Stable heterologous expression of hydroxysteroid sulphotransferase in Chinese hamster V79 cells and their use for toxicological investigations

Chemico-Biological Interactions ELSEVIER

Chemico-Biological Interactions 92 (1994) 119-128

Stable heterologous expression of hydroxysteroid sulphotransferase in Chinese hamster V79 cells and their use for toxicological investigations Andreas Czich *a, Ingrid Bartsch bt, Satish Dogra b*, . b~t, Hansruedi R. Glatt a'° Sabine Ho r nharclt aDepartment of Toxicology, Deutsches Institut J~r Erniihtungsforschung, Arthur-Scheunert-Allee 114116. D-14558 Potsdam-Rehbriieke, Germany bDepartment of Toxicology, University of Mainz, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany

Received 6 August 1993; revision received 3 January 1994; accepted 5 January 1994

Abstract

Various benzylic alcohols are metabolically activated to electrophilic, potentially mutagenic and carcinogenic sulphuric acid esters. The involved sulphotransferases are not expressed in the cell lines in culture which are commonly used for mutagenicity testing. The liver of adult female rats is very efficient in the bioactivation of 1-hydroxymethylpyrene. The major enzyme involved was purified and identified as hydroxysteroid sulphotransferase a. Its cDNA was stably expressed in Chinese hamster V79 cells, which are particularly suited for the quantitative detection of various types of mutations and other genotoxic and cytotoxic effects. The mRNA, protein and enzyme activity levels in the constructed cell lines (V79rSTa-1 and V79rSTa-2) were measured, and the cells were also used in mutagenicity and cytotoxicity investigations with benzylic alcohols. 1-Hydroxymethylpyrene, 9-hydroxymethylanthracene and 6-hydroxymethylbenzo[a]pyrene showed enhanced cytotoxicity in V79rSTa-1 and V79rSTa-2 cells, as compared with sulphotransferase-deficient control cells. In addition, l-hydroxymethylpyrene induced sister chromatid exchanges, and 6-hydroxymethylbenzo[a]pyrene induced * Corresponding author. tPresent address: Department of Biochemistry, University of Adelaide, Sdelaide, South Australia, Australia. ~tPresent address: Institut fiir Toxikologie, GSF Miinchen, lngolst/idter Landstrasse 1, D-85758 Oberschleissheim, Germany. Abbreviations: HMA, 9-hydroxymethylanthracene; HMBP, 6-hydroxymethylbenzo[a]pyrene; HMP, I-hydroxymethylpyrene; SMP, l-hydroxymethylpyrene sulphate (l-sulphooxymethylpyrene); ST, sulphotransferase(s); STa, rat hydroxysteroid sulphotransferase a. 0009-2797/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009-2797(94)03295-J

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gene mutations in V79rSTa-1 cells. We intend carrying out more investigations with other chemicals on these cell lines. Their advantages, as compared with systems with external metabolising systems, include the formation of the active metabolites within the target cell, as in ST-proficient cells in vivo, eliminating the problems which may result from restricted intercellular transport of reactive and ionized sulphuric acid conjugates. Furthermore, cells expressing other sulphotransferases, including human enzymes, may be constructed and used for comparative investigations.

Keywords: Benzylic alcohols; Chinese hamster V79 cells; Heterologous expression; Hydroxysteroid sulphotransferase; Mutagenicity; Polycyclic aromatic hydrocarbons; Sulphotransferase-mediated activation I. Introduction

Numerous toxicological effects are not caused by the administered compound itself, but by metabolites, frequently by chemically reactive intermediates. Many reactive intermediates are, in addition, subject to metabolic detoxification. Xenobiotic-metabolising enzymes therefore are host factors which are critically involved in the toxicological effects of chemicals. In order to study their role in toxification and detoxification processes, heterologous cytochromes P450 (see for example Refs. 1-3), UDP-glucuronosyltransferases [4], microsomal epoxide hydrolase [5] and glutathione transferase 7r (T. Friedberg, B. L611mann and H.R. Glatt, unpublished data) were stably expressed in mammalian cell lines in culture which are suitable for toxicological investigations. We now report on the application of this approach to sulphotransferases (ST). The construction of ST-proficient cell lines which are suitable for toxicological investigations is of special interest for several reasons: (i)

(ii)

