiquitin in a process distinct from classical ubiquitination pathway.

We characterize a novel microsome system that forms high-molecular-mass (HMM) CYP3A, CYP2E1, and ubiquitin conjugates, but does not alter CYP4A or most other microsomal proteins. The formation of the HMM bands was observed in hepatic microsomes isolated from rats treated 1 week or more with high doses (50 mg/kg/day) of nicardipine, clotrimazole, or pregnenolone 16alpha-carbonitrile, but not microsomes from control, dexamethasone-, nifedipine-, or diltiazem-treated rats. Extensive washing of the microsomes to remove loosely attached proteins or cytosolic contaminants did not prevent the conjugation reaction. In contrast to prototypical ubiquitination pathways, this reaction did not require addition of ubiquitin, ATP, Mg(2+), or cytosol. Addition of cytosol did result in the degradation of the HMM CYP3A bands in a process that was not blocked by proteasome inhibitors. Immunoprecipitated CYP3A contained HMM ubiquitin. Even so, mass spectrometric analysis of tryptic peptides indicated that the HMM CYP3A was in molar excess to ubiquitin, suggesting that the formation of the HMM CYP3A may have resulted from conjugation to itself or a diffuse pool of ubiquitinated proteins already present in the microsomes. Addition of CYP3A substrates inhibited the formation of the HMM CYP3A and the cytosol-dependent degradation of HMM CYP3A. These results suggest that after extended periods of elevated CYP3A expression, microsomal factors are induced that catalyze the formation of HMM CYP3A conjugates that contain ubiquitin. This conjugation reaction, however, seems to be distinct from the classical ubiquitination pathway but may be related to the substrate-dependent stabilization of CYP3A observed in vivo.

Differential induction of rat hepatic cytochromes P450 3A1, 3A2, 2B1, 2B2, and 2E1 in response to pyridine treatment.

Pyridine (PY) effects on rat hepatic cytochromes P450 (CYP) 3A1 and 3A2 expression were examined at the levels of metabolic activity, protein, and mRNA and were compared with those of CYP2B1/2 and CYP2E1. CYP3A metabolic activity as well as CYP3A protein and mRNA levels increased following treatment of rats with PY. CYP3A1 and CYP3A2 were differentially affected by PY treatment in terms of induction levels, dose dependence, and stability of mRNA. CYP3A1 mRNA levels maximally increased ~42-fold after PY treatment, whereas CYP3A2 mRNA level increased ~4-fold. Moreover, CYP3A1 mRNA levels decreased more rapidly than those of CYP3A2 as determined following inhibition of transcription with actinomycin D or cordycepin. Treatment of rats with PY resulted in a dose-dependent increase in CYP3A1, CYP3A2, and CYP2B1/2B2 protein levels. In contrast to the effects of PY treatment on CYP3A1 and 2B, CYP2E1 protein levels increased in the absence of a concomitant increase in CYP2E1 mRNA levels. Treatment of rats with PY at 200 mg/kg/day for 3 days increased both protein and mRNA levels of CYP3A2, whereas treatment with higher than 200 mg/kg/day for 3 days increased CYP3A2 protein levels without an increase in CYP3A2 mRNA levels. These data demonstrated that PY regulates the various CYPs examined in this study at different levels of expression and that PY regulates CYP3A1 expression through transcriptional activation and CYP3A2 expression through transcriptional and post-transcriptional activation at a low- and high-dose PY treatment, respectively.

Cytotoxicity of trichloroethylene and S-(1, 2-dichlorovinyl)-L-cysteine in primary cultures of rat renal proximal tubular and distal tubular cells.

