Biomedical subjects

F Oesch

Publications and source records attributed to F Oesch.

At least 145 records · Page 8Linked to original sources

PubMed1994

The anticonvulsant FCE 26743 is a selective and short-acting MAO-B inhibitor devoid of inducing properties towards cytochrome P450-dependent testosterone hydroxylation in mice and rats.

The effects of the potent anticonvulsant FCE 26743 ((S)-2-(4-3-fluorobenzyloxy)benzylamino)propionamide) on monoamine oxidase (MAO) activity were measured in-vitro and ex-vivo using rat tissue homogenates. In-vitro, FCE 26743 showed potent and selective inhibitory properties towards liver MAO-B, with IC50 values about 10(-7) M for MAO-B and higher than 10(-5) M for MAO-A. When determined ex-vivo in brain, the ED50 value for the inhibition of MAO-B was 1.1 mg kg-1 (p.o.) 1 h post-dosing, whereas MAO-A remained virtually unaffected after administration of 60 mg kg-1. Similar effects were seen in liver. Following oral administration of 5 mg kg-1 FCE 26743 to rats, brain MAO-B inhibition was 79% after 1 h and 13% after 24 h, indicating that FCE 26743 behaves as a short-acting MAO-B inhibitor. The ability of FCE 26743 to act as a MAO substrate was assessed in mice by measuring the urinary excretion of alaninamide, a potential metabolite of FCE 26743 which would result from the action of MAO. No alaninamide was detectable in the 0-8 h urines after administration of a 119 mg kg-1 dose, suggesting that FCE 26743 is not, or only to a small degree, a substrate of MAO. The effects of FCE 26743 on cytochrome P450 enzymes involved in testosterone hydroxylation were determined in rats after repeated administration. No induction of the cytochrome P450 system was noted.

Alanine↗

PubMed1994

The effect of indobufen on the activities of selected rat liver phase I and phase II drug metabolizing enzymes, peroxisomal beta-oxidation and hepatic glutathione status.

Oral administration of indobufen to male rats for three days at daily doses of 5, 10 and 20 mg kg-1 resulted in no changes in liver total glutathione, cytosolic glutathione S-transferases or microsomal epoxide hydrolase. Reduced glutathione appeared slightly diminished to about 84% of control at the highest dose level. Microsomal cytochrome P450-dependent ethoxyresorufin O-de-ethylase and pentoxy-resorufin de-alkylase activities were decreased to 64% (not significantly) and 67% of control at the lowest dose level. 6 alpha- and 7 alpha-Hydroxytestosterone activities were decreased to 67 and 68% of control at the highest dose level. Cyanide-insensitive peroxisomal fatty acid beta-oxidation was increased to 223, 261 and 232% of control at doses of 5, 10, and 20 mg kg-1, respectively. The results obtained in this study are indicative of the action of indobufen as a weak peroxisome proliferator in male rat liver, and suggest a slight but toxicologically insignificant inhibitory action of this drug on microsomal cytochrome P450-dependent enzyme activities.

Animals↗

PubMed1994

Mechanism-based predictions of interactions.

Exposure to more than one toxic compound is common in real life. The resulting toxic effects are often more than the simple sum of the effects of the individual compounds. It is unlikely that it will ever be possible to test all combinations. It is therefore highly desirable to improve or develop means for reasonably approximating predictions of interactions. In order to be valid and extrapolatable, these predictions are most promising if they are mechanism-based. Examples will be given for possibilities of mechanism-based predictions of interactions which exceed trivialities of simple increases by enzyme induction of enzymatic rates of a given biotransformation pathway leading to a toxic metabolite. Instead, examples will be provided where competition between various enzymes for shunting the same substrate into divergent pathways can lead to predictable dramatic changes in toxicity by shifting the metabolic routes under conditions of no significant changes of overall metabolism. Further examples are given on predictable interactions between chemicals which need bioactivation for exerting their toxicity and chemicals which effect hormonal status and other endogenous factors which in turn modify enzymes involved in the control of toxic metabolites.

Animals↗

PubMed1994

Oval cell lines OC/CDE 6 and OC/CDE 22 give rise to cholangio-cellular and undifferentiated carcinomas after transformation.

