Andreas Czich

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

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, 1-hydroxymethylpyrene induced sister chromatid exchanges, and 6-hydroxymethylbenzo[a]pyrene induced 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.

Activation of benzylic alcohols to mutagens by rat and human sulfotransferases expressed in Escherichia coli

Human hydroxysteroid sulfotransferase, human phenol-sulfating form of phenol sulfotransferase, rat hydroxysteroid sulfotransferase a and rat phenol sulfotransferase IV were expressed in Escherichia coli. Cytosol preparations of transformed bacteria were used as activating systems in mutagenicity tests with Salmonella typhimurium TA98. All test compounds, 1-hydroxymethylpyrene, 2-hydroxymethylpyrene, 1-(1-pyrenyl)ethanol, 9-hydroxymethylanthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene and 4H-cyclopenta[def]chrysen-4-ol, were activated by both hydroxysteroid sulfotransferases investigated. However, 1-(1-pyrenyl)ethanol was 67-fold more efficiently activated by the human enzyme, whereas 7-hydroxymethyl-12-methylbenz[a]anthracene was 27-fold more efficiently activated by the rat enzyme. The phenol sulfotransferases showed relatively low activities with the benzylic alcohols investigated. The only exception was 4H-cyclopenta[def]chrysen-4-ol, which was activated efficiently by rat phenol sulfotransferase IV. We had previously tested the ability of rat and human hepatic cytosol preparations to activate the same compounds. The results of a statistical analysis suggest that the activities of human hydroxysteroid sulfotransferase, rat hydroxysteroid sulfotransferase a and phenol sulfotransferase IV can account for a substantial portion of the activation of benzylic alcohols in human, female rat and male rat liver, respectively.

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.

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.

Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats

Sixteen training compounds selected in the IMI MIP-DILI consortium, 12 drug-induced liver injury (DILI) positive compounds and 4 non-DILI compounds, were assessed in cryopreserved primary human hepatocytes. When a ten-fold safety margin threshold was applied, the non-DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes (n=13 donors) in suspension and 14-days following repeat dose exposure (3 treatments) to an established 3D-microtissue co-culture (3D-MT co-culture, n=1 donor) consisting of human hepatocytes co-cultured with non-parenchymal cells (NPC). In contrast, only 5/12 DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes in suspension. Exposure of the 2D-sandwich culture human hepatocyte monocultures (2D-sw) for 3days resulted in the correct identification of 11/12 DILI-positive compounds, whereas exposure of the human 3D-MT co-cultures for 14days resulted in identification of 9/12 DILI-compounds; in addition to ximelagatran (also not identified by 2D-sw monocultures, Sison-Young et al., 2016), the 3D-MT co-cultures failed to detect amiodarone and bosentan. The sensitivity of the 2D human hepatocytes co-cultured with NPC to ximelagatran was increased in the presence of lipopolysaccharide (LPS), but only at high concentrations, therefore preventing its classification as a DILI positive compound. In conclusion (1) despite suspension human hepatocytes having the greatest metabolic capacity in the short term, they are the least predictive of clinical DILI across the MIP-DILI test compounds, (2) longer exposure periods than 72h of human hepatocytes do not allow to increase DILI-prediction rate, (3) co-cultures of human hepatocytes with NPC, in the presence of LPS during the 72h exposure period allow the assessment of innate immune system involvement of a given drug.

Predictive in silico off-target profiling in drug discovery

Drug action can be rationalized as interaction of a molecule with proteins in a regulatory network of targets from a specific biological system. Both drug and side effects are often governed by interaction of the drug molecule with many, often unrelated, targets. Accordingly, arrays of protein-ligand interaction data from numerous in vitro profiling assays today provide growing evidence of polypharmacological drug interactions, even for marketed drugs. In vitro off-target profiling has therefore become an important tool in early drug discovery to learn about potential off-target liabilities, which are sometimes beneficial, but more often safety relevant. The rapidly developing field of in silico profiling approaches is complementing in vitro profiling. These approaches capitalize from large amounts of biochemical data from multiple sources to be exploited for optimizing undesirable side effects in pharmaceutical research. Therefore, current in silico profiling models are nowadays perceived as valuable tools in drug discovery, and promise a platform to support optimally informed decisions.

Species-specific developmental toxicity in rats and rabbits: Generation of a reference compound list for development of alternative testing approaches

For regulatory information requirements, developmental toxicity testing is often conducted in two mammalian species. In order to provide a set of reference compounds that could be used to explore alternative approaches to supersede testing in a second species, a retrospective data analysis was conducted. The aim was to identify compounds for which species sensitivity differences between rats and rabbits are not caused by maternal toxicity or toxicokinetic differences. A total of 330 compounds were analysed and classified according to their species-specific differences. A lack of concordance between rat and rabbit was observed in 24% of the compounds, of which 10% were found to be selective developmental toxicants in one of the species. In contrast to previously published analyses the presented comparison is based entirely on publically data allowing validating and comparing alternative approaches for developmental toxicity testing. Furthermore, this list could be useful to identify mechanisms leading to species differences.

Stable Expression of Heterologous Sulfotransferase in V79 Cells: Activation of Primary and Secondary Benzylic Alcohols

Abstract A sulfotransferase (ST) capable of activating 1-hydroxymethylpyrene (HMP) and 9-hydroxymethylanthracene (HMA) to mutagens was purified from rat liver. This enzyme appeared to be identical with hydroxysteroid STa, whose cDNA was cloned and stably expressed in Chinese hamster V79 cells. Several primary and secondary benzylic alcohols derived from polycyclic aromatic hydrocarbons induced gene mutations, sister chromatid exchanges (SCE) and/or cytotoxicity in these cells.