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.

Salmonella strains and mammalian cells genetically engineered for expression of sulfotransferases.

Rat and human sulfotransferases (STs) were expressed in his- S. typhimurium strains. These new bacterial strains detected various mutagens which are difficult to recognize in traditional test systems, including benzylic alcohols derived from polycyclic aromatic hydrocarbons, hycanthone and 1'-hydroxysafrole. STs were also stably expressed in V79 Chinese hamster cells, which do not express endogenous ST and are suitable for the detection of genotoxic effects. Positive responses in these test systems were observed with various benzylic alcohols, including benzo[a]pyrene-7,8,9,10-tetrols. We demonstrate that a few reactive sulfuric acid conjugates are efficiently detected as genotoxicants only when generated directly within the indicator cell.

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.

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.

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.

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.