Activities of several glutathione-dependent enzymes, expression of cytochrome P450 isoenzymes, and time- and concentration-dependent cytotoxicity of trichloroethylene (TRI) and S-(1, 2-dichlorovinyl)-L-cysteine (DCVC) were evaluated in primary cultures of proximal tubular (PT) and distal tubular (DT) cells from rat kidney. These cells exhibited cytokeratin staining and maintained activities of all glutathione-dependent enzymes measured. Of the cytochrome P450 isoenzymes studied, only CYP4A expression was detected. CYP4A mRNA and protein expression were higher in primary cultures of DT cells than in PT cells and were increased in DT cells by ciprofibrate treatment. Incubation of cells for 6 h with concentrations of TRI as high as 10 mM resulted in minimal cytotoxicity, as determined by release of lactate dehydrogenase (LDH). In contrast, marked cytotoxicity resulted from incubation of PT or DT cells with DCVC. Addition to cultures of TRI (2-10 mM) for 24 or 72 h resulted in modest, but significant time- and concentration-dependent increases in LDH release. Treatment of cells with DCVC (0.1-1 mM) for 24 h caused significant increases in LDH release and alterations in cellular protein and DNA content. Finally, exposure of primary cultures to TRI or DCVC for 72 h followed by 3 h of recovery caused a slight increase in the expression of vimentin, consistent with cellular regeneration. These studies demonstrate the utility of the primary renal cell cultures for the study of CYP4A expression and mechanisms of TRI-induced cellular injury.

Comparative metabolism of carbon tetrachloride in rats, mice, and hamsters using gas uptake and PBPK modeling.

No study has comprehensively compared the rate of metabolism of carbon tetrachloride (CCl4) across species. Therefore, the in vivo metabolism of CCl4 was evaluated using groups of male animals (F344 rats, B6C3F1 mice, and Syrian hamsters) exposed to 40-1800 ppm CCl4 in a closed, recirculating gas-uptake system. For each species, an optimal fit of the family of uptake curves was obtained by adjusting Michaelis-Menten metabolic constants Km (affinity) and Vmax (capacity) using a physiologically based pharmacokinetic (PBPK) model. The results show that the mouse has a slightly higher capacity and lower affinity for metabolizing CCl4 compared to the rat, while the hamster has a higher capacity and lower affinity than either rat or mouse. A comparison of the Vmax to Km ratio, normalized for milligrams of liver protein (L/h/mg) across species, indicates that hamsters metabolize more CCl4 than either rats or mice, and should be more susceptible to CCl4-induced hepatotoxicity. These species comparisons were evaluated against toxicokinetic studies conducted in animals exposed by nose-only inhalation to 20 ppm 14C-labeled CCl4 for 4 h. The toxicokinetic study results are consistent with the in vivo rates of metabolism, with rats eliminating less radioactivity associated with metabolism (14CO2 and urine/feces) and more radioactivity associated with the parent compound (radioactivity trapped on charcoal) compared to either hamsters or mice. The in vivo metabolic constants determined here, together with in vitro constants determined using rat, mouse, hamster, and human liver microsomes, were used to estimate human in vivo metabolic rates of 1.49 mg/h/kg body weight and 0.25 mg/L for Vmax and Km, respectively. Normalizing the rate of metabolism (Vmax/Km) by milligrams liver protein, the rate of metabolism of CCl4 differs across species, with hamster > mouse > rat > human.

Post-transcriptional regulation of rat CYP2E1 expression: role of CYP2E1 mRNA untranslated regions in control of translational efficiency and message stability.

Altered expression of hepatic CYP2E1 by xenobiotic or physiological stimuli is largely mediated through post-transcriptional mechanisms that may include altered CYP2E1 mRNA translation and/or protein degradation. Examination of the polyribosomal distribution of rat hepatic P450 mRNAs indicated that, whereas nearly all of the CYP2B, CYP3A, and CYP4A mRNAs were recovered in the polysomal fractions, indicating active translation, approximately 30-40% of CYP2E1 mRNA was not associated with polysomes and therefore not actively engaged in protein synthesis. To examine the CYP2E1 mRNA molecule for sequences that might affect its translational efficiency, a series of CYP2E1 recombinant RNAs (rcRNAs) with modified 5' or 3' untranslated regions (UTRs) was translated in vitro using the rabbit reticulocyte lysate system. Deletion of most of the CYP2E1 5' UTR, which was predicted to contain secondary structure, increased in vitro CYP2E1 protein synthesis. Polysomal distribution analyses of 5'-modified rcRNAs demonstrated that, as seen for hepatic CYP2E1 mRNA, a substantial fraction of each CYP2E1 rcRNA was not associated with polysomes. The polysomal distribution analyses of the CYP2E1 rcRNAs also confirmed that the observed changes in CYP2E1 protein synthesis were associated with altered ribosomal loading. Deletion of the poly(A) tail, and partial or complete deletion of the 3' UTR, decreased CYP2E1 protein synthesis. These changes in protein synthesis were accompanied by increased degradation of the CYP2E1 rcRNAs. Incubation with translational inhibitors, but not increased levels of RNase inhibitor, decreased the degradation of the rcRNAs during in vitro translation. In conclusion, these studies suggest that secondary structure in the 5' UTR of CYP2E1 mRNA is at least partially responsible for the inefficient translation of this mRNA. The poly(A) tail and sequences contained within the 3' UTR appear to be important for protecting CYP2E1 mRNA from RNase activity associated with the translation machinery.

Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes.

We examined which human CYP450 forms contribute to carbon tetrachloride (CCl(4)) bioactivation using hepatic microsomes, heterologously expressed enzymes, inhibitory antibodies and selective chemical inhibitors. CCl(4) metabolism was determined by measuring chloroform formation under anaerobic conditions. Pooled human microsomes metabolized CCl(4) with a K(m) of 57 microM and a V(max) of 2.3 nmol CHCl(3)/min/mg protein. Expressed CYP2E1 metabolized CCl(4) with a K(m) of 1.9 microM and a V(max) of 8.9 nmol CHCl(3)/min/nmol CYP2E1. At 17 microM CCl(4), a monoclonal CYP2E1 antibody inhibited 64, 74 and 83% of the total CCl(4) metabolism in three separate human microsomal samples, indicating that at low CCl(4) concentrations, CYP2E1 was the primary enzyme responsible for CCl(4) metabolism. At 530 microM CCl(4), anti-CYP2E1 inhibited 36, 51 and 75% of the total CCl(4) metabolism, suggesting that other CYP450s may have a significant role in CCl(4) metabolism at this concentration. Tests with expressed CYP2B6 and inhibitory CYP2B6 antibodies suggested that this form did not contribute significantly to CCl(4) metabolism. Effects of the CYP450 inhibitors alpha-naphthoflavone (CYP1A), sulfaphenazole (CYP2C9) and clotrimazole (CYP3A) were examined in the liver microsome sample that was inhibited only 36% by anti-CYP2E1 at 530 microM CCl(4). Clotrimazole inhibited CCl(4) metabolism by 23% but the other chemical inhibitors were without significant effect. Overall, these data suggest that CYP2E1 is the major human enzyme responsible for CCl(4) bioactivation at lower, environmentally relevant levels. At higher CCl(4) levels, CYP3A and possibly other CYP450 forms may contribute to CCl(4) metabolism.

Effect of calcium channel antagonists nifedipine and nicardipine on rat cytochrome P-450 2B and 3A forms.

Calcium channel antagonists are widely prescribed for treatment of hypertension. In this study, we examined whether treatment with the calcium channel antagonists, nicardipine, nifedipine or diltiazem, alters cytochrome P-450 2B or 3A (CYP2B or CYP3A, respectively) expression in rat liver. Western blot analyses were undertaken using antibodies specific for one or several members of these cytochrome P-450 subfamilies. Nicardipine was found to be an effective inducer of CYP3A; in particular, CYP3A23 was increased approximately 36-fold following treatment with 100 mg of nicardipine/kg/day. Nicardipine induced CYP2B forms up to approximately 3.1-fold. Nifedipine did not alter CYP3A expression but did increase CYP2B expression such that total CYP2B, CYP2B1, and CYP2B2v (a splice variant of CYP2B2) were increased approximately 5- to 15-fold after treatment with 100 mg of nifedipine/kg/day, with increases in benzyloxyresorufin O-dealkylase and erythromycin N-demethylase activities, respectively. The distinct differences in cytochrome P-450 induction profile induced by nicardipine and nifedipine suggest that they may enhance cytochrome P-450 expression by different mechanisms unrelated to their effects on calcium channels.

Cellular distribution of cytochromes P-450 in the rat kidney.

