Search

The quantitation of mRNA, essential for assessing mechanisms of enzyme regulation, is normally carried out using reverse transcriptase-polymerase chain reaction (RT-PCR). An alternative method uses a signal-amplification nucleic acid probe assay, which measures RNA directly by the QuantiGene Expression Kit and incorporates branched DNA technology from Bayer and luminometer-based readings of a chemilumigenic alkaline phosphatase substrate. To broaden the utility of this assay, we investigated substitution of a fluorescent substrate, 2'-(2-benzothiazol)-6'-hydroxybenzothiazole phosphate and a fluorometer, and applied the method to quantitation of CYP1A1 and 1B1 mRNA in human T-47D and HepG2 cells following induction by benzo[a]pyrene (B[a]P) and dibenzo[a,h]anthracene (DB[a,h]A). The fluorescence response increased linearly for 200 min without photobleaching and increased linearly (r2=0.997) up to at least 0.2 microg total RNA. The data revealed that at 0.5 and 1.0 microM inducing agent, the induction of CYP1A1 mRNA in HepG2 cells by DB[a,h]A exceeded that by B[a]P by 18- and 6-fold, respectively. In T-47D cells B[a]P induced CYP1A1 mRNA by 23-fold and CYP1B1 mRNA by 3.9-fold. A B[a]P cocontaminant in the environment, arsenite, did not affect B[a]P-induced levels of CYP1A1 or 1B1 mRNA in these cells. The modified analytical system provides a rapid-throughput, reproducible, and less labor-intensive method than RT-PCR for quantifying cellular mRNA levels.

Drugs and carcinogens are excreted from the body after metabolic conversion involving enzymes mediating oxidative metabolism and conjugation. Many of the corresponding genes exhibit functional polymorphisms that contribute to individual cancer susceptibility. To increase the efficiency and to facilitate genotyping, we developed a combined approach (PCR-ASO) which includes multiplex PCR and allele-specific oligonucleotide (ASO) hybridization. PCR primer pairs were used to amplify the following alleles/variants: CYP1A1*1, *2A, *2B; CYP2D6*3, *4; NAT1*4, *3, *10, *11, *14, *15; and NAT2*4, *5A, *5B, *5C, *6A, *7B. The products were dot-blotted and polymorphisms were detected by hybridization with ASO probes for both wild-type and variant sites in parallel. This approach was validated by genotyping DNA samples from a French-Canadian population that was previously analyzed by PCR-RFLP. The variants frequencies were compared with the data on other populations available in the literature. The PCR-ASO assay appears to be simple, efficient, and cost-effective, particularly if a large number of samples are to be screened for several DNA variants. This approach has potential for automation with microplates and robotic workstations for high throughput.

The aim of this work was to develop a procedure for the lipid-mediated transfection of DNA into normal adult human hepatocytes in culture. Cells were plated in a serum-free culture medium at various cell densities, on plastic or collagen-coated dishes, both in the absence and in the presence of epidermal growth factor (EGF). The cells were incubated for various periods of time with mixtures of DNA-lipofectin or DNA-3 beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-chol) liposomes, and the efficiency of transfection was assessed by measuring the activity of reporter genes, beta-galactosidase or chloramphenicol acetyl-transferase (CAT). For comparison, similar experiments were carried out with human cell lines including HepG2, Caco-2, and WRL68. The efficiency of transfection (in percentage of cells) was not significantly different after transfection with lipofectin or DC-chol and comprised between 0.04 and 1.7% (extreme values) for different cultures. The efficiency of transfection decreased as the age or density of the culture increased and increased in cultures treated with EGF. Direct measurement of the rate of DNA synthesis suggested that the efficiency of transfection was related to the number of cells entering the S phase. Under the same conditions, the efficiency of transfection was one to two orders of magnitude greater in the three cell lines. A plasmid harboring 660 bp of the 5'-flanking region of CYP1A1 (containing two xenobiotic enhancer elements) fused upstream of the promoter of thymidine kinase and the CAT reporter gene was constructed. When this plasmid was transfected in human hepatocytes, CAT activity was induced as expected. We conclude that normal adult human hepatocytes can be transfected with exogenous DNA and that the transfected construct is regulated in the manner expected from in vivo studies.

An efficient method for measuring polychlorinated biphenyl-mediated induction of cytochrome P4501A, using the ethoxyresorufin-O-deethylase (EROD) assay, and total porphyrins in chicken embryo hepatocyte cultures is described. Hepatocytes were cultured in 48-well plates, assays were carried out within the wells, and concentrations of the product of the EROD reaction (resorufin), porphyrins, and total proteins were measured in the same wells with a fluorescence plate reader. The method differs from previous methods developed in this laboratory in that three fluorophors were measured in the same well rather than two.