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Chemicals belonging to different classes - - including aromatic amine, amide and nitro compounds, primary and secondary alcohols derived from polycyclic aromatic hydrocarbons and alkenylbenzenes, ~-hydroxylated nitrosamines, and secondary nitro alkanes - - may be metabolised to chemically reactive, mutagenic and/or carcinogenic sulphuric acid conjugates (for references, see [61). Cells in continuous culture show only rudimentary xenobiotic metabolism [7]. Many ST activities are low or absent. This is especially true for those ST which are involved in the activation of hydroxymethylarenes [8], the class of compounds investigated in the present study. The use of external metabolising systems, e.g. purified ST, in tests with cultured cells may not always yield conclusive results, since some reactive sulphuric acid esters are short-lived and ionized and may not readily penetrate the target cell membrane, as demonstrated by a comparison of 1-chloromethylpyrene and l-hydroxymethylpyrene sulphate (SMP). Both compounds form similar levels and patterns of adducts when reacted with free DNA [9,10], but only the lipophilic 1-chloromethylpyrene is a potent mutagen in bacteria [1 i,12]. In this particular case, SMP is spontaneously converted to

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l-chloromethylpyrene in the presence of physiological levels of chloride anions, allowing the detection of potent mutagenic effects in bacteria of externally added SMP in the presence of chloride [11]. This mechanism may not function with all sulphuric acid conjugates, indicating the need for ST-proficient target cells. The enhancement of the mutagenicity of SMP in the presence of chloride anions (400-fold) [13] was employed to develop assays which detect ST activity toward benzylic alcohols (e.g. 1-hydroxymethylpyrene (HMP) and 9-hydroxymethylanthracene (HMA)) based on mutagenicity in Salmonella typhimurium TA98 [6]. The mutagenic response was usually linear to the amount of enzyme over a large range (> 100-fold) (as illustrated for example in Fig. 3). Furthermore, the assay is extraordinarily sensitive, since 0.1 #g ofcytosolic hepatic protein from female rats was sufficient to activate HMP measurably. The assay was used for monitoring the fractions in the purification of a HMP ST, and for quantifying heterologously expressed enzyme activity. 2. Purification of a suiphotransferase which activates l-hydroxymethylpyrene

The liver of adult female rats shows about three times higher HMP ST activity than that of males, as determined in bacterial mutagenicity assays [6]. Similar sex differences were reported for the sulphonation of other benzylic alcohols, 5-hydroxymethylchrysene [14], 7-hydroxymethyl-12-methylbenz[a]anthracene [15] and 6hydroxymethylbenzo[a]pyrene (HMBP) [16]. These sex differences suggested that hydroxysteroid ST were involved in the activation. We therefore followed a procedure designed for the purification of hydroxysteroid ST [17], but we measured the activation of HMP and HMA in addition to the ST activity towards dehydroepiandrosterone in the chromatographic fractions. The major proportion of all three activities co-eluted in two peaks from the anion exchange column (Fig. 1, left panel). The first peak was further purified on a hydroxylapatite column, from which about 90% of the three activities co-eluted in a single sharp peak (Fig. 1, right panel). The enrichment factors and yields in this peak, compared with liver homogenate, were similar for the dehydroepiandrosterone ST activity and the activation of HMP (190fold and 9%), and were slightly lower for the activation of HMA. The protein homogeneity of the peak, as determined by SDS gel electrophoresis, was > 90%. The major band (apparent Mr = 29 000) was eluted electrophoretically for N-terminal sequencing and for raising an antibody in rabbits. The antibody recognized a single band, with the electrophoretic behaviour of the purified protein, in liver cytosol of adult female rats. Thirty-two amino acids of the N-terminus could be sequenced and agreed with the 20 N-terminal amino acids of the STa protein [18] and the nucleotide sequence of ST-40 [19], which presumably encodes STa. These findings together with the similarities in the purification procedure and kinetic properties (data not shown) strongly indicated that our purified enzyme corresponds to STa of Ogura and coworkers [18,19]. Our purified ST is therefore termed STa in the subsequent sections of this paper.