The distribution of several cytochrome P-450 (P-450) isoenzymes between proximal tubular (PT) and distal tubular (DT) cells of the rat kidney was determined. Western blot analysis of microsomes prepared from liver and kidney cortical homogenates revealed that CYP2E1 protein was expressed in rat kidney microsomes at approximately 10% of hepatic levels. Microsomes from renal cortical, PT, and DT cells all expressed CYP2E1, with DT microsomes expressing slightly higher levels than PT microsomes. In contrast, chlorzoxazone hydroxylation activity was markedly higher in microsomes from PT cells than in those from DT cells. Northern blot analysis of total RNA from PT and DT cells exhibited a pattern of CYP2E1 mRNA distribution similar to that of CYP2E1 protein. CYP2C11 protein expression in renal cortical microsomes was approximately 10% of that in liver microsomes but was significantly higher in microsomes from PT cells than in those from DT cells. CYP3A1/2 was not detected in microsomes from either cortical, PT, or DT cells, but was detected in microsomes isolated from total liver or kidney cortical homogenates. CYP2B1/2 expression was detected in all tissues tested. The peroxisomal proliferator clofibrate enhanced the level of CYP2B1/2 in microsomes from both total liver and kidney cortical homogenates but not in microsomes from cortical, PT, or DT cells. CYP4A2/3 protein and CYP4A mRNA expression were detected in microsomes from total liver and kidney cortical homogenates and from renal cortical, PT, and DT cells using Western and Northern blot analyses, respectively. Lauric acid hydroxylation activity, an indicator of CYP4A, was comparable in PT and DT cells. Clofibrate elevation of CYP4A in cortical, PT, and DT microsomes was not as great as that detected in total kidney cortical microsomes. These results establish the distribution of several P-450 isoenzymes between different cell populations of the rat kidney. Furthermore, these results present evidence that the level of induction of certain P-450 isoenzymes in the kidney is cell type-specific.

Posttranslational elevation of cytochrome P450 3A levels and activity by dimethyl sulfoxide.

The molecular mechanisms by which dimethyl sulfoxide (DMSO) enhances CYP3A protein in phenobarbital-treated primary cultured rat hepatocytes were examined. DMSO treatment rapidly increased CYP3A protein levels in the absence of an increase in CYP3A mRNA levels or an increase in CYP2B protein or mRNA levels. CYP3A levels were increased approximately 3.7- and 9-fold following 0.1% DMSO treatment for 6 and 48 h, respectively. Analyses of the polysomal distribution of CYP3A mRNA suggested that DMSO treatment did not significantly alter the translational efficiency of the CYP3A mRNA. Comparative analyses of immunodetectable protein levels following treatment with cycloheximide showed that DMSO clearly decreased the rate of CYP3A protein turnover but not that of CYP2B. Examination of testosterone metabolism in hepatocyte cultures revealed that DMSO pretreatment increased CYP3A-catalyzed 2 beta- and 6 beta-testosterone hydroxylation. When DMSO was in the culture medium, no inhibitory affect on CYP3A-catalyzed testosterone metabolism was observed, although a slight (15-21%) inhibitory effect was noted for CYP2B-catalyzed 16 alpha- and 16 beta-testosterone hydroxylation. These data provide evidence that DMSO increased CYP3A protein levels as a result of decreased protein degradation. DMSO increased both immunodetectable CYP3A protein levels and catalytic activity, in contrast to compounds that have been reported to stabilize CYP3A protein and inhibit activity.

Posttranscriptional elevation of cytochrome P450 3A expression.

Human CYP3A, the most abundant hepatic and intestinal cytochrome P450, catalyzes the metabolism of a diverse array of xenobiotics. Dimethyl sulfoxide is a commonly used solvent which has been used therapeutically. Dimethyl sulfoxide effects on CYP3A, CYP2E1, CYP2B and NADPH cytochrome P450 reductase expression in rat liver and in primary cultured rat hepatocytes were examined. Dimethyl sulfoxide increased immunodetectable hepatic CYP3A and CYP2E1 levels approximately 2.5 to 3-fold in the absence of any change in the respective mRNA levels. No change in CYP2B or P450 reductase expression was observed, indicating that dimethyl sulfoxide effects were selective. Dimethyl sulfoxide also increased CYP3A protein in rats pretreated with dexamethasone. In primary cultured rat hepatocytes, dimethyl sulfoxide increased CYP3A and CYP2E1 protein without increasing the respective mRNA levels. These results show that dimethyl sulfoxide, at levels relevant to human exposure, enhances CYP3A and CYP2E1 expression by posttranscriptional mechanisms.

Effects of fatty acids and ketone bodies on cytochromes P450 2B, 4A, and 2E1 expression in primary cultured rat hepatocytes.

