Peter H. Roos

A novel CYP3 gene from female rats

A cDNA library constructed from adult female Sprague Dawley rat liver was screened with polyclonal anti CYP3A-IgG. One of the positive clones, cUT, was found to contain the complete coding sequence of a new gene more similar to hamster gene CYP3A10 (cDNA: 85%, deduced amino acid sequence: 79%) than to the known rat CYP3A genes (cDNA: 75-76%, amino acid sequences: 66-69%). The deduced sequence of the first 28 amino acids is identical to the N-terminus of the testosterone 6 beta-hydroxylase, 6 beta-2 (Nagata, K., Gonzalez, F.J., Yamazoe, Y. and Kato, R. (1990) J. Biochem. 107, 718-725). Northern blots show the presence of cUT mRNA in livers of adult female and male rats. The transcription of the new gene is enhanced in either sex by pregnenolone-alpha-carbonitrile, dexamethasone, phenobarbital, and triacetyloleandomycin, known inducers of CYP3A gene expression.

FORMATION OF LIGAND AND METABOLITE COMPLEXES AS A MEANS FOR SELECTIVE QUANTITATION OF CYTOCHROME P450 ISOZYMES

The suitability of triacetyloleandomycin (TAO) metabolite complex formation and metyrapone binding to reduced cytochrome P450 as a means for selective isozyme quantitation has been studied. Although isozymes of both subfamilies bind metyrapone in the reduced state, selective quantitation of 2B isozymes through the metyrapone complex is possible after complex formation of P450 3A with a TAO metabolite. Thus, consecutive application of both reactions allows the spectroscopic quantitation of P450 3A and 2B isozymes. Complete conversion of P450 3A into the complex, a precondition for P450 3A quantitation, requires NADH in addition to NADPH. A precise collective quantitation of 3A + 2B isozymes as metyrapone complexes alone is not possible because the corresponding complexes possess different molar extinction coefficients, i.e 71.5 and 52 mM-1 cm-1 at 446-490 nm, respectively. The formation of the TAO complex appears to be quite specific, since it correlates well with 3A-specific enzymatic activities, i.e. TAO N-demethylation and formation of 2 beta-hydroxy-, 15 beta-hydroxy- and 6-dehydrotestosterone. P450 3A levels in liver microsomes of male rats either untreated or treated with TAO, dexamethasone (DEX), phenobarbital or hexachlorobenzene amount to 13%, 78%, 66%, 24% and 11% of total P450, respectively. Good correlation between these values and P450 3A-specific enzymatic activities is obtained. By the spectroscopic method, P450 2B isozymes could not be detected in microsomes of untreated rats. With TAO, DEX and hexachlorobenzene the microsomal 2B level is elevated to about 20% of total P450, i.e. to 0.8, 0.4 and 0.4 nmol P450/mg protein, respectively. 2B levels of about 60% of total P450 (0.75 nmol P450/mg protein) are obtained by phenobarbital treatment. Immunoblotting with anti-P450 2B shows that the ratio of expressed 2B1 and 2B2 differs depending on the type of inducer. DEX predominantly leads to induction of 2B2, which may explain the low pentoxyresorufin O-depentylase activity in these microsomes.

Extrahepatic metabolism at the body's internal–external interfaces

Abstract In general, xenobiotic metabolizing enzymes (XMEs) are expressed in lower levels in the extrahepatic tissues than in the liver, making the former less relevant for the clearance of xenobiotics. Local metabolism, however, may lead to tissue-specific adverse responses, e.g. organ toxicities, allergies or cancer. This review summarizes the knowledge on the expression of phase I and phase II XMEs and transporters in extrahepatic tissues at the bodys internal–external interfaces. In the lung, CYPs of families 1, 2, 3 and 4 and epoxide hydrolases are important phase I enzymes, while conjugation is less relevant. In skin, phase I-related enzymatic reactions are considered less relevant. Predominant skin XMEs are phase II enzymes, whereby glucuronosyltransferases (UGT) 1, glutathione-S-transferase (GST) and N-acetyltransferase (NAT) 1 are important for detoxification. The intestinal epithelium expresses many transporters and phase I XME with high levels of CYP3A4 and CYP3A5 and phase II metabolism is mainly related to UGT, NAT and Sulfotransferases (SULT). In the kidney, conjugation reactions and transporters play a major role for excretion processes. In the bladder, CYPs are relevant and among the phase II enzymes, NAT1 is involved in the activation of bladder carcinogens. Expression of XMEs is regulated by several mechanisms (nuclear receptors, epigenetic mechanisms, microRNAs). However, the understanding why XMEs are differently expressed in the various tissues is fragmentary. In contrast to the liver – where for most XMEs lower expression is demonstrated in early life – the XME ontogeny in the extrahepatic tissues remains to be investigated.