BACKGROUND: There is compelling evidence for a parenchymal origin of the predominant cell lineage leading from preneoplastic clear and acidophilic glycogen storage foci through mixed and basophilic cell populations to hepatocellular carcinomas in the rat. However, a controversial question remains to be answered: Do the basophilic cell foci invariably originate from parenchymal cells or do oval cells also have the potential to give rise to this type of focus and progress to hepatocellular neoplasms? Oval cells are nonparenchymal epithelial cells with scant cytoplasm and ovoid nuclei that first appear in the periportal areas of the liver lobules and thereafter invade the whole parenchyma when animals are exposed to high doses of a wide range of chemical carcinogens. EXPERIMENTAL DESIGN: Two oval cell lines, OC/CDE 6 and OC/CDE 22, which had been established from rats fed a choline-deficient/DL-ethionine-supplemented diet for 6 or 22 weeks, were transformed either by leaving the cells in confluence for a long time period (OC/CDE 6) or by treating the cells with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. The transformed cells were injected subcutaneously in newborn rats and the tumors developing in these animals were analyzed histopathologically, ultrastructurally, and immunohistochemically. RESULTS: The two transformed oval cell lines gave rise to carcinomas, in which cholangiocellular, adenoid and solid tumor formations were observed. Subpopulations of these tumors expressed cytokeratins 7, 8, 18, and 19, but were albumin- and alpha-fetoprotein-negative. Areas within the carcinomas derived from transformed OC/CDE 22 cells representing undifferentiated liver tumor formations were also identified. Cells within these areas had lower nucleus/cytoplasm ratios than cells in the solid growing tumor formations, stained positive for cytokeratins 8 and 18 and were cytokeratin 7- and 19-, albumin- and alpha-fetoprotein-negative. Ultrastructurally, these cells did not resemble those of differentiated hepatocellular carcinomas. CONCLUSIONS: It has been shown that oval cells are precursor cells of carcinomas containing cholangiocellular, adenoid and solid formations which may be largely undifferentiated. However, the transformed OC/CDE 6 or OC/CDE 22 cells do not serve as precursor cells of differentiated hepatocellular carcinomas.

Animals↗

PubMed1993

Isolation and characterization of a cDNA encoding rat liver cytosolic epoxide hydrolase and its functional expression in Escherichia coli.

A cDNA of 1992 base pairs encoding the complete rat liver cytosolic epoxide hydrolase has been isolated using a polymerase chain reaction-derived DNA fragment (Arand, M., Knehr, M., Thomas, H., Zeller, H. D., and Oesch, F. (1991) FEBS Lett. 294, 19-22) known to represent the 3'-end of the cytosolic epoxide hydrolase mRNA. Sequence analysis revealed an open reading frame of 1662 nucleotides corresponding to 554 amino acids (M(r) = 62,268). The DNA sequence obtained did not display significant homology to the sequences of microsomal epoxide hydrolase or leukotriene A4 hydrolase or to any other DNA included in the EMBL Data Bank (release 32). On Northern blotting of rat liver RNA, a single mRNA species was detected that was strongly induced on treatment of the animal with fenofibrate, a potent peroxisome proliferator. The most significant structure of the deduced protein is a modified peroxisomal targeting signal (Ser-Lys-Ile) at the carboxyl terminus that is regarded to be responsible for the unusual dual localization of the cytosolic epoxide hydrolase in peroxisomes as well as in the cytosol. In addition, a leucine zipper-like motif was identified at the amino terminus. Its possible implication for the observed dimeric structure of cytosolic epoxide hydrolase is discussed. The isolated cDNA was expressed in bacteria to yield a catalytically active enzyme. Specific activity of the crude lysate obtained exceeded that of rat liver cytosols from maximally induced animals by a factor of 8.

Amino Acid Sequence↗

PubMed1993

Evidence for the involvement of a bis-diol-epoxide in the metabolic activation of dibenz[a,h]anthracene to DNA-binding species in mouse skin.