CYP2B, CYP4A, and CYP2E1 mRNA levels are elevated in response to pathophysiological conditions, such as diabetes, high-fat diet, and fasting, in which lipids and ketone bodies are increased. In order to avoid confounding hormonal effects, we utilized primary rat hepatocytes to examine whether ketone bodies or fatty acids altered CYP2B, CYP4A, or CYP2E1 expression. Ketone bodies increased CYP2B mRNA and protein levels, but failed to alter CYP4A or CYP2E1 expression. Straight-chain saturated fatty acids, C8 to C16, increased levels of CYP2B and CYP4A mRNA, but not CYP2E1 mRNA. Treatment with octanoylcarnitine, a mitochondrial beta-oxidation inhibitor, in combination with hexadecanoate increased CYP2B and CYP4A expression approximately 1.4-fold over that observed with hexadecanoate alone, suggesting that mitochondrial conversion of fatty acids to ketone bodies was not required for enhanced CYP2B expression and that mitochondrial beta-oxidation decreased intracellular fatty acid levels and thereby lowered CYP2B expression. Undecynoic acid or aminobenzotriazole treatment increased CYP2B mRNA levels, consistent with these compounds inhibiting the initial CYP4A-catalyzed step in the conversion of monocarboxylic to dicarboxylic acids and thereby decreasing peroxisomal beta-oxidation and increasing intracellular fatty acid levels. Addition of glycerol, which suppresses fatty acid synthesis by inhibiting conversion of lactate to pyruvate, decreased basal expression of CYP2B and CYP4A but did not alter CYP2E1 expression. Pyruvate, but not lactate, completely prevented the glycerol-mediated decrease in CYP2B expression. These results provide evidence that intracellular levels of fatty acids and ketone bodies regulate the expression of CYP2B but not CYP2E1.

Differential effects of ciprofibrate on renal and hepatic cytochrome P450 2E1 expression.

Since cytochrome P450 2E1 (CYP2E1) mRNA levels have been reported to be increased during physiological states in which peroxisomes are increased, we examined the effects of the peroxisome proliferator, ciprofibrate (CIPRO), on renal and hepatic CYP2E1 expression, as well as other enzymes associated with peroxisome proliferation, including CYP4A, CYP2B, fatty acyl CoA oxidase (FACO), and peroxisomal thiolase (PT). Male rats were treated with CIPRO for 18, 48, or 120 hr. Northern blot or immunoblot analyses were used to determine mRNA or protein levels, respectively, relative to levels in vehicle controls. CIPRO elevated renal CYP2E1 and CYP4A mRNA levels approximately 3- to 4-fold at 18 hr, and these levels remained elevated to 120 hr. CIPRO progressively increased renal CYP2E1 and CYP4A protein levels, so that approximately 7- and 4-fold increases, respectively, were observed at 120 hr of treatment. CIPRO treatment increased renal peroxisomal FACO mRNA levels approximately 2-fold and PT mRNA levels approximately 4- to 6-fold at all time points. In contrast to results observed in the kidney, hepatic CYP2E1 mRNA and protein levels were unchanged by CIPRO treatment. Hepatic CYP4A mRNA levels were increased approximately 100-fold at all time points. Hepatic CYP2B mRNA levels were elevated approximately 5-fold, but only at the 120-hr time point. Hepatic CYP4A and CYP2B protein levels were elevated in proportion to the respective mRNA levels. Hepatic FACO mRNA levels were increased approximately 5-, 9-, and 20-fold, and hepatic PT mRNA levels were increased approximately 15-, 24-, and 39-fold, at 18, 48, and 120 hr, respectively. These results show that CIPRO-mediated, tissue-specific changes in CYP2E1 expression are not obligatorily associated with elevations in peroxisomal enzymes.

Effects of hydrazine, phenelzine, and hydralazine treatment on rat hepatic and renal drug-metabolizing enzyme expression.