Differential induction of CYP1A1 in duodenum, liver and kidney of rats after oral intake of soil containing polycyclic aromatic hydrocarbons

Abstract. We have analyzed the induction of the cytochrome P450 enzyme CYP1A1 as a biomarker of effect in duodenum, liver and kidney of rats after oral intake of contaminated soil particles. The soil samples originated from industrial sites and were contaminated with polycyclic aromatic hydrocarbons (PAH) to variable extents, ranging from 60 to 4700xa0mg PAH/kg soil. Soil samples were administered for one week as a mixture with commercial rodent diets. After exposure, microsomes of several organs were prepared and analyzed for CYP1A1, enzymatically and by Western blots. All contaminated soils led to induction of CYP1A1 in duodenal mucosa cells, regardless of their extent of contamination, showing that relevant doses were mobilized in the gastrointestinal tract and adsorbed. Subsequent distribution of non-metabolized compounds is indicated by induction of CYP1A1 in the liver. However, some samples did not lead to a response in the liver, due to their quantitative and qualitative contaminant composition. In accordance with previous results, there is a sigmoidal dose-response relationship between induction of hepatic CYP1A1 levels and the soil contamination with higher condensates of PAH. In contrast, the response in the duodenum appeared to be hyperbolic and correlated well with the amounts of total PAH. Highly contaminated soil, being nearly devoid of higher condensates of PAH, led to pronounced induction in the duodenum but failed to induce CYP1A1 in the liver. Successful passage of contaminants through the intestinal barrier and the liver compartment is shown by increased CYP1A1 expression in the kidney. Compared with enzyme levels induced in the liver, those of the kidney are much lower and amount to only about 1/20 of the liver values for soils with high induction potential. Hence, oral PAH intake leads to differential induction patterns of CYP1A1 in duodenum, liver and kidney of rats. The observations raise questions concerning the role of the primary duodenal PAH metabolism in preventing contaminant-dependent hazardous effects, and of the significance of differential CYP1A1 expressions for carcinogenic processes in several tissues.

A multi-parametric microarray for protein profiling: simultaneous analysis of 8 different cytochromes via differentially element tagged antibodies and laser ablation ICP-MS

The paper presents a new multi-parametric protein microarray embracing the multi-analyte capabilities of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The combination of high throughput reverse phase protein microarrays with element tagged antibodies and LA-ICP-MS makes it possible to detect and quantify many proteins or biomarkers in multiple samples simultaneously. A proof of concept experiment is performed for the analysis of cytochromes particularly of cytochrome P450 enzymes, which play an important role in the metabolism of xenobiotics such as toxicants and drugs. With the aid of the LA-ICP-MS based multi-parametric reverse phase protein microarray it was possible to analyse 8 cytochromes in 14 different proteomes in one run. The methodology shows excellent detection limits in the lower amol range and a very good linearity of R(2) ≥ 0.9996 which is a prerequisite for the development of further quantification strategies.

Induction of cytochrome P450 1A1 in multiple organs of minipigs after oral exposure to soils contaminated with polycyclic aromatic hydrocarbons (PAH).

Abstract. We have used the minipig as a prospective animal model for human risk characterization to study primary biochemical alterations upon oral contaminant intake. The effects of three orally administered soils containing polycyclic aromatic hydrocarbons (PAH) on the expression pattern of the cytochrome P450 enzyme CYP1A1 in various organs have been analyzed. Dependent on the soil sample, subchronic daily oral PAH doses ranged from 0.38 to 1.90xa0mg PAHEPA/kg body weight. In all cases, soil administration lead to significant CYP1A1 induction in several organs of minipigs to a different extent, following the order liver ≈ duodenum >lung >kidney ≈ spleen. Hepatic ethoxyresorufin-O-deethylase activities were elevated to 310, 1250 and 1780 compared with a background level of 200xa0pmol resorufin/mg protein per min. Induced duodenal activities appear to be even higher than in the liver, namely 405, 1280 and 2500 compared with a basal activity of 11xa0pmol resorufin/mg protein per min. CYP1A1 induction in several organs is clear evidence for successful contaminant mobilization and absorption in the duodenum and subsequent distribution of contaminant into diverse body compartments. As is shown in one case, impairment of CYP1A1 induction in the liver and thus breakdown of its PAH-metabolizing activity appears to have no effect on induced CYP1A1 levels in other organs. It appears important with respect to risk assessment that induction of CYP1A1 is particularly sensitive in the duodenum of minipigs and is achieved with soil doses which are in the range of amounts ingested by playing children due to hand-to-mouth activities. Induced duodenal CYP1A1 activities obtained in minipigs by oral exposure to PAH largely exceed maximal duodenal activities so far observed in rats. This is equally relevant for risk assessment and for selection of a suitable animal model that reflects effects of PAH exposure in humans.

