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Biomedical subjects

A Seidel

Publications and source records attributed to A Seidel.

At least 109 records · Page 6Linked to original sources
PubMed1995

Malignant transformation of the liver tumour precursor cell line OC/CDE 22 by the four stereoisomeric fjord region 3,4-dihydrodiol 1,2-epoxides of benzo[c]phenanthrene.

In previous work we established the rat liver oval cell line OC/CDE 22 in order to study in vitro mechanisms of liver cell transformation. We have now exposed OC/CDE 22 cells to each of the four optically active fjord region dihydrodiol epoxides of benzo[c]phenanthrene to investigate their capacity for malignant transformation of liver cells. All four configurational isomers, which are among the most potent carcinogenic metabolites of polycyclic aromatic hydrocarbons tested in murine tumour models, malignantly transform OC/CDE 22 cells at a 2 microM dose level, resulting in a similar colony-forming efficiency in soft agar. Inoculation of the transformed cells into newborn syngeneic rats produced an extremely high incidence of carcinomas with a short latency period. The induced carcinomas displayed cholangiocellular, adenoid and solid growing structures. Neither cell growth in soft agar nor induction of tumor formation in newborn rats were achieved if confluent OC/CDE 22 cell cultures were exposed to each of the four stereoisomers and left in the confluent state for 4 weeks. In contrast, if confluent cells were exposed to the four stereoisomers, immediately split and then subcultured as usual, full transformation was accomplished. Our results indicate that the fjord region dihydrodiol epoxides of benzo[c]phenanthrene are highly efficient transforming agents for rat liver cells and that proliferation plays a pivotal role in the liver cell transformation process induced by polycylic aromatic hydrocarbons.

Animals↗
PubMed1995

Induction of chromosomal aberrations and spindle disturbances in Chinese hamster epithelial liver cells in culture by pyrene and benzo[a]pyrene quinones.

Regioisomers of pyrene and benzo[a]pyrene quinones were tested for their ability to induce structural and numerical aberrations and spindle disturbance in Chinese hamster epithelial liver (CHEL) cells in culture. All quinones tested were clastogenic. Pyrene-1,8-quinone (P-1,8-Q) and benzo[a]pyrene-3,6-quinone (BP-3,6-Q) induced strikingly high levels of triradials. In addition, dicentrics and ring chromosomes were very common in BP-3,6-Q-treated cultures. Isomers of these compounds, pyrene-1,6-quinone (P-1,6-Q) and benzo[a]pyrene-1,6-quinone (BP-1,6-Q), induced unobtrusive patterns of chromosomal aberrations. We suspect that the P-1,8-Q and BP-3,6-Q moieties bound to the DNA were still reactive, and formed crosslinks and/or underwent redox cycling leading to high local concentrations of reactive oxygen species. In addition, P-1,8-Q and BP-3,6-Q induced c-mitoses, hyperdiploidies and polyploidies, in particular endoreduplications. These effects were not seen with the other two test compounds, or they were only detected at the highest concentrations used, which were strongly cytotoxic (c-mitoses with P-1,6-Q, polyploidies with BP-1,6-Q).

Animals↗
PubMed1995

Cytochrome P450 mediated reactions studied in genetically engineered V79 Chinese hamster cells.

V79 Chinese hamster cells genetically engineered for stable expression of rat and human CYP have been shown to serve as analytical tools for studying metabolism related problems in toxicology and pharmacology. Here, the application of rat and human CYP1A1 and CYP1A2 is demonstrated for comparative studies on the oxidation of polycyclic aromatic hydrocarbons, such as phenanthrene, benz[a]anthracene, and benzo[a]pyrene. Live cells were cultivated for 2 days in the presence of these chemicals. Thereafter, the supernatant medium was checked for metabolites by gas chromatography and mass spectrometry. Marked cytochromes P450 and species dependent differences in the metabolite profiles were observed. Most important was the finding, that human cytochrome P450 1A1 almost exclusively oxidized benzo[a]pyrene in the 7,8,9,10-position, yielding the ultimate carcinogen 7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene whereas the rat cytochrome P450 1A1 oxidized benzo[]pyrene in the 4,5-position and 7,8,9,10-position. The importance of this finding is underlined by results from cytotoxicity studies. Benzo[a]pyrene was twice as cytotoxic in the human cytochrome P450 1A1 than in the rat cytochrome P450 1A1 expressing V79 cells. Species and cytochrome P450 specific metabolite profiles were also observed for phenanthrene and benz[a]anthracene.

