In vitro - in vivo correlation of gene expression alterations induced by liver carcinogens.

Although cultivated hepatocytes are widely used in the studies of drug metabolism, their application in toxicogenomics is considered as problematic, because previous studies have reported only little overlap between chemically induced gene expression alterations in liver in vivo and in cultivated hepatocytes. Here, we identified 22 genes that were altered in livers of rats after oral administration of the liver carcinogens aflatoxin B1 (AB1), 2-nitrofluorene (2-NF), methapyrilene (MP) or piperonyl-butoxide (PBO). The functions of the 22 genes have been classified into two groups. Genes related to stress response, DNA repair or metabolism and genes associated with cell proliferation, respectively. Next, rat hepatocyte sandwich cultures were exposed to AB1, 2-NF, MP or PBO for 24h and expression of the above mentioned genes was determined by RT-qPCR. Significant correlations between the degree of gene expression alterations in vivo and in vitro were obtained for the stress, DNA repair and metabolism associated genes at concentrations covering a range from cytotoxic concentrations to non-toxic/in vivo relevant concentrations. In contrast to the stress associated genes, no significant in vivo/in vitro correlation was obtained for the genes associated with cell proliferation. To understand the reason of this discrepancy, we compared replacement proliferation in vivo and in vitro. While hepatocytes in vivo, killed after administration of hepatotoxic compounds, are rapidly replaced by proliferating surviving cells, in vitro no replacement proliferation as evidenced by BrdU incorporation was observed after washing out hepatotoxic concentrations of MP. In conclusion, there is a good correlation between gene expression alterations induced by liver carcinogens in vivo and in cultivated hepatocytes. However, it should be considered that cultivated primary hepatocytes do not show replacement proliferation explaining the in vivo/in vitro discrepancy concerning proliferation associated genes.

Toxicogenomic analysis of N-nitrosomorpholine induced changes in rat liver: comparison of genomic and proteomic responses and anchoring to histopathological parameters.

A common animal model of chemical hepatocarcinogenesis was used to examine the utility of transcriptomic and proteomic data to identify early biomarkers related to chemically induced carcinogenesis. N-nitrosomorpholine, a frequently used genotoxic model carcinogen, was applied via drinking water at 120 mg/L to male Wistar rats for 7 weeks followed by an exposure-free period of 43 weeks. Seven specimens of each treatment group (untreated control and 120 mg/L N-nitrosomorpholine in drinking water) were sacrificed at nine time points during and after N-nitrosomorpholine treatment. Individual samples from the liver were prepared for histological and toxicogenomic analyses. For histological detection of preneoplastic and neoplastic tissue areas, sections were stained using antibodies against the placental form of glutathione-S-transferase (GST-P). Gene and protein expression profiles of liver tissue homogenates were analyzed using RG-U34A Affymetrix rat gene chips and two-dimensional gel electrophoresis-based proteomics, respectively. In order to compare results obtained by histopathology, transcriptomics and proteomics, GST-P-stained liver sections were evaluated morphometrically, which revealed a parallel time course of the area fraction of preneoplastic lesions and gene plus protein expression patterns. On the transcriptional level, an increase of hepatic GST-P expression was detectable as early as 3 weeks after study onset. Comparing deregulated genes and proteins, eight species were identified which showed a corresponding expression profile on both expression levels. Functional analysis suggests that these genes and corresponding proteins may be useful as biomarkers of early hepatocarcinogenesis.

Primary rat hepatocytes as in vitro system for gene expression studies: comparison of sandwich, Matrigel and 2D cultures.

