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Protocol no. 61
AN IN VITRO MODEL FOR STUDIES OF PROSTAGLANDIN H SYNTHASE (PHS) - MEDIATED GENOTOXICITY OF XENOBIOTICS

This protocol describes the use of SEMV cells (a cell line derived from ram seminal vesicles) in studies into prostaglandin H synthase-mediated metabolism of xenobiotics in intact cells.

CONTACT

Dr Gisela H. Degen
Institut für Arbeitsphysiologie an der Universitat Dortmund
Ardeystr. 67 W-Dortmund 1 Germany
Tel: Germany - 231 1084 351 or Germany 231 1084 342
Fax: Germany - 231 1084 308

RATIONALE

The monooxygenases are present at the highest levels in hepatic cells, and are responsible for the bioactivation of numerous xenobiotics to harmful metabolic products. However, extrahepatic cells that have low or undetectable monooxygenase activity are also capable of metabolising xenobiotics; in many such cells this activity is due to prostaglandin H synthase (PHS). In particular, PHS is suggested to be responsible for the metabolic activation of stilbene oestrogens and steroid oestrogens to their genotoxic metabolites. PHS activity is found in many mammalian tissues. Ram and bull seminal vesicles, in particular, contain high concentrations of PHS. A cell line derived from ram seminal vesicles (SEMV cells) has been produced to permit studies on the bioactivation of various carcinogens by PHS in the absence of monooxygenase activity. SEMV cells have no detectable monooxygenase activity, a characteristic making them suitable for studying the metabolic profiles of compounds metabolised specifically by PHS. Recent studies have also shown that SEMV cells produce micronuclei as a result of treatment with diethylstilbestrol (DES), a synthetic oestrogen, (Foth et al, 1992). Micronuclei formation may prove to be a suitable endpoint to the genotoxic assay using SEMV cells, but is still at the developmental stage.

BASIC PROCEDURE

S Xenobiotic metabolism studies SEMV cells are plated out and cultured for 20 hours. The labelled test compound is then added and the incubation is terminated after 4, 12 or 24 hours. Initially, the distribution of radioactivity between the cellular fraction and medium is determined. HPLC analysis of metabolites A working up procedure is then followed to prepare the samples for HPLC analysis. The cells and media are removed from the dishes and the cells lysed. Absolute ethanol is used to precipitate protein from the medium, cellular fraction, and the wash liquid resulting from the cell lysis procedure. Parent compound and metabolites are analyzed by reverse phase chromatography with on-line detection of radioactivity in the supernatant. In addition, protein-bound (radioactive) material can be determined by liquid scintillation counting in the precipitated protein fraction dissolved in 1M NaOH (1 hour, at 70øC) or after combustion in a sample oxidizer. Micronucleus genotoxicity assay SEMV cells are plated out onto slides and exposed to test compounds after overnight incubation. After 5 hours, the exposure is terminated and the cells are incubated in fresh medium for up to 24 hours post-treatment. At intervals during this period, the cells are fixed in methanol, incubated for 30 minutes in S”rensen buffer and stained with propidium iodide. The slides are then scored for micronucleus formation by fluorescence microscopy. Cytotoxicity assays Cytotoxic effects will affect the results of the genotoxicity assay, therefore it is necessary to determine the concentrations at which cytotoxicity will become significant. Cytotoxicity is assessed by the Neutral Red (NR) uptake method, and total cell protein is determined by the Kenacid blue method on the same plates, subsequent to the NR assay.