Animals↗
PubMed1995

Cytokine production and serum proteins in depression.

One, as yet unemployed, approach to investigating immunology in depression is the assessment of the cytokine production by leucocytes, which would allow the determination of immune response under standardized conditions. Thus we measured the production of mitogen-induced cytokines (IL-1 beta, IL-2, IL-6, IL-10, interferon-gamma) and sIL-2R in a whole blood assay, and serum protein levels such as C-reactive protein (CRP), haptoglobin (Hp) and alpha 2-macroglobulin (alpha 2 M) in a longitudinal 6-week study in an attempt to assess leucocyte function during and after acute clinical stage of depression in 39 patients. Shortly after admission to hospital we found higher levels of all measured cytokines in the patients. Serum protein levels were significantly higher in the patients than in controls, and decreased over the study period. Whereas slightly elevated monokine levels in patients tended to reach control values, lymphokines showed a significant decrease over the 6 weeks as compared to baseline. These results suggest that the increase in immune activity seen at the beginning of the study may be followed by a suppressed cell-mediated immune function.

Acute-Phase Proteins↗
PubMed1994

Fjord-region diol-epoxides of benzo[c]chrysene are potent inducers of micronuclei in murine bone marrow.

Vicinal diol-epoxides are the best established carcinogenic metabolites of polycyclic aromatic hydrocarbons. Numerous studies have demonstrated their high genotoxic activity in various in vitro test systems. However, in vivo mutagenicity data are not available. The fjord-region diol-epoxides of benzo[c]chrysene combine high mutagenic activity in vitro with high hydrolytic stability. They were tested for the induction of micronuclei in the bone marrow following intraperitoneal administration to NMRI mice. The anti diastereomer of the diol-epoxide enhanced the frequency of micronucleated polychromatic erythrocytes strongly (7-19-fold above the value in untreated controls) over a very wide dose range (2.5-300 mumol/kg body weight). The syn diastereomer demonstrated similar effects, but required about 5 times higher doses. The corresponding proximate mutagen, benzo[c]chrysene-trans-9,10-dihydrodiol, was only moderately active, whereas the positive control substance, benzo[a]pyrene-trans-7,8-dihydrodiol, was strongly active. The study indicates that intraperitoneally administered diol-epoxides of benzo[c]chrysene may reach the bone marrow. Therefore, it will be possible to study the influence of metabolic modulation, e.g. by enzyme induction, on the effects of these ultimate mutagens and their metabolic precursors in vivo.

Animals↗
PubMed1994

The potent carcinogen dibenzo[a,l]pyrene is metabolically activated to fjord-region 11,12-diol 13,14-epoxides in human mammary carcinoma MCF-7 cell cultures.

Dibenzo[a,l]pyrene (DB[a,l]P), an environmental hydrocarbon and very potent carcinogen in rodent bioassays, could be activated to DNA-binding intermediates in cells through formation of three different regioisomeric bay- or fjord-region diol-epoxides or other more highly oxidized metabolites. The mechanism of metabolic activation of DB[a,l]P in the human mammary carcinoma cell line MCF-7 was elucidated by analyzing the DB[a,l]P-DNA adducts formed by [35S]phosphorothioate postlabeling, immobilized boronate chromatography, and high-performance liquid chromatography. Six DB[a,l]P-DNA adducts were detected. Comparison with those formed in cells by DB[a,l]P-11,12-diol and by reaction of DNA with syn- and anti-(benzylic hydroxyl and epoxide oxygen cis and trans, respectively) DB[a,l]P-11,12-diol-13,14-epoxide (DB[a,l]PDE) demonstrated that all DB[a,l]P-DNA adducts in MCF-7 cells were formed by these diol-epoxide isomers. Cellular DNA contained large amounts of two syn- and one anti-DB[a,l]PDE-DNA adducts and small amounts of one syn- and two anti-DB[a,l]PDE-DNA adducts. The ability of human cells to activate DB-[a,l]P to its fjord-region 11,12-diol 13,14-epoxides suggests that environmental exposure to DB[a,l]P could pose a risk for humans.