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3. Cloning of cDNA and expression in Chinese hamster V79 cells We prepared two 42 meric oligonucleotides which represent the 3'- and 5'-coding sequences of ST-40, and used them for screening a female rat liver c D N A library in ~,gtl 1. We isolated a full-length c D N A containing 998 base pairs. The sequence was identical to that of ST-40 [19], except for two silent mutations at positions 175 (A -- G) and 640 (A - G), and a T -- A transversion at position 503, implying a difference in the encoded amino acid, tyrosine in the clone of Ogura and coworkers and asparagine in our clone. The c D N A was cloned into pMPSV [20], a eukaryotic expression vector carrying the LTR promoter of the myeloproliferative sarcoma virus, as previously described for a c D N A encoding rat microsomal epoxide hydrolase [5]. This construct was co-transfected, in a 20-fold excess, together with pBSpacAp, which encodes the selection marker puromycin acetyl transferase (conferring resistance to puromycin), into Chinese hamster V79 cells, using the techniques which were successful in the transfection of cytochrome P450 cDNAs [1]. Puromycinresistant clones were screened using the antibody raised against STa (Fig. 2, left panel). Two transfectant clones, V79rSTa-I and V79rSTa-2, expressed an antigen with the electrophoretic behaviour of STa purified from rat liver. No signal was detected in parental V79 cells and in a control clone (V79p) into which the puromycin resistance marker only was transfected. The same pattern was found at the R N A level (Fig. 2, right panel).

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Preliminary results of a Southern analysis suggest that both clones have integrated a single copy of the plasmid into their chromosomal DNA. In order to study the expression at the functional level, we used cytosol preparations for the activation of HMP in the bacterial mutagenicity assay (Fig. 3). The transfectant clones V79rSTa-1 and V79rSTa-2 showed about 10% and 20%, respectively, of the activity in hepatic cytosol of adult female rats. These activities are respectable, when taking into account that different enzymes other than STa contribute to the activation in the liver of female rats (by about 50%, as estimated from the activity profiles shown in Fig. 1) and also that the liver of female rats has the highest activity among all the investigated tissues of male and female rats [11]. Cytosol from V79p control cells was fully inactive, with an estimated limit of detection of 0.02% and 0.1% of the activities in liver and V79rSTa-2 cytosol, respectively. Although the expression of HMP ST activity was higher in V79rSTa-2 cells, we preferred V79rSTa-1 cells for subsequent investigations. The reason is that this clone is indistinguishable in its morphology and growth characteristics from parental V79 cells, whereas clone V79rSTa-2 showed reduced growth rates. The expression of STa in V79rSTa-I cells was stable for at least 35 passages (approximately 150 population doublings) (Fig. 3, right panel).

4. Mutagenicity and cytotoxicity of benzylic alcohols in V79-derived cells expressing STa Benzylic alcohols were tested for their mutagenicity in V79 and V79-derived cells, using the acquisition of resistance to 6-thioguanine as a marker of mutations in the hprt locus. HMP and HMA showed only marginal mutagenic effects in V79rSTa-1 cells (data not shown), although the cytosol of these cells activated both compounds

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A. Czich et al. ~Chem.-Biol. Interact. 92 (1994) 119-128

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to potent bacterial mutagens. This finding correlates with the low mutagenicity of the authentic sulphuric acid ester, SMP, and 1-chloromethylpyrene in parental V79 cells [6]. However, these compounds were strongly cytotoxic. This finding is reflected in the enhanced cytotoxicity of HMP and HMA in V79rSTa-1 and V79rSTa-2 cells, as compared with V79 and V79p cells (Fig. 4). Moreover, HMP produced another genotoxic effect, namely the induction of sister chromatid exchanges in V79rSTa-1 cells, an effect not seen in V79 and V79p cells [6]. Another benzylic alcohol, the hepatocarcinogen HMBP [16], showed not only enhanced cytotoxicity (Fig. 4), but also pronounced mutagenicity (Fig. 5) in V79rSTa-1 cells. 5. Conclusions

We stably expressed rat STa in Chinese hamster V79 cells. This cell line was used for the transfection, since (i) it does not appear to express any endogenous ST enzymes and thus the transfectants are exactly defined according to their ST form; (ii) it does not appear to express any cytochromes P450, enzymes which may catalyse alternative activation pathways; (iii) V79-derived cell lines are available which express defined heterologous cytochromes P450 [1,7], and may be used as recipient cells for ST, allowing the reconstruction of complex activation pathways; (iv) V79 cells allow the detection of many toxicological endpoints, including cytotoxicity, gene mutations, chromosomal aberrations, polyploidies and sister chromatid exchanges. We have shown that benzylic alcohols may induce various genotoxic and cytotoxic effects in ST-expressing transfectant lines. Interestingly, the three investigated benzylic alcohols exhibited different patterns of activities. Only the hepatocarcinogen