Dibenz[a,h]anthracene (DB[a,h]A) and its microsomal metabolites, trans-3,4-dihydro-3,4-dihydroxydibenz[a,h]anthracene (DBA-3,4-diol), trans,trans-3,4:8,9-tetrahydro-3,4:8,9-tetrahydroxydibenz[a,h]anth racene, trans,trans-3,4:10,11-tetrahydro-3,4:10,11-tetrahydroxydibenz[a,h] - anthracene (DBA-3,4,10,11-bis-diol) and trans,trans-3,4:12,13-tetrahydro-3,4:12,13- tetrahydroxydibenz[a,h]anthracene were each applied topically to mouse skin and the epidermal DNA isolated 24 h later. 32P-postlabeling analysis of each of the DNA samples was performed. DNA from mice treated with DB[a,h]A produced an adduct map on TLC consisting of one major and three minor adduct spots. A similar pattern of spots was produced by DBA-3,4-diol. No detectable DNA adducts were produced by trans,trans-3,4:12,13-tetrahydro-3,4:12,13-tetrahydroxy- dibenz[a,h]anthracene, although a single, minor adduct spot was produced by trans,trans-3,4:8,9-tetrahydro-3,4:8,9-tetrahydroxydibenz[a,h]- anthracene. However, DBA-3,4,10,11-bis-diol was found to produce a major single adduct that comigrated on thin layer chromatography with the major adduct produced by both DB[a,h]A and DBA-3,4-diol. In addition, this adduct was present at a level 10 times higher than the corresponding adduct produced by treatment with the parent hydrocarbon. Coelution of the major adducts formed from DB[a,h]A and DBA-3,4-diol with that formed from DBA-3,4,10,11-bis-diol was also demonstrated on reverse-phase high performance liquid chromatography. Thus, we propose that, in mouse skin, the major pathway of DB[a,h]A activation to DNA binding products is via a 3,4-diol to the 3,4,10,11-bis-diol and ultimately to a bis-diol-epoxide (potentially the 3,4,10,11-bis-dihydrodiol-1,2-oxide).

Animals↗

PubMed1993

A method for the cryopreservation of liver parenchymal cells for studies of xenobiotics.

An optimized computer-controlled freezing protocol for the cryopreservation of rat liver parenchymal cells was developed. The best survival rates were obtained when a slow cooling rate was used and when the supercooling was interrupted with a shock cooling to initiate ice nucleation. Ten percent dimethyl sulfoxide was added and removed gradually for best results. Thawed rat liver parenchymal cells had a viability, as judged by trypan blue exclusion, of 69% (SD = 6) versus 82% (SD = 7) for freshly isolated cells. The content and activities of the xenobiotic metabolizing enzymes, cytochrome P450, UDP-glucuronosyl transferase, and microsomal and cytosolic epoxide hydrolase, were not affected, whereas a slight reduction of glutathione S-transferase and sulfotransferase occurred. If cryopreserved cells were purified by a Percoll centrifugation after thawing the enzyme activities were not significantly different from those of freshly isolated parenchymal cells and also the viability was 86% (SD = 3). Cryopreserved rat liver parenchymal cells only metabolized about 50% of benzo(a)pyrene compared to freshly isolated cells. It is less likely that the reduction in enzyme activities was due to the cryopreservation procedure than that it was due to the loss of NADPH as a cofactor for cytochrome P450 which then resulted in the decreased xenobiotic metabolism. This cryopreservation protocol was also suitable for a variety of liver parenchymal cells from other species when trypan blue exclusion was used as a viability marker.

Animals↗

PubMed1993

Isolation, biochemical characterization, long-term culture, and phenotype modulation of oval cells from carcinogen-fed rats.

Oval cells are liver epithelial cells that proliferate during hepatocarcinogenesis and chemically induced severe liver injury. It has been suggested that these cells represent hepatic stem cells which might play an important role in the histogenesis of cholangiocellular as well as hepatocellular carcinomas. In order to test this hypothesis highly purified oval cell preparations and propagable oval cell lines are needed. In the present study the isolation, biochemical characterization, and long-term culture of oval cells from rats fed a choline-deficient/DL-ethionine-supplemented diet for 6, 14, or 22 weeks are described. The freshly isolated oval cells were gamma-glutamyltranspeptidase-positive, cytokeratin 7-, 8-, 18-, and 19-positive, albumin-positive, peroxidase-negative, and alpha-fetoprotein-negative and expressed lactate dehydrogenase isoenzymes 1-5. In addition, low but clearly measurable glucose-6-phosphatase and high gamma-glutamyltranspeptidase and alkaline phosphatase activities (when compared to activities in untreated liver parenchymal cells) were measured in oval cells. Three oval cell lines, OC/CDE 6, OC/CDE 14, and OC/CDE 22, were established. They contained small and large epithelial cells replicating to form uniform monolayers with a cobblestone appearance; furthermore, a very low number of mononucleated giant cells were also present in the three cell lines. OC/CDE 6, OC/CDE 14, and OC/CDE 22 cells were gamma-glutamyltranspeptidase-negative, were transiently albumin-positive, maintained the glucose-6-phosphatase activity levels measured in freshly isolated oval cells, and expressed lactate dehydrogenase isoenzymes 2-5. After exposure of the cultured oval cells to dimethyl sulfoxide or sodium butyrate, 35-40% of the cells reexpressed albumin, and glucose-6-phosphatase activity was enhanced; in addition, sodium butyrate strongly increased gamma-glutamyltranspeptidase and alkaline phosphatase activities. In conclusion, oval cells express phenotypic markers of liver parenchymal as well as bile duct epithelial cells and possess a certain intrinsic plasticity. In order to test if the oval cells indeed represent an intermediate step in the differentiation of certain cells within the bile duct and ductular epithelial cell compartment to parenchymal cells, the three cell lines described herein will be transformed in vitro and their potential to give rise to cholangiocellular and/or hepatocellular carcinomas will be verified in vivo.