Benzopyrenes↗
PubMed1994

Different enzyme kinetics during the glutathione conjugation of the four stereoisomers of the fjord-region diolepoxides of benzo[c]phenanthrene by the mu-class rat liver glutathione S-transferase HTP II.

The enzyme-catalysed conjugation of each of the four stereoisomers of trans-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (B[c]PhDE) with glutathione (GSH) by HTP II, a novel isolated mu-class GSH transferase from the liver of untreated rat, was studied. All four stereoisomers were substrates for GSH transferase HTP II. The enzymatic reaction shows three different types of enzyme kinetics: substrate inhibition for (-)-anti-B[c]PhDE with (R,S,S,R)-absolute configuration, allosteric behavior using (+)-anti-B[c]PhDE with (S,R,R,S)-absolute configuration and Henri-Michaelis-Menten kinetics with both the (-)-syn- and (+)-syn-enantiomers, with (S,R,S,R)- and (R,S,R,S)-absolute configuration, respectively. When the concentration of these diolepoxides was varied (using 2 mM GSH), the apparent Vmax values were 1975 nmol/min x mg for (-)-anti-B[c]PhDE and about 60 nmol/min x mg for both (-)-syn- and (+)-syn-B[c]PhDE, with the corresponding Km values of 1.05 and 0.20 mM. The reaction of (+)-anti-B[c]PhDE determined by applying the Hill equation had an estimated Vmax value of 930 nmol/min x mg. On varying the concentration of GSH, linear Lineweaver-Burk plots were obtained. No competitive effect could be observed using a mixture of (-)-anti- and (+)-anti-enantiomers, indicating that their binding sites are different and independent. It was also shown, that the binding sites of (+)-anti- and both syn-enantiomers were different and independent of each other, while there was a small effect on the binding of the syn-enantiomers caused by (-)-anti-B[c]PhDE. All products of the reaction between GSH and the dihydrodiol epoxides of benzo[c]phenanthrene could be resolved by HPLC and were identified and quantitated using the corresponding synthetic GSH conjugates.

Animals↗
PubMed1994

Complementary Mössbauer and EPR studies of iron(III) in diferric human serum transferrin with oxalate or bicarbonate as synergistic anions.

The spectral and magnetic properties of iron(III) bound in the metal binding sites of human serum transferrin with oxalate or bicarbonate as synergistic anions has been studied with Mössbauer spectroscopy and electron paramagnetic resonance (EPR). The Mössbauer spectra of the iron(III) in diferric transferrin with oxalate have been described using a spin Hamiltonian with the values of the zero field splitting parameter, D = 0.55 +/- 0.05 cm-1, and the rhombicity of the crystal field, E/D = 0.045 +/- 0.005. The EPR spectrum can be described with D = 0.58 cm-1 and E/D = 0.057, using a g-strain model for the lineshape that is based on a Gaussian distribution of D and E/D with Gaussian widths sigma(D) = 0.35 cm-1 and sigma(E/D) = 0.013, respectively. The rhombicity of the iron surroundings for the transferrin-oxalate complex is almost one order of magnitude smaller than for the bicarbonate complex and the zero field splitting parameter is twice as large in the oxalate as in the bicarbonate complex. We conclude that the crystal field symmetry of the iron site is almost tetragonal in the oxalate complex but rhombohedral in the bicarbonate complex, reflecting the different geometries of the oxalate and bicarbonate coordination. The isomer shift delta = 0.56 +/- 0.01 mm s-1 and the quadrupole splitting delta EQ = 0.2 +/- 0.1 mm s-1, on the other hand, are very close to the values found for the bicarbonate complex. No differences between the Mössbauer spectra of the two iron(III) ions in diferric transferrin with oxalate were found. The homogeneity of the diferric transferrin samples was controlled by capillary zone electrophoresis in the presence of urea.