An assay was characterized that facilitated per-cell estimation of cytochrome P-450-dependent monooxygenase activities in whole-cell suspensions and cultures of murine epidermal keratinocytes (MEKs). 7-Ethoxycoumarin O-deethylase (7-ECD), 7-ethoxyresorufin O-deethylase (7-ERD), and 7-pentoxyresorufin O-deethylase (7-PRD) activities were monitored by fluorescent detection of their products. MEKs were made permeable by a freeze-thaw cycle, and xenobiotic metabolism occurred in situ. Analyses of cultured MEKs were made with the cells attached to the culture dishes. Product formation was proportional with MEK cell number and assay time and was dependent upon a NADPH-generating system. The three monooxygenase activities were inhibited to various degrees, in a dose-dependent manner, by the P-450 inhibitors α-naphthoflavone and metyrapone. The number of MEKs obtained from a single mouse was sufficient for multiple analyses. The assay was also used to determine monooxygenase activities in whole-cell suspensions of rat hepatocytes. Consitutive per hepatocyte 7-ECD, 7-PRD, and 7-ERD activities were 357-, 96-, and 1926-fold greater, respectively, than the activities measured in suspensions of dorsal MEKs.

We have previously reported the synthesis of four alpha-cyano-containing ethers based on 2-naphthaldehyde (2-NA) as cytochrome P450 (P450) fluorescent substrates. Activity detection was based on the formation of fluorescent 2-NA following substrate hydrolysis. A major limitation of these substrates was the need to remove NADPH, a required cofactor for P450 oxidation, before measuring 2-NA fluorescence. In this article, we report the synthesis of a new series of novel P450 substrates using 6-dimethylamino-2-naphthaldehyde (6-DMANA), which has a green fluorescent emission that is well separated from the NADPH spectrum. A major advantage of the 6-DMANA substrates is that NADPH removal is not required before fluorescence detection. We used eight alpha-cyano ether-based substrates to determine the O-dealkylation activity of human, mouse, and rat liver microsomes. In addition, substrate activities were compared with the commercial substrate 7-ethoxyresorufin (7-ER). The catalytic turnover rates of both the 6-DMANA- and 2-NA-based substrates were in some cases threefold faster than the catalytic turnover rate of 7-ER. The 2-NA-based substrates had greater turnover than did the 6-DMANA-based substrates. Murine and rat liver microsomes prepared from animals that had been treated with various P450 inducers were used to examine for isozyme-selective turnover of the substrates. The vastly improved optical properties and synthetic flexibility of the alpha-cyano ether compounds suggest that they are possibly good general P450 substrates.

In this article, we describe a highly sensitive biosensing system, DRESSA, for detection of dioxin and dioxin-like chemicals. Tandem copies of the dioxin-responsive element (DRE) fused to a minimal viral promoter were subcloned into an expression plasmid upstream of a secreted alkaline phosphatase (SEAP) gene. When murine hepatoma cell line Hepa-1c1c7 was stably transfected with this construct, established sensor clones secreted SEAP following stimulation with 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). A clone HeDS49 was found to be extremely sensitive; it secreted SEAP in response to TCDD in dose- and time-dependent manners, and the minimal detection limit was 100 fM. To detect more than 6 pM of TCDD, the whole assay time (from cell seeding to measurement of SEAP activity) could be reduced to 4 h. Secretion of SEAP was induced selectively by other activators of DRE (3-methylcholanthrene, benzo[ a ]pyrene, and β-naphthoflavone) but not by activators of unrelated responsive elements. These data suggested that because of the rapidity, easiness, specificity, and high sensitivity of DRESSA, it is more suitable than currently available detection systems for dioxin and dioxin-like chemicals and would be of great advantage to high-throughput screening of these pollutants in environmental samples.

A method for determining monohydroxybenzo[a]pyrene (OHBaP) isomers using column-switching high-performance liquid chromatography with fluorescence detection was developed. Eleven of 12 isomers of OHBaP (all except 6-OHBaP) were separated on an alkylamide-type reversed-phase column and, via column-switching, on a beta-cyclodextrin-bonded silica gel column. The detection limits for the OHBaPs were in the range 0.3-8 pg/injection (S/N=3). By using this method, 1-, 3-, and 9-OHBaPs were identified as major metabolites of benzo[a]pyrene in vitro by human recombinant p450 1A1. The method was used to determine OHBaPs in the urine of a nonsmoker subject. After enzymatic hydrolysis of the conjugated metabolites by beta-glucuronidase/aryl sulfatase, the analytes were selectively adsorbed on blue rayon (a cellulose-supported copper phthalocyanine) from the urine matrix. Methanol as the eluting solvent from the rayon gave the best recoveries of OHBaPs and 1-hydroxypyrene (1-OHP) in the range of 91-103%, which was superior to that of the solid-phase extraction method. 1-OHP, a well-known biomarker of the exposure to polycyclic aromatic hydrocarbons, was simultaneously analyzed. Intra- and interday accuracy values for the determination of 3-OHBaP in 200 ml of urine were 95.5 and 100.9%, and those for 1-OHP were 96.4 and 103.6%, respectively. The intra- and interday precision values were 3.9 and 2.4% for 3-OHBaP and 2.4 and 3.2% for 1-OHP, respectively. In 11 kinds of isomers, only 3-OHBaP was detected in the human urine. Urinary concentration of 3-OHBaP was quantified at 0.5 ng/g creatinine concentration and the 3-OHBaP/1-OHP ratio was approximately 1/130.