The hepatic and renal toxicity associated with hydrazine treatment has been linked to free radical damage resulting from oxidative metabolism by cytochrome P4502E1 (CYP2E1). Despite this association, there has been little characterization of the effects of hydrazine treatment on the expression of hepatic and renal CYP2E1 or glutathione-S-transferase-alpha (GST-alpha), an enzyme responsible for catalyzing the conjugation of free radicals with reduced glutathione. Therefore, the effects of treatment with hydrazine or one of the therapeutic hydrazines phenelzine and hydralazine on rat hepatic and renal CYP2E1 and GST-alpha expression were investigated. Adult male Sprague-Dawley rats were treated with 0.9% saline vehicle (1 dose ip), hydrazine (100 mg/kg ip), phenelzine (100 mg/kg ip), or hydralazine (25 mg/kg ip). CYP2E1 mRNA and protein levels were monitored by Northern and immunoblot analyses, respectively, and GST-alpha Ya and Yc subunit levels were determined by immunoblot analysis. Hydralazine administration caused a significant (approximately 159%) increase in renal GST-alpha subunit expression. In addition, hydrazine and phenelzine treatment produced substantial elevations (approximately 464% and 566%, respectively) in renal CYP2E1 protein, whereas hydralazine administration did not alter renal CYP2E1 expression. Changes in rat hepatic GST-alpha Ya or Yc subunit levels after treatment with hydrazines phenelzine, or hydralazine were not statistically significant. Similarly, hepatic CYP2E1 levels were not significantly altered after treatment with hydrazine, phenelzine, or hydralazine. Northern blot analysis revealed that the observed increases in renal CYP2E1 protein levels after treatment with hydrazine or phenelzine were not accompanied by concomitant increases in CYP2E1 mRNA. These results suggest that treatment with hydrazine or the therapeutic hydrazine phenelzine significantly increases the expression of rat renal CYP2E1 protein, and that the molecular mechanism responsible for these effects are posttranscriptional in nature.

Gender-specific and developmental differences in protein kinase C isozyme expression in rat liver.

Protein kinase C (PKC) activity is important in regulating cellular growth and differentiation and is believed to play a role in the promotional stage of carcinogenesis. To determine the effects of age and gender on PKC expression, isozyme-specific antibodies and immunoblot analyses were employed to examine the expression of PKC alpha, PKC beta, PKC gamma, PKC delta and PKC epsilon in the cytosolic and membrane fractions isolated from hepatic tissue of 1, 3, 5 and 12 week old male or female rats. PKC alpha levels were comparable at 1 week of age in the respective male and female hepatic fractions. In contrast, at 3, 5 and 12 weeks of age, cytosolic PKC alpha levels were approximately 2-, 5- and 7-fold greater, respectively, in females than in males. At 5 weeks of age, cytosolic levels of PKC delta were approximately 2-fold higher in females than in males. Other PKC isozymes were either below the limit of detection (PKC gamma), or failed to exhibit any gender-related differences (PKC beta and PKC epsilon). At 12 weeks of age, PKC activity was 1.7- or 2.4-fold greater in hepatic cytosol and membrane fractions, respectively, from females than in male samples. These results show distinct gender-specific and developmental differences in hepatic PKC isozyme expression, which may play a role in susceptibility to cancer.

Xenobiotic-enhanced expression of cytochrome P450 2E1 and 2B1/2B2 in primary cultured rat hepatocytes.

Investigation of the posttranscriptional mechanisms involved in the xenobiotic-mediated enhancement of cytochrome P450 2E1 (CYP2E1) expression has been limited by a lack of a functional primary hepatocyte cell culture system. We examined the effects of ciprofibrate (CIPRO) and pyridine (PYR) treatment on the expression of CYP2E1, P450 4A (CYP4A), and P450 2B (CYP2B) in primary rat hepatocytes cultured on Vitrogen or Matrigel substratum and in the presence of Chee's medium. Cells were cultured for 72 hr or longer before initiation of treatment. Northern blot analyses indicated that 24-hr CIPRO treatment enhanced the expression of CYP4A, and CYP2B mRNA in a concentration-dependent manner, with maximal induction of CYP2E1 mRNA (2- to 3-fold) and CYP4A1 mRNA (up to approximately 15-fold) monitored at 30-300 microM CIPRO. Maximal CYP2B mRNA levels (7- to 8-fold) were monitored at 300-1000 microM CIPRO. Treatment of hepatocytes for 24, 48, and 72 hr with 30 microM CIPRO showed progressive increases in CYP2B and CYP4A mRNA levels, with approximately 13- and 60-fold elevations in the respective mRNAs occurring at 72 hr posttreatment. In contrast, CYP2E1 mRNA levels were maximally elevated between 2- and 3-fold at both 24 and 48 hr and were returning to basal levels by 72 hr. Western blot analyses revealed that 24-hr PYR (25 mM) treatment of CIPRO-treated cells, in the absence of any further increase in CYP2E1 mRNA levels, increased CYP2E1 protein levels approximately 6- to 8-fold. PYR treatment also increased CYP2B mRNA and CYP2B1/2B2 protein levels approximately 16-fold relative to cells treated only with CIPRO.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of nasal carboxylesterase in F344 rats following inhalation exposure to pyridine.