CRITICAL ASSESSMENT

Degen et al (1991) have used diethylstilbestrol (DES), a synthetic oestrogen known to be a human carcinogen, to assess the suitability of using SEMV cells in PHS-mediated carcinogenicity assays. PHS has both cyclooxygenase and peroxidase activity; it catalyses the bisoxygenation of arachidonic acid (ARA) to the endoperoxide hydroperoxide PGG2, and the reduction of PGG2 to the corresponding alcohol PGH2. PHS and/or peroxyl radicals, generated from ARA during this reaction, oxidise various cosubstrates (xenobiotics). DES is oxidised to Z,Z-dienestrol (Z,Z-DIES) in this way. To assess the PHS activity of SEMV cells DES-treated SEMV cells were incubated with factors such as ARA and indomethacin (an inhibitor), known to increase or decrease PHS activity, respectively, (Degen & Foth, 1991; 1992; Foth & Degen, 1992). As expected, an increased amount of available ARA increased Z,Z-DIES formation. However, the inhibitor, indomethacin, did not completely inhibit DES oxidation. This is because indomethacin inhibits the cyclooxygenase, but not the peroxidase, activity of PHS and, therefore, prevents the PGG2-dependent cooxidation of xenobiotic cosubstrates such as DES. PHS peroxidase can also utilise other hydroperoxides, which can be generated by lipoxygenases or by lipid peroxidation. Therefore, in intact cells, or in vivo, the inhibitor approach can underestimate the overall contribution of hydroperoxide-dependent oxidation of a given compound. This is particularly significant as the role of PHS in carcinogen activation is currently usually assessed using inhibitors. Other studies are based on measuring the differences in monooxygenase-catalysed and PHS catalysed product pattern of a few suitable substrates. The SEMV cell culture system has the advantage over these other methods due to its lack of detectable monooxygenase activity, which makes it possible to study PHS-catalysed co-oxidation of xenobiotics in the absence of monooxygenase-catalysed metabolic activation. An advantage of the use of SEMV cells over other cell lines for studying PHS metabolising properties, is that prostaglandin production by SEMV cells is higher than in cells such as Syrian hamster embryo (SHE) cells studied under the same conditions. This has been shown to be due to greater PHS activity in SEMV cells, rather than to an increased availability of arachidonic acid. For example, Western immunoblots have shown that the SEMV cells have higher levels of PHS protein. Also, when SEMV and SHE cells are incubated under the same conditions with DES, the SEMV cells produce greater quantities of Z,Z-DIES (Foth, J. 1992). ARA conversion to prostaglandins is comparable in early and late passages of SEMV cells, suggesting that PHS expression is not lost during culture (Foth et al, 1992). Another promising feature is that DES can induce micronuclei in SEMV cells. This indicates a potential endpoint for determining the genotoxicity of test compounds in the assay. In preliminary investigations by Foth et al, (1992), the background of spontaneous micronucleated cells (MNC) was found to be lower in SEMV cell cultures than in SHE cell cultures. This suggests that the SEMV cells represent a more sensitive system in which to measure MNC as an endpoint of genotoxicity. Another feature that makes SEMV cells a sensitive genotoxicity testing system is the fact that the DES concentration found to induce the highest frequency of MNC is 3-5 fold lower (10mM) in SEMV cells than in SHE cells (30-50 mM). This greater sensitivity of SEMV cells means that it is possible to use lower concentrations of test compound in order to measure a genotoxic effect and therefore avoid confounding cytotoxic effects. A DES concentration of 10mM only marginally affected Neutral Red, and cellular protein content in SEMV cells exposed for 45 hours; and with shorter treatments these parameters were affected only at the higher DES concentrations tested (Foth et al, 1992). Cytotoxicity became apparent at 100mM DES, and the reduced MNC observed at the highest DES conc. was believed to be due to this cytotoxic effect. It should be borne in mind that results have suggested that doses that are genotoxic are not necessarily the same as those that are cytotoxic. It is therefore important that a cytotoxicity test be performed in conjunction with the genotoxicity assay in order to make a reliable interpretation of the level of MNC induced. It can therefore be concluded from the above mentioned features that SEMV cells show potential as a good model for further investigations of the role of PHS in mediating genotoxicity of DES and other xenobiotics.

TEST STATUS

In-house development.

CHEMICALS TESTED

Diethylstilbestrol (DES)
Paracetamol (acetaminophen)

REFERENCES

  1. Arlett, C.F., Ashby, J., Fielder, P.J. & Scott, D. (1989) Micronuclei: origins, applications and methodologies. Mutagenesis, 4, 482-485.
  2. Borenfreund, E., Babich, H. & Martin-Alguacil, N. (1988) Comparisons of two in vitro cytotoxicity assays-the neutral red (NR) and tetrazolium MTT tests. Toxic. in Vitro, 2, 1-6
  3. Degen, H. & Foth, J. (1991) Peroxidative metabolism of diethylstilbestrol in ram seminal vesicle cell cultures, an in vitro model for studies of oestrogen-induced genotoxicity. Proceedings of the First International Symposium on Hormonal Carcinogenesis, 19-23
  4. DeGrassi, F & Tanzarella, C. (1988) Immunofluorescent staining of kinetochores in micronuclei: a new assay for aneuploidy. Mutation Research, 203, 339-345.
  5. Eastmond, D.A. & Tucker, J.D. (1989) Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocytes and an antikinetochore antibody. Environmental and Molecular Mutagenesis, 13, 34-43.
  6. Foth, J. & Degen, G.H. (1991) Prostaglandin H synthase dependent metabolism of diethylstilbestrol by ram seminal vesical cell cultures. Arch. Toxicol., 65, 344-347
  7. Foth, J. (1992) Untersuchungen zur Kooxidation von Fremdstoffen in einer Prostaglandin-H-synthase kompetenten Zellinie. Phd Thesis, University of Würzburg Foth, J., Schnitzler, M., Koob, M. & Degen, G.H. (1992) Characterisation of sheep seminal vesicle cells-a new tool for studying genotoxic effects in vitro Toxic in Vitro, 6(3), 219-225
  8. Freyberger, A., Schnitzler, R., Schiffmann, D. & Degen, G.H. (1987) Prostaglandin-H-synthase competent cells derived from ram seminal vesicles: A tool for studying cooxidation of xenobiotics. Molecular Toxicology, 1, 503-512
  9. Freyberger, A. & Degen, G.H. (1989) Covalent binding to proteins of reactive intermediates resulting from prostaglandin H synthase-catalyzed oxidation of stilbene and steroid estrogens. J.Biochem Toxicology, 4(2), 95-103.
  10. Riddell, R.J., Clothier, R.H. & Balls, M. (1986) An evaluation of three in vitro cytotoxicity assays. Food and Chemical Toxicology, 24, 469-471.

IP-61 © October 1992