Animals↗

PubMed1993

Benzene metabolism by reconstituted cytochromes P450 2B1 and 2E1 and its modulation by cytochrome b5, microsomal epoxide hydrolase, and glutathione transferases: evidence for an important role of microsomal epoxide hydrolase in the formation of hydroquinone.

Benzene metabolism was investigated using two purified rat hepatic MFO systems containing either cytochrome P450 2B1 or cytochrome P450 2E1. Studies performed over a wide substrate concentration range indicate that cytochrome P450 2B1 represents a relatively low-affinity form of cytochrome P450 with respect to benzene metabolism while cytochrome P450 2E1 is substantially more efficient at low benzene concentrations (apparent Km value 0.17 mM). Cytochrome b5 stimulated benzene metabolism by both cytochromes P450 2B1 and P450 2E1. With cytochrome P450 2E1 the stimulation of benzene metabolism by cytochrome b5 was very pronounced (up to 6-fold) at low concentrations of benzene and was most effective (up to 15-fold) with respect to formation of hydroquinone. The metabolites observed in these studies were phenol and hydroquinone. Cytochrome P450 2E1 metabolized phenol with an affinity and capacity comparable to those of benzene. Hydroquinone was the major product formed at all substrate concentrations, while some catechol was formed at all substrate concentrations, while some catechol was formed at higher concentrations of phenol. Phenol metabolism was also stimulated by cytochrome b5. The metabolism of benzene by cytochrome P450 2E1 in the presence of the major microsomal epoxide hydrolase, mEHb, yielded phenol, hydroquinone, and benzene dihydrodiol. Interestingly, the addition of mEHb did not lead to a decrease of the toxicologically important metabolite hydroquinone as might be expected from sequestration of the intermediate benzene oxide to the vicinal dihydrodiol pathway but rather led to a marked (more than 4-fold) increase in the formation of hydroquinone, suggesting catalysis by mEHb of a predominant attack at the homoallylic position rather than at a carbon atom which forms the epoxide ring of benzene oxide. The addition of glutathione transferases plus glutathione did not yield GSH conjugates during benzene metabolism. However, metabolism of phenol by cytochrome P450 2E1 in the presence of glutathione yielded a nonenzymatically formed glutathione conjugate derived from hydroquinone or from an oxidative product of hydroquinone.

Animals↗

PubMed1993

Some substrates and inhibitors of cytosolic epoxide hydrolase induce sister-chromatid exchanges in mammalian cells, but do not induce gene mutations in Salmonella typhimurium and V79 cells.

Trans-stilbene oxide, trans-beta-methylstyrene 7,8-oxide, trans-beta-ethylstyrene 7,8-oxide, trans-beta-propylstyrene 7,8-oxide and 4-fluorochalcone oxide were investigated for genotoxic activity in bacterial and mammalian cells, in the absence of external xenobiotic-metabolising systems. All compounds strongly enhanced the frequency of sister-chromatid exchanges (SCE) in cultured human lymphocytes. None of them was mutagenic in Salmonella typhimurium (reversion of the his- strains TA98, TA100 and TA104). The limit of detection was 1/20,000 to 1/10(6) of the activity of the positive control, benzo[a]pyrene 4,5-oxide, depending on the compound and the bacterial strain. Trans-beta-methylstyrene 7,8-oxide and 4-fluorochalcone oxide were additionally tested for induction of SCE and gene mutations in the same target cells, namely Chinese hamster V79 cells. Their influence on the level of SCE was similar to that observed in human lymphocytes, whilst gene mutations (at the hprt locus) were not induced. The four investigated styrene oxide derivatives are known to be excellent substrates for a mammalian enzyme, cytosolic epoxide hydrolase (cEH). 4-Fluorochalcone oxide is a potent selective inhibitor of this enzyme and is structurally similar to the investigated styrene oxide derivatives. These properties of the test compounds however cannot explain the observed discrepancies in the results, since the genetic end point (SCE versus gene mutations) was decisive, and SCE were induced in cEH-proficient human lymphocytes as well as in cEH-deficient V79 cells.