Carboxylesterases (CEs) in the nasal mucosa metabolize some inhaled esters, including industrially important acrylates and acetates, to toxic acid metabolites that produce site-specific lesions in the nasal epithelium. The metabolic capacity of CEs in the normal nasal mucosa is theoretically sufficient to protect the lower respiratory tract from toxicant-induced injury at concentrations of acrylates and acetates likely to be inhaled in industrial environments. Thus, alterations in the metabolism and toxicity of these substrates would be predicted with changes in the amount or activity of CE in the nasal mucosa. Although many other nasal enzymes have been reported to be relatively refractory to induction, the amount of CE in the nasal mucosa can be increased by inhalant exposure. In the liver, expression of CEs may be elevated in response to exposure to P450 inducers. To examine this phenomenon in the nose with the widely used industrial solvent pyridine, we examined the effect of pyridine inhalation at the threshold limit value concentration of 5 ppm, or at 444 ppm, 6 hr/day for 4 days on the localization and amount of immunoreactive CE in olfactory mucosas of F344/N rats. CE immunoreactivity was increased in Bowman's glands following exposure to 5 or 444 ppm pyridine, and in sustentacular cells most notably following the 5 ppm exposure. Quantitative densitometry showed a statistically significant, dose-related increase in the density of immunoreactive CE in Bowman's glands of pyridine-exposed rats. These results indicate pyridine, and possibly other solvents, can induce nasal CE, an enzyme not directly involved in the metabolism of those solvents, following low-dose, short-term exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal exposure to xenobiotics alters adult hepatic protein kinase C alpha levels and testosterone metabolism: differential effects by diethylstilbestrol and phenobarbital.

Hepatic enzymes that metabolize endogenous and xenobiotic compounds have been shown to be altered in adult rats that had been exposed to xenobiotics as neonates. Protein kinase C (PKC) is important in intracellular signaling and has been implicated in the regulation of hepatic monooxygenases. Therefore, we examined the effects of neonatal exposure to diethylstilbestrol (DES) and phenobarbital (PB) on hepatic microsomal testosterone metabolism and on the alpha form of protein kinase C (PKC alpha) in adult rats. In adult males, neonatal exposure to DES altered adult testosterone metabolism such that 7 alpha-hydroxylation was increased by 58% but 2 alpha-, 16 alpha-, and 6 beta-hydroxylations and conversion to androstenedione were decreased 31-44%. In contrast, adult males neonatally exposed to PB showed increased (20-27%) testosterone 2 alpha- and 16 alpha-hydroxylations and androstenedione formation, but no effect was observed in the rate of 6 beta- or 7 alpha-hydroxylations. Western blot analyses indicated that cytosolic PKC alpha levels in male rats neonatally exposed to PB were decreased by approximately 63% relative to the vehicle control group but were not significantly altered in the DES males. The PKC alpha levels generally correlated (r = -.75) with 16 alpha-hydroxytestosterone formation in all samples. These results show that neonatal treatment with DES or PB differentially alters hepatic monooxygenase enzyme activities and PKC alpha levels in adult rats.

Determination of the poly(A) tail lengths of a single mRNA species in total hepatic RNA.

Poly(A) tail length is important in the stability and translation of mRNA. We describe procedures for the rapid and reproducible analysis of poly(A) tail length of a single mRNA species contained in a sample of total hepatic RNA. A short 3' fragment of a specific mRNA is prepared by RNase H digestion of the targeted mRNA region annealed to a short DNA oligonucleotide. The length of the poly(A) tail of the 3' fragment is then determined by running the sample on a polyacrylamide gel, by electrophoretic transfer, by probing with a radiolabeled cDNA and by comparing the size of the detected region with a specific RNA ladder or a DNA ladder.