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H M B P showed p r o n o u n c e d mutagenicity, whereas the predominant toxicological effect of H M P in ST-proficient mammalian cells was its cytotoxicity. Relatively little is known about the carcinogenicity o f this compound. Investigations in an in vivo short-term test, with ATPase-negative, putatively preneoplastic lesions as the endpoint, indicated that H M P is a p r o m o t o r rather than an initiator [6,21]. Initiation is usually associated with mutations, whereas promotion appears to involve other mechanisms such as selection and cytotoxicity. Thus, the observed patterns of activities of H M P and H M B P in ST-expressing cells matches the available data on their carcinogenic properties. N o results appear to be available on the carcinogenicity o f H M A . In ST-proficient cells, it was less mutagenic than HMBP, and less cytotoxic than H M P (Fig. 4). Due to its low cytotoxicity, its mutagenic activity could be unambiguously demonstrated, despite its weakness (data not shown). Investigations will continue, with other chemicals, on these cell lines. Their advantages, as compared with systems with external metabolising systems, include the formation o f the active metabolites within the target cell, as is the case in ST-proficient cells in vivo, eliminating the problems which may result from restricted intercellular transport of reactive and ionized sulphuric acid conjugates. Furthermore, cells expressing other ST, including h u m a n enzymes, may be constructed and used for comparative investigations.

6. Acknowledgements We thank Ms Karin Pauly for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 302) and the Commission o f the European C o m m u n i t y (Contract No. BMH1-CT92-0097).

7. References 1 J. Doehmer, C. W61fel,S. Dogra, C. Doehmer, A. Seidel, K.L. Platt, F. Oesch and H.R. Glan, Applications of stable V79-derived cell lines expressing rat cytochromes P4501AI, IA2, and 2B1, Xenobiotica, 22 (1992) 1093-1099. 2 R. Langenbach, P.B. Smith and C. Crespi, Recombinant DNA approaches for the development of metabolic systems used in in vitro toxicology, Mutat. Res., 277 (1992) 251-275. 3 L.H. Thompson, R.W. Wu and J.S. Felton, Introduction of cytochrome-P4501A2 metabolic capability into cell lines genetically matched for DNA repair proficiency/deficiency,Proc. Natl. Acad. Sci. USA, 88 (1991) 3827-3831. 4 T. Ebner and B. Burchell, Substrate specificities of 2 stably expressed human liver UDPglucuronosyltransferases of the UGTI gene family, Drug Metab. Dispos., 21 (1993) 50-55. 5 H.R. Glatt, R. Becker, A. Pi6e, F. Oesch and T. Friedberg, Stable expression of heterologous epoxide hydrolase in BHK21 cells: influence on the mutagenicity of benzo[a]pyrene-4,5-oxide, in: N. Seemayer and W. Hadnagy (Eds.), Environmental hygiene II1, Springer-Verlag, Heidelberg, 1992, pp. 67-70. 6 H.R. Glatt, G. Werle-Schneider, N. Enders, S. Monnerjahn, J. Pudil, A. Czich, A. Seidel and M. Schwarz, 1-Hydroxymethylpyrene and its sulfuric acid ester: toxicological effects in vitro and in vivo, and metabolic aspects, Chem.-Biol. Interact. 92 (1994) 305-319. 7 H.R. Glan, I. Gemperlein, G. Turchi, H. Heinritz, J. Doehmer and F. Oesch, Search for cell culture systems with diverse xenobiotic-metabolising activities and their use in toxicological studies, Mol. Toxicol., I (1987) 313-333.