A method for simultaneous measurement of the cytochrome P4501A-associated enzyme, ethoxyresorufin-O-deethylase (EROD), and total protein concentration in liver microsomes is described. EROD assays were carried out in multiwell plates, and the fluorescent product (resorufin) and total proteins were quantified within the same wells with a fluorescence plate reader. Fluorescamine was used for protein assays.

The formation of resorufin from 7-ethoxyresorufin by microsomal mixed-function oxidase may be assayed rapidly by precipitating the protein in incubation mixtures with methanol and measuring the fluorescence of resorufin in the supernatant after centrifugation. Conditions are described for minimizing the effects of the instability of resorufin and of inhibition by the product. Higher activity and linear plots of microsomal protein concentration versus activity are obtained by including bovine serum albumin or ethylenediamine-tetraacetate in the incubation mixture.

We have developed a sensitive and nonradiometric assay of estradiol 2- and 16 alpha-hydroxylase activities using reverse-phase high-performance liquid chromatography with voltametric detector. The 2- and 16 alpha-hydroxylated estrogens produced by the incubation of estradiol with rat liver microsomes were initially separated into the catechol and phenolic fractions using a QAE-Sephadex A-25 borate column. The metabolites were detected in quantities as low as 0.5-1 ng using 3-methoxy-1,3,5(10)-estratriene-2,16 alpha,17 beta-triol or 4-hydroxyestrone 17-oxime as an internal standard. Apparent Km and Vmax of the 2- and 16 alpha-hydroxylases were 41.9 microM and 1.3 nmol/mg protein/min, and 82 microM and 480 pmol/mg protein/min, respectively.

A procedure to directly measure the cytochrome P-450-dependent 7-ethoxyresorufin O-deethylase activity with a visible spectrophotometer is described and compared to the standard fluorometric method. The two assays yielded identical results with both β-naphthoflavone-treated mammalian (rat) and fish (scup, Stenotomus chrysops) liver microsomes. The assay takes advantage of a clean distinction in visible absorption spectra obtained for highly purified 7-ethoxyresorufin (substrate) and resorufin (enzymatic product). The purification and characterization of resorufin, the enzymatic product, are detailed, and its extinction coefficient (e572 = 73 m m −1 cm−1) provides for an accurate quantitation of enzyme activity. The large visible extinction coefficient of the product chromophore provides a high sensitivity for low-activity samples. The application of this enzyme assay in a visible spectrophotometer, along with the considerable evidence that a single aromatic hydrocarbon-inducible cytochrome P-450 isozyme is responsible for the catalysis, enhances the utility of this substrate in microsomal monooxygenase assays. The utility of the visible assay is further demonstrated by the simple determinations of the coupling ratio for 7-ethoxyresorufin oxidation in scup liver microsomes and the KI for 7,8-benzoflavone and phenylimidazole inhibition of the enzymatic reaction.

A new method for the simultaneous assay of estrogen 2- and 4- hydroxylases consists of determining the enzymatically produced catechol estrogens by means of high-performance liquid chromatography with electrochemical detection. Estradiol was incubated with the enzyme preparation in the presence of NADPH. 2-Hydroxyestrone, 2-hydroxyestradiol, 4-hydroxyestrone, and 4-hydroxyestradiol formed were purified by an extraction method with boric acid and then separated by high-performance liquid chromatography. The peaks on the chromatogram were characterized by gas chromatography-mass spectrometry together with microchemical reactions. The amounts of the four catechol estrogens could be determined using 2-hydroxy-16-ketoestradiol 17-acetate as an internal standard with satisfactory accuracy and precision. The assay method was found to be superior in simplicity, sensitivity, and versatility.

[2- 3 H]Estradiol was synthesized from 2-iodoestradiol by reduction with sodium borotritiride in the presence of palladium chloride as a catalyst. When the labeled substrate was incubated with the rat liver and kidney microsomes, the tritium label was liberated quantitatively depending upon the 2-hydroxylase activity. Tritiated water produced in the incubation medium was recovered as a satisfactory rate by passage through a column of Amberlite XAD-2 resin without any interference due to the labeled substrate. The present method for the assay of estradiol 2-hydroxylase activity was found to be simple, reliable, and sensitive (detection limit:29 pmol of 2-hydroxyestradiol).