Bicarbonates↗
PubMed1994

Secondary combined resistance to the multidrug-resistance-reversing activity of cyclosporin A in the cell line F4-6RADR-CsA.

Multidrug-resistant tumor cells can be resensitized by combined application of the selecting cytostatic drug and a chemosensitizer, such as cyclosporin A (CsA) or a calcium channel blocker. Since clinical trials on the circumvention of multidrug resistance (MDR) with chemosensitizers report disparate results, we investigated whether tumor cells of the MDR phenotype can develop additional resistance to the cytostatic chemosensitizer combination. Thus, the Adriamycin(ADR)-selected, P-glycoprotein-positive MDR Friend leukemia cell line F4-6RADR was exposed to stepwise increased concentrations of CsA at a constant level of 0.05 microgram/ml ADR. The initial CsA concentration (plus 0.05 microgram/ml ADR) to inhibit cell growth of F4-6RADR cells by 50% (IC50) was 0.04 microgram/ml. By continuous incubation for more than 6 months, the IC50 for CsA (at constant ADR) was elevated to 3.6 micrograms/ml (90-fold), thus generating the variant F4-6RADR-CsA. The F4-6RADR-CsA cells were cross-resistant for cyclosporin H (CsH), a non-immunosuppressive derivative of CsA. As shown by immunocytochemistry as well as by the polymerase chain reaction and by Western blotting including densitometry, P-glycoprotein was preserved in the F4-6RADR-CsA variant and was expressed at a 4-fold higher level than in F4-6RADR cells. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis could detect no new proteins in F4-6RADR-CsA as compared to F4-6RADR. Interestingly, resistance of F4-6RADR-CsA cells remained reversible for the calcium antagonists verapamil and dihydropyridine B859-35 (dexniguldipine-HCl), indicating that CsA and these compounds interfere with the P glycoprotein function by different pharmacodynamic mechanisms. Transport studies with [14C]ADR, performed in the presence and absence of chemosensitizers, confirmed the good correlation of P-glycoprotein function with the pattern of resistance found in proliferation assays. Cellular accumulation of [3H]cyclosporin was reduced to 71% of that of the F4-6 controls in F4-6RADR-CsA cells, but remained at the level of controls in F4-6RADR cells. Results indicate that increased amounts of the P-glycoprotein--besides other, perhaps more important mechanisms that are as yet unknown--partially mediate CsA resistance in F4-6RADR-CsA cells. We have designated this new form of resistance "secondary combined resistance" (SCR). The results suggest that at least some clinical cases of insensitivity to chemosensitizers or of relapse after reversing therapy could be explained by SCR, and that resensitizing treatment of tumor patients should be based on the consideration of several chemosensitizers of different pharmacodynamics.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

1-Hydroxymethylpyrene and its sulfuric acid ester: toxicological effects in vitro and in vivo, and metabolic aspects.

1-Hydroxymethylpyrene (HMP) is activated to a potent mutagen, detectable in Salmonella typhimurium, in the presence of hepatic cytosol, cofactor for sulfotransferases, and chloride anions. The number of induced mutations is linear to the amount of cytosol used over a wide range, allowing for the quantification of this activity. The activity is expressed with high selectivity in certain tissues and cell types. In adult rats, the highest level is found in the liver, the activity in females exceeding that in males about threefold. About half of the activity in the liver of females is provided by hydroxysteroid sulfotransferase a (STa), whereas other enzymes may be more important in males on account of their very low level of STa. The expression of STa is decreased in ATPase-negative, presumably preneoplastic, hepatic foci in female rats. In contrast to its high mutagenicity in bacteria, SMP shows only weak mutagenic activity in mammalian cells (Chinese hamster V79 cells), independently of whether it is externally added, or generated from HMP within the cells by heterologously expressed STa. Sulfation, however, strongly enhances the cytotoxicity of HMP in mammalian cells. The high cytotoxicity and low mutagenicity in mammalian cells in culture have possible correlates in vivo: while HMP is only a weak initiator of ATPase-negative hepatic foci in newborn rats, it shows substantial promoting activity with regard to such foci in female, but not in male rats. We postulate that this promotion results from selective toxification by STa in the normal hepatic parenchyma of female rats, and resistance of ATPase/STa-negative foci.