Animals↗

PubMed1993

Genotoxicity characteristics of reverse diol-epoxides of chrysene.

Trans-3,4-dihydroxy-3,4-dihydrochrysene (chrysene-3,4-diol), a major metabolite of chrysene, is further metabolized by rat liver enzymes to products which effectively revert the his- Salmonella typhimurium strain TA98 to histidine prototrophy, but are only weakly mutagenic in strain TA100 and in Chinese hamster V79 cells (acquisition of resistance to 6-thioguanine). The liver enzyme mediated mutagenicity of chrysene-3,4-diol is substantially enhanced in the presence of 1,1,1-trichloropropene 2,3-oxide, an inhibitor of microsomal epoxide hydrolase. The predominant metabolites of chrysene-3,4-diol, namely the anti- and syn-isomers of its 1,2-oxide (termed reverse diol-epoxides), proved to be extraordinarily effective mutagens in S.typhimurium strain TA98, but were only moderately active in strains TA100 and TA104, and in the SOS induction in Escherichia coli PQ37. These genotoxicity spectra in bacteria are completely different from those observed with the bay-region diol-epoxides of chrysene and 3-hydroxychrysene. In V79 cells, the reverse diol-epoxides formed low levels of DNA adducts and were very weak inducers of gene mutations. In M2 mouse prostate cells, however, high numbers of transformed foci were induced by chrysene-3,4-diol and its diastereomeric 1,2-oxides. Chrysene-3,4-diol was somewhat more potent than chrysene-1,2-diol. The potency of both reverse diol-epoxides was similar to that of the syn-diastereomers of the bay-region diol-epoxides of chrysene and 3-hydroxychrysene, but lower than that of their anti-diastereomers. The reverse diol-epoxides of chrysene, unlike the bay-region diol-epoxides, were inactivated by purified microsomal epoxide hydrolase. Noteworthy findings were also made with regard to the chemical stability of the diol-epoxides in buffer, determined from the decline in mutagenicity after preincubation in the absence of the target cells. Despite its lower delta Edeloc/beta value for the formation of the benzylic carbocation, anti-chrysene-3,4-diol 1,2-oxide was shorter-lived (t1/2 = 46 min) than anti-chrysene-1,2-diol 3,4-oxide (t1/2 = 74 min). Unlike other investigated diastereomeric pairs of diol-epoxides, it was also shorter-lived than its syn-diastereomer (t1/2 = 340 min).

Animals↗

PubMed1993

Effects of sodium butyrate on DNA content, glutathione S-transferase activities, cell morphology and growth characteristics of rat liver nonparenchymal epithelial cells in vitro.

The effects of sodium butyrate, which has been shown to act as a differentiation promoting agent in several different tumor cell lines, were studied in a rat liver nonparenchymal epithelial cell line. Exposure of these cells to 3.75 mM butyrate resulted in an inhibition of cell proliferation and, at the same time, an increase in cell diameter (2- to 6-fold) and size of the nuclei (approximately 2-fold) after 3 days in culture. Binucleated cells arose, comprising approximately 12% of the cells investigated, and the number of cells with an abnormal set of chromosomes was increased. Intercellular communication, measured by dye transfer of Lucifer Yellow, was unchanged. From the various xenobiotic metabolizing enzyme activities measured, only those of glutathione S-transferases were significantly altered (increases of 4- to 9-fold) by butyrate treatment. These increases were mainly due to the predominant rise in the pi class isoenzyme which is a well-known tumour marker in rat hepatocarcinogenesis. Thus, our results cannot be interpreted as being either due to promotion of differentiation or due to transformation. The state and type of cell under study has to be considered and investigations of further differentiation parameters are needed to obtain a deeper insight into the biological activity and the underlying mechanisms of cell state modifying agents like butyrate.