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H.R. Glatt, S. Hornhardt, K. Pauly, A. Pi6e-Staffa, A. Seidel and A. Czich, Activation of promutagens by endogenous and heterologous sulfotransferases expressed in continuous cell cultures, Toxicol. Lett. (in press). S. Monnerjahn, A. Seidel, P. Steinberg, F. Oesch, M. Hinz, J.J. Stezowsky, A. Hewer, D.H. Phillips and H.R. Glatt, Formation of DNA adducts from 1-hydroxymethylpyrene in liver cells in vivo and in vitro, in: D.H. Phillips, M. Castegnaro and H. Bartsch (Eds.), Postlabelling methods for detection of DNA adducts, International Agency for Research on Cancer, Lyon, 1993, pp. 189-193. Y.-J. Surh, J.C. Blomquist, A. Liem and J.A. Miller, Metabolic activation of 9-hydroxymethyl-10methylanthracene and l-hydroxymethylpyrene to electrophilic, mutagenic and tumorigenic sulfuric acid esters by rat hepatic sulfotransferase activity, Carcinogenesis, 11 (1990) 1451-1460. H.R. Glatt, R. Henschler, D.H. Phillips, J.W. Blake, P. Steinberg, A. Seidel and F. Oesch, Sulfotransferase-mediated chlorination of l-hydroxymethylpyrene to a mutagen capable of penetrating indicator cells, Environ. Health Perspect., 88 (1990) 43-48. H.R. Glatt, R. Henschler, H. Frank, A. Seidel, C. Yang, E. Abu-Shqara and R.G. Harvey, Sulfotransferase-mediated mutagenicity of l-hydroxymethylpyrene and 4H-cyclopenta[deJ]chrysen-4-ol and its enhancement by chloride anions, Carcinogenesis, 14 (1993) 599-602. N. Enders, A. Seidel, S. Monnerjahn and H.R. Glatt, Synthesis of 11 benzylic sulfate esters, their bacterial mutagenicity and its modulation by chloride, bromide and acetate anions, Polycyclic Aromatic Comp., 3 (1993) Suppl. 887-894. H. Okuda, H. Nojima, N. Watanabe and T. Watabe, Sulphotransferase-mediated activation of the carcinogen 5-hydroxymethylchrysene: species and sex differences in tissue distribution of the enzyme activity and a possible participation of hydroxysteroid sulphotransferases, Biochem. Pharmacol., 38 (1989) 3003-3009. Y.-J. Surh, A. Liem, E.C. Miller and J.A. Miller, Age- and sex-related differences in activation of the carcinogen 7-hydroxymethyl-12-methylbenz[a]anthracene to an electrophilic sulfuric acid ester metabolite in rats: possible involvement of hydroxysteroid sulfotransferase activity, Biochem. Pharmacol., 41 (1991) 213-221. Y.-J. Surh, A. Liem, E.C. Miller and J.A. Miller, Metabolic activation of the carcinogen 6hydroxymethylbenzo[a]pyrene: formation of an electrophilic sulfuric acid ester and benzylic DNA adducts in rat liver in vivo and in reactions in vitro, Carcinogenesis, 10 (1989) 1519-1528. C.J. Marcus, R.D. Sekura, E.S. Lyon and W.B. Jakoby, A hydroxysteroid sulfotransferase from rat liver, Anal. Biochem., 107 (1980) 296-304. K. Ogura, T. Sohtome, A. Sugiyama, H. Okuda, A. Hiratsuka and T. Watabe, Rat liver cytosolic hydroxysteroid sulfotransferase (sulfotransferase a) catalyzing the formation of reactive sulfate esters from carcinogenic polycyclic hydroxymethylarenes, Mol. Pharmacol., 37 (1990) 848-854. K. Ogura, J. Kajita, H. Narihata, T. Watabe, S. Ozawa, K. Nagata, Y. Yamazoe and R. Kato, cDNA cloning of the hydroxysteroid sulfotransferase STa sharing a strong homology in amino acid sequence with the senescence marker protein SMP-2 in rat livers, Biochem. Biophys. Res. Commun., 166 (1990) 1494-1500. P. Artelt, C. Morelle, M. Ausmeier, M. Fitzek and H. Hauser, Vectors for efficient expression in mammalian fibroblastoid, myeloid and lymphoid cells via transfection or infection, Gene, 68 (1988) 213-219. G. Werle-Schneider, M. Schwarz and H.R. Glatt, Development of hydroxysteroid sulfotransferasedeficient lesions during hepatocarcinogenesis in rats, Carcinogenesis, 14 (1993) 2267-2270. S.S. Singer and S. Sylvester, Enzymatic sulfation of steroids. 11: the control of hepatic cortisol sulfotransferase activity and of the individual hepatic steroid sulfotransferase by gonads and gonadal hormones, Endocrinology, 99 (1976) 1346-1352.