Adenosine Triphosphatases↗
PubMed1994

Regiospecific reduction of polycyclic aromatic quinones by rabbit liver dihydrodiol dehydrogenases.

Dihydrodiol dehydrogenase (DDH) isoenzymes were purified from rabbit liver (Klein et al., Eur. J. Biochem., 205 (1992) 1155), and the major forms CF-1, CF-5 and CM-2 were tested for their substrate specificity with dihydrodiol and quinone metabolites of polycyclic aromatic hydrocarbons. CF-5, which was shown to correspond to aldehyde reductase in rabbit liver, was found to efficiently oxidize aromatic dihydrodiol metabolites (phenanthrene-1,2-dihydrodiol, benz[a]anthracene-3,4-dihydrodiol) while CF-1, corresponding to carbonyl reductase, and CM-2 were much less active. All three enzyme forms were found to reduce polycyclic K-region o-quinones of benz[a]anthracene, chrysene and benzo[a]pyrene. CF-1 was the least active, and CM-2 was the most active form with reaction velocities of > 10 mumol/min.mg protein. Among a range of synthetic quinones tested, benz[a]anthracene-8,9-quinone and benzo[a]pyrene 9,10-quinone were also good substrates for the three enzymes, as well as p-benzoquinone and naphthalene-1,4-quinone. The reduction of polycyclic o-quinones, but not of p-benzoquinone, by enzyme CM-2 was accompanied by the oxidation of large amounts of NADPH and the consumption of molecular oxygen which is indicative of a redox-cycling process. Thus, the formation of catechol metabolites from dihydrodiols and o-quinones may be catalyzed by the same enzymes in rabbit liver, and the reaction rate of the enzymatic reduction is strongly dependent on the structural type of the polycyclic quinone.

Alcohol Oxidoreductases↗
PubMed1994

Activation of promutagens by endogenous and heterologous sulfotransferases expressed in continuous cell cultures.

Various environmental chemicals are metabolised to chemically reactive sulfuric acid esters, which may covalently bind to cellular macromolecules and induce mutations and tumours. This activation pathway is usually not taken into account in external xenobiotic-metabolising systems used in short-term tests. We therefore analysed the abilities of cytosols from mammalian cell lines to activate benzylic alcohols (1-hydroxymethylpyrene and 9-hydroxymethylanthracene) to mutagens detectable in Salmonella typhimurium TA98. No activation was observed in cell lines which are commonly used in mutagenicity and cell transformation assays, and only low activities were found in epithelial cell lines in culture. We have therefore constructed Chinese hamster V79-derived cell lines which stably express a heterologous sulfotransferase, rat hydroxysteroid sulfotransferase a. Cytosol of these cells effectively activated 1-hydroxymethylpyrene and 9-hydroxymethylanthracene to mutagens detected in S. typhimurium. The hepatocarcinogen 6-hydroxymethylbenzo[a]pyrene induced gene mutations in sulfotransferase-expressing V79-derived cells, whereas it elicited only marginal effects in sulfotransferase-deficient control cells. The new cell lines may allow the detection of novel classes of mutagens, since some externally generated reactive sulfuric acid esters may not readily penetrate target cells due to their short life span and their ionization.

Animals↗
PubMed1994

Combined influence of quartz dust, ozone and NO2 on chemotactic mobility, release of chemotactic factors and other cytokines by macrophages in vitro.