Animals↗

PubMed1993

Characterization of DNA adducts at the bay region of dibenz[a,h]anthracene formed in vitro.

Bay region diolepoxide-DNA adducts of dibenz[a,h]anthracene (DBA) formed in vitro were identified and their absolute stereochemistry was assigned. After activation of [5,12-14C]DBA with liver microsomes obtained from Aroclor 1254 treated male Sprague-Dawley rats in the presence of calf thymus DNA for 1 h, the amount of DNA adducts was found to be 9.9 +/- 2.4 pmol/mg DNA, calculated on the basis of the portion of radioactivity eluted from the HPLC reversed-phase column with a water/acetonitrile gradient. Bay region diolepoxide-DNA adducts represented 27.5% of radioactivity associated with DNA adducts. The absolute configuration of the various adducts was determined from the reaction of the (+)- and (-)-3,4-dihydrodiol after metabolic activation and the reaction of the anti- and syn-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracen e with DNA or with the individual deoxyribonucleotides. The main bay region adduct was identified as a deoxyguanosine adduct of (anti)-3S,4R-dihydroxy-1R,2S-epoxy-1,2,3,4-tetrahydrodibenz [a,h]anthracene, a metabolite of (-)-3,4-dihydroxy-3,4-dihydrodi- benz[a,h]anthracene. Anti bay region diolepoxide-deoxyguanosine adducts of DBA contributed to 17.7% and syn diolepoxide-derived deoxyguanosine adducts to 5.8% of adduct-associated radioactivity. The amount of bay region deoxyadenosine adducts was calculated to be 4%. For six of probably eight different deoxyadenosine adducts absolute stereochemistry could be assigned. 32P-Postlabelling experiments revealed a binding of 23 +/- 6 pmol/mg DNA for (-)-3,4-dihydrodiol and of 1.5 +/- 0.4 pmol/mg DNA for (+)-3,4-dihydrodiol of DBA.

Animals↗

PubMed1993

Characterization of highly polar bis-dihydrodiol epoxide--DNA adducts formed after metabolic activation of dibenz[a,h]anthracene.

Dibenz[a,h]anthracene as well as a biologically important metabolite of dibenz[a,h]anthracene, namely the M-region dihydrodiol trans-3,4-dihydroxy-3,4-dihydrodibenz[a,h]anthracene were in addition to further metabolism to a bay region diol epoxide, extensively transformed to a distal bisdihydrodiol, 3,4,10,11-tetrahydroxy-3,4,10,11-tetrahydro-dibenz[a,h]anthracene, which exhibited after renewed metabolic activation high DNA binding efficiency, leading to a new class of very polar DNA adducts. After incubation of dibenz[a,h]anthracene with DNA in the presence of liver microsomes from Aroclor 1254 treated male Sprague-Dawley rats highly polar DNA adducts probably originating from 3R,4R,10R,11R-tetrahydroxy-3,4,10,11-tetrahydrodibenz[a,h]an thracene and 3R,4R,10S,11S-tetrahydroxy-3,4,10,11-tetrahydrodibenz[a,h]anthr ace ne were identified by reversed phase HPLC and by the 32P-postlabeling method. The adducts obtained were further characterized by comparing their fluorescence spectra with those obtained from 3,4,10,11-tetrahydroxy- 3,4,10,11-tetrahydrodibenz[a,h]anthracene and from 3,4-dihydroxy-3,4-dihydrodibenz[a]anthracene, the putative chromophore of the polar adduct. DNA adducts formed via 3S,4S,10S,11S-tetrahydroxy-3,4,10,11-tetrahydro- dibenz[a,h]anthracene were not found. After incubation of 14C-labelled dibenz[a,h]anthracene highly polar DNA adducts derived from the bisdihydrodiol contributed 38% to the adducts found in the HPLC profile. Bay region diol epoxide adducts represented a fraction of 25%. Using the 32P-postlabelling technique a higher DNA binding yield for the racemic bisdihydrodiol (38 +/- 12 pmol/mg DNA) was calculated than for the most active 3,4-dihydrodiol enantiomer, 3R,4R-dihydroxy-3,4- dihydrodibenz[a,h]anthracene (23 +/- 6 pmol/mg DNA).