Liver microsomal levels of cytochrome P450IA1 as biomarker for exposure and bioavailability of soil-bound polycyclic aromatic hydrocarbons

The bioavailability of soil-bound polycyclic aromatic hydrocarbons (PAHs) for mammalian species was studied with rats fed with a diet containing contaminated soil preparations. The extent of cytochrome P450IA1 (CYP1A1) induction in the liver correlated with the amount of 5- and 6-ring PAHs in the soil samples but not with the total PAH content. Other cytochromes P450 were much less affected by the soil-contaminants. The highest induction of CYP1A1 was obtained with a sample containing 274 mg 5- and 6-ring PAH/kg soil, resulting in a nearly 360-fold increase in the ethoxyresorufin deethylase (EROD) activity. In a semilogarithmic plot, a linear correlation was found between the 5- and 6-ring PAH concentration in the soil and the microsomal CYP1A1 content. As a model for the action of intestinal fluids, soil samples were extracted by bile acid solution. In these experiments, the selectivity in the solubilization of individual PAHs parallels that of toluene extraction, although the yield is lower than the latter and varies with the soil sample. The bioavailability of PAHs for microorganisms, but not for mammals, was shown to be considerably reduced in the presence of high total organic carbon (TOC) values of the soil samples. This may have implications for decontamination strategies, diminishing the effectiveness of biological decontamination in cases with high TOC values. The data suggest that CYP1A1 induction in rats is a parameter that may be useful in risk assessments of contaminated soils for mammalian species.

Chromatographic separation and behavior of microsomal cytochrome P450 and cytochrome b5

The methods used for separation of the multiple mammalian cytochrome P450 enzymes by liquid chromatography are reviewed. In addition to the chromatographic techniques, preparation and handling of samples and prefractionation procedures are considered. Conditions that affect stability and chromatographic resolution of cytochromes P450 are also discussed. Special emphasis is put on useful methods which are not routinely used for P450 separation, such as immobilized metal affinity or hydrophobic-interaction chromatography. Applications of low- and high-pressure methods with regard to preparative and analytical separations are compared. It is shown that high- and medium-pressure ion-exchange chromatography are suitable tools for separation of closely related P450 enzymes, especially when specific detection methods are available. In addition to fractionation of cytochromes P450, the isolation and chromatographic behavior of cytochrome b5 is discussed.

Metabolite complex formation of orphenadrine with cytochrome P450: Involvement of CYP2C11 and CYP3A isozymes

Expression and inhibition of cytochrome P450 (CYP) isozymes capable of forming an orphenadrine metabolite complex were studied in microsomes of untreated and inducer-treated male and female rats. High levels of complex-forming isozymes were found in microsomes of untreated male as compared to female rats. Treatment of male rats with several P450 inducers did not considerably increase the extent of in vitro complex formation. In female rats, however, phenobarbital or dexamethasone treatments led to pronounced induction. The isozyme specificity of complex formation was investigated by several approaches including: 1. inhibition by orphenadrine of isozyme-specific P450 activities, such as hydroxylation of testosterone, O-dealkylation of pentoxy-and ethoxyresorufin and complex formation with triacetyloleandomycin (TAO), 2. inhibition of orphenadrine complex formation by metyrapone, TAO, and cimetidine, and 3. correlation of complex levels with immunochemically, enzymatically, or spectroscopically determined amounts of P450 isozymes. Our data suggest that CYP2C11, a CYP3A isozyme and an unidentified P450 species are involved in complex formation with orphenadrine, but exclude the involvement of CYP1A1/2 and CYP2B1/2. The capability of CYP2C11 to form a metabolite complex with orphenadrine is strongly suggested for the following reasons: 1. Efficient inhibition of testosterone 2 alpha- and 16 alpha-hydroxylation by complex formation with orphenadrine in microsomes of untreated male rats, 2. high expression of orphenadrine-complexing isozymes in untreated male compared to female rats, 3. specific inhibition of in vitro complex formation by cimetidine, 4. suppression of complex-forming isozymes by 3-methylcholanthrene and beta-naphthoflavone, and 5. concomitant induction of complex-forming isozymes, immunodetectable CYP2C11, and testosterone 2 alpha-hydroxylase by stanozolol. That at least one, but not all, CYP3A isozymes is involved in complex formation is concluded from inhibition experiments with TAO that show that orphenadrine complexation can be significantly inhibited in microsomes of dexamethasone-treated, but not in microsomes of untreated rats. Furthermore, complex formation with TAO is not inhibited by orphenadrine in microsomes of phenobarbital (PB)-treated rats. In PB-treated female rats, a further unidentified complex-forming isozyme can be detected that is not inhibited by complex formation with TAO.

Analytical fractionation of microsomal cytochrome P-450 isoenzymes from rat liver by high-performance ion-exchange chromatography

Ion-exchange Fast Protein Liquid Chromatography (FPLC) on Mono Q and Mono S was optimized for the analytical separation of microsomal cytochrome P-450 species from rat liver. The effects of detergent, pH, gradient profile and column load on resolution are demonstrated. Successive application of anion- and cation-exchange chromatography leads to eleven separated P-450 fractions. The altered microsomal P450 pattern after treatment of rats with various inducers is reflected by distinct elution profiles. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and enzymatic analysis imply that several FPLC fractions contain more than one P-450 species. Preliminary results are presented showing the suitability of immobilized metal affinity chromatography (MAC) for general P-450 fractionation and thus for the further resolution of Mono Q and Mono S fractions. Scale-up for preparative P-450 fractionation is easily done by adapting the optimized analytical FPLC procedures to Q- and S-Sepharose Fast Flow.