In this study the single as well as combined effects of quartz, ozone and nitrogen dioxide (NO2) on some immunofunctions of bovine alveolar macrophages (BAM) were investigated. After incubation with 10 micrograms/ml of particles the chemotactic response of BAM is increased nonspecifically, whereas after incubation with 100 micrograms/ml of quartz chemotaxis is specifically decreased. In addition, quartz induces tumor necrosis factor alpha (TNF-alpha) and chemokines to be released dependent on the concentration. Ozone by itself is also a very potent inducer of the release of chemokines and TNF-alpha, but in combination with ozone, quartz has not more than an additive effect. NO2 alone suppresses drastically the release of TNF-alpha. The results show that quartz, ozone and NO2 alter some immunofunctions of BAM and that by combining toxic particles such as quartz with these gases, additive but not synergistic effects might be expected.

Animals↗
PubMed1994

Synthesis and mutagenicity of the diastereomeric fjord-region 11,12-dihydrodiol 13,14-epoxides of dibenzo[a,l]pyrene.

Extensive tumorigenicity studies in rodents revealed that dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogen among all polycyclic aromatic hydrocarbons (PAHs) tested so far. The structure of the genotoxic metabolite(s) responsible for this exceptional carcinogenicity is unknown. The fjord-region syn- and anti-DB[a,l]P-11,12-dihydrodiol 13,14-epoxides (syn- and anti-DB[a,l]PDE) were synthesized to clarify their role as possible ultimate mutagenic and carcinogenic metabolites of DB[a,l]P.9-Formyl-11,12-dimethoxybenzo[g] chrysene was prepared from 9-phenanthrylacetic acid by a photochemical route. After reaction of the aldehyde with trimethylsulfonium iodide to generate an oxiranyl side-chain, treatment with boron trifluoride produced the key intermediate 11,12-dimethoxy-DB[a,l]P in 14% overall yield. From 11,12-dimethoxy-DB[a,l]P the syn- and anti-DB[a,l]PDE were stereoselectively prepared via the trans-11,12-dihydrodiol. The mutagenicity of the syn- and anti-DB[a,l]PDE was examined in four his- strains of Salmonella typhimurium and in Chinese hamster V79 cells. In all five test systems, the new dihydrodiolepoxides were more potent than any of the previously investigated dihydrodiolepoxides. The specific mutagenicity observed with anti-DB[a,l]PDE in strain TA104 exhibited the highest value ever found with any compound in any his- strains of S.typhimurium. The same appears to be true for the activity observed with this compound in V79 cells. In all five systems, syn-DB[a,l]PDE was only moderately less active than its anti-diastereomer (approximately 2-fold). The exceptional mutagenic activities of these dihydrodiolepoxides may be one of the reasons for the exceptional carcinogenic activity of DB[a,l]P.

Animals↗
PubMed1994

Substance-dependent sex differences in the activation of benzylic alcohols to mutagens by hepatic sulfotransferases of the rat.

Six primary and 10 secondary benzylic alcohols derived from polycyclic aromatic hydrocarbons were tested for mutagenicity in Salmonella typhimurium TA98 in the presence of varying amounts of hepatic cytosol from adult male and female rats and 3'-phosphoadenosine-5'-phosphosulfate, the cofactor for sulfotransferases. With the exception of 1-(9-anthryl)ethanol, 4H-cyclopenta[def]-phenanthren-4-ol and 10-hydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene, all the benzylic alcohols were activated to mutagens. For 1-(1-pyrenyl)ethanol (1-HEP), 1-(2-pyrenyl)ethanol (2-HEP), 6-hydroxymethylanthanthrene (6-HMAA), 2-hydroxymethylpyrene (2-HMP), 10H-indeno[1,2,7,7a-bcd]pyren-10-ol (OH-IP), 3-hydroxy-3,4-dihydrocyclopenta[cd]pyrene (3-OH-H2-CPcdP) and 1-(6-benzo[a]pyrenyl)ethanol (6-HEBP), this is the first observation of a mutagenic activity. The primary alcohols 1-hydroxymethylpyrene, 2-HMP, 9-hydroxymethylanthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene and 6-hydroxymethylbenzo[a]pyrene, as well as the secondary alcohols 1-HEP and 3-OH-H2-CPcdP, were more efficiently activated by hepatic cytosol from females than by preparations from males (2.6- to 8-fold). A further compound, 6-HEBP showed significant, but relatively weak, effects in the presence of cytosol from females, whereas it was inactive in the presence of hepatic cytosol from males. The reverse sex difference was observed in the activation of 4H-cyclo-penta[def]chrysen-4-ol, the activity of cytosol from males amounting to about four times that from females. Four other compounds, 2-HEP, 7-hydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene, 6-HMAA and OH-IP, were activated with similar efficiency by hepatic cytosol from both sexes (< two-fold differences). The study indicates that different sulfotransferases are involved in the bioactivation of benzylic alcohols, including forms preferentially expressed in females as well as forms preferentially expressed in males, and that these enzymes qualitatively differ in their substrate tolerance for benzylic alcohols.