Animals↗

PubMed1993

Formation of DNA adducts from 1-hydroxymethylpyrene in liver cells in vivo and in vitro.

The binding of 1-hydroxymethylpyrene (HMP) and its active metabolites, 1-hydroxymethylpyrene sulfate (SMP) and 1-chloromethylpyrene (CMP), to DNA was studied. In the liver of rats, maximum adduct levels were observed 1.5 h after i.p. injection of HMP, followed by a relatively rapid decrease. Separate exposure of different liver cell types in vitro to HMP led to high adduct levels in parenchymal cells, intermediate levels (1/10) in endothelial cells and low levels (1/200) in Kupffer cells. The adduct patterns were similar in the different cell types. The same pattern was also obtained when isolated DNA was incubated with SMP or CMP. One of the four major spots co-chromatographed on TLC with a dG adduct, one with a dA adduct and one with both dG and dA adducts. The fourth spot did not co-chromatograph with any of the adducts observed in reactions with any nucleic acid homopolymer.

Animals↗

PubMed1992

Colorimetric quantitation of trace amounts of sodium lauryl sulfate in the presence of nucleic acids and proteins.

A fast and sensitive procedure for the colorimetric detection of sodium lauryl sulfate (SDS) is presented. The assay is based upon the formation of a chloroform-extractable ion pair between lauryl sulfate and methylene blue that is quantified spectrophotometrically with an estimated detection limit of 150 ng of SDS. The method is suitable for the monitoring of contaminating traces of SDS in protein or nucleic acid samples that have the potential to interfere with enzymatic manipulations such as proteolytic digest, restriction analysis, or reverse transcription. Since the procedure is extremely simple and no special equipment is required it is accessible to every researcher concerned with SDS contamination.

Animals↗

PubMed1992

Improved sample preparation for the testosterone hydroxylation assay using disposable extraction columns.

The preparation of samples for injection into a high-performance liquid chromatograph from assay mixtures for the determination of cytochrome P-450-dependent testosterone hydroxylation has been substantially facilitated. By replacing the multiple cumbersome extraction steps of the conventional method with a single column extraction the time for sample preparation was reduced from hours to minutes. The new procedure also yields better recoveries for most of the testosterone metabolites than the original protocol. The use of extraction columns for sample preparation allows the simultaneous treatment of a large number of samples or even the automation of the whole assay procedure. The modified procedure is a straightforward, easy-to-perform method that should greatly facilitate the implementation of the testosterone hydroxylation assay for sharply discriminating between many individual cytochrome P-450 species in routine enzyme diagnostics.

Chromatography↗

PubMed1992

Sequence of a novel cytochrome CYP2B cDNA coding for a protein which is expressed in a sebaceous gland, but not in the liver.

The major phenobarbital-inducible rat hepatic cytochromes P-450, CYP2B1 and CYP2B2, are the paradigmatic members of a cytochrome P-450 gene subfamily that contains at least seven additional members. Specific oligonucleotide probes for these genomic members of the CYP2B subfamily were used to assess their tissue-specific expression. In Northern-blot analysis a probe specific to gene 4 (which is designated now as CYP2B12) hybridized to a single mRNA present in the preputial gland, an organ which is used as a model for sebaceous glands, but did not hybridize to mRNA isolated from the liver or from five other tissues of untreated or Aroclor 1254-treated rats. The cDNA sequence for the CYP2B12 RNA was determined from overlapping cDNA clones and contained a long open reading frame of 1476 bp. The nucleotide sequence of the CYP2B12 cDNA was 85% similar to the sequence of the CYP2B1 cDNA in its coding region and was different from any CYP2B cDNA characterized until now. The cDNA-derived primary structure of the CYP2B12 protein contains a signal sequence for its insertion into the endoplasmic reticulum and the putative haem-binding site characteristic of cytochromes P-450. A part of the potential haem pocket of CYP2B12 was identical with a similar structure in a bacterial protocatechuate dioxygenase. In immunoblot analysis of preputial-gland microsomes, antibodies against CYP2B1 recognized a single abundant protein with a lower apparent molecular mass than that of CYP2B1. Our results demonstrate that the CYP2B12 protein has the potential to be enzymically active and are the first demonstration that a member of the CYP2B subfamily is expressed exclusively and at high levels in an extrahepatic organ.

Amino Acid Sequence↗