Animals↗
PubMed1994

Activation of benzylic alcohols to mutagens by human hepatic sulphotransferases.

Four primary and five secondary benzylic alcohols derived from polycyclic aromatic hydrocarbons were tested for mutagenicity in Salmonella typhimurium TA98 in the presence of 3'-phosphoadenosine-5'-phosphosulphate, the cofactor for sulphotransferases, and varying amounts of hepatic cytosol from three or four different human subjects, a 3-year-old child, an adult female, an adult male and one unknown. All compounds except one, 4H-cyclopenta[def]phenanthren-4-ol, were activated to mutagens. The interindividual variation in the activities was at most 3-fold and the individual activities towards the different substrates were correlated with each other. The same compounds had previously been tested in the presence of hepatic cytosol from rats and all compounds activated in one species were also activated in the other species. However, there were marked quantitative differences, which were further complicated by the observation of a substantial sex difference in the rat. Male and female rat liver cytosol showed higher sulphotransferase activities towards 1-hydroxymethylpyrene, 9-hydroxymethylanthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene and 4H-cyclopenta[def]chrysen-4-ol than human liver cytosol. The largest difference in activity was seen with 7-hydroxymethyl-12-methylbenz[a]anthracene, reaching a factor of approximately 100 between human and female rat. However, with other benzylic alcohols, the activity in human liver cytosol was in the range of that found in the less active sex of rat (3-hydroxy-3,4-dihydrocyclopenta[cd]pyrene, 2-hydroxymethylpyrene) or the more active sex of rat [1-(1-pyrenyl)ethanol].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Stable expression of human cytochrome P450 1A1 cDNA in V79 Chinese hamster cells and metabolic activation of benzo[a]pyrene.

A V79 Chinese hamster cell line stably expressing human cytochrome P450 1A1 (CYP1A1) was obtained by chromosomal integration of the human CYP1A1 cDNA under the control of the SV40 early promoter. Chromosomal integration was verified by Southern analysis, and effective transcription of the human CYP1A1 cDNA was demonstrated by Northern analysis. The CYP1A1 cDNA-encoded protein was characterized by Western analysis using anti-rat CYP1A1. Intracellular association of CYP1A1 with the endoplasmic reticulum could be visualized by in situ immunofluorescence. Crude cell lysates of the V79 derived cell line was able to catalyze 7-ethoxyresorufin-O-deethylation (EROD) with an activity of about 50 pmol min-1 mg-1 total protein, and an aryl hydrocarbon hydroxylase activity (AHH) of 25 pmol min-1 mg-1. CYP1A1 dependent cytotoxicity, measured by neutral red uptake, and genotoxicity, determined by the frequency of micronucleus formation, of benzo[a]pyrene (B[a]P) and trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) could be demonstrated at substrate concentrations as low as 10 nM. Thus, this cell line presents a sensitive tool for studying CYP1A1 mediated metabolism of polycyclic aromatic hydrocarbons (PAH). B[a]P and the purified (+)- and (-)-enantiomers of B[a]P-7,8-diol were compared for their mutagenicity. The (-)-enantiomer was found to be 3-5-fold more mutagenic than the (+)-enantiomer.

Animals↗