Recent studies have presented evidence that in vivo obtained gene expression data can be used for carcinogen classification, for instance to differentiate between genotoxic and non-genotoxic carcinogens. However, although primary rat hepatocytes represent a well-established in vitro system for drug metabolism and enzyme induction, they have not yet been systematically optimized for toxicogenomic studies. The latter may be confounded by the fact that cultured hepatocytes show strong spontaneous alterations in gene expression patterns. Therefore, we addressed the following questions: (1) which culture system is optimal, comparing sandwich, Matrigel and 2D cultures, (2) how critical is the impact of culture period on substance-induced alterations in gene expression and (3) do these substance-induced alterations in cultured hepatocytes occur already at in vivo relevant concentrations? For this purpose we analyzed the expression of four genes, namely Abat, Gsk3beta, Myd116 and Sult1a1 that recently have been reported to be influenced by the antihistamine and non-genotoxic carcinogen methapyrilene (MPy). The most reproducible effects of MPy were observed in sandwich cultures. Induction factors of Gsk3beta and Myd116 at 100 microM MPy were 2 and 4 (medians), respectively, whereas expression of Abat and Sult1a1 were inhibited by factors of 7 and 5, respectively. Similar results were observed in hepatocytes maintained for 24 h or 3 weeks in sandwich culture with respect to the influence of MPy on the expression of Abat, Gsk3beta, Myd116 and Sult1a1. To determine whether MPy influences gene expression at in vivo relevant concentrations, 3.5 mg/kg MPy were administered to male Wistar rats intraperitoneally, resulting in plasma concentrations ranging between 1.72 and 0.32 microM 5 and 80 min after injection. Inhibition of Abat and Sult1a1 expression in vitro already occurred at in vivo relevant concentrations of 0.39 microM MPy. Induction of Myd116 was observed at 6.25 microM which is higher but in the same order of magnitude as in vivo relevant concentrations. In conclusion, the presented data strongly suggest that sandwich cultures are most adequate for detection of MPy-induced gene expression alterations and the effect of MPy was detected at in vivo relevant concentrations.

Toxicity of waters from two streams to early life stages of brown trout (Salmo trutta f. fario L.), tested under semi-field conditions.

The development of brown trout (Salmo trutta f. fario L.) in water of two differently polluted streams and in a control situation was monitored in order to get insights into the impact of anthropogenic chemical stressors on the reproductive success of this fish species indigenous to both streams. The test streams, situated in the south of Stuttgart, Germany, were the complexly polluted Körsch stream and the less polluted Krähenbach stream. Bypass systems connected to the streams and a laboratory control system were used for continuous exposure of early brown trout stages shortly after fertilisation up to the end of the embryonic development. Temperature and oxygen conditions were standardised in all test series in order to minimise unspecific effects. The examined endpoints were: (1) mortality, (2) developmental rate, (3) time course of hatching, (4) malformations, and (5) growth. A retarded development, reduced growth rates and higher mortality rates of Körsch stream water exposed embryos indicated an embryotoxic potential for the more polluted stream. High infection-related mortality rates of embryos suggested the presence of confounding factors also in the less polluted Krähenbach stream. In parallel to the exposure experiment, physicochemical and limnochemical parameters as well as concentrations of organic contaminants and heavy metals were monitored. Analytical data confirm the different degrees of pollution of both streams.

The use of toxicodynamics in risk assessment.

Risk assessment of xenobiotics is a qualitative and quantitative assessment of toxic properties conventionally based on data resulting from tests in animals exposed to the substance. The assessment of dose-effect relationship includes evaluation of exposure at the site of action. More recently, emphasis is put on understanding the relationship between exposure at the site of action and the resulting effect, i.e. toxicodynamic. In this respect, results from genotoxicity studies may be a measure for exposure and at the same time of an effect. Results of toxicodynamic endpoints such as binding to receptors or release of hormones have been used when replacing default values for interspecies extrapolation. It may also be envisaged to use toxicodynamic endpoints in order to get an estimate of intraspecies variability. It was demonstrated that this approach may be helpful only if the relationship between the toxicodynamic endpoint and the definite endpoint is known by using the example of bisphenol A. Whereas there are clear effects of bisphenol A in in vitro and ex vivo studies, the classical two generation study has not been able to detect an effect on reproduction and/or fertility. Looking in the future development of toxicodynamic endpoints, gene profiling and the analysis of proteins ('proteomics') may be helpful tools employed in screening and being related to the mode of action are explored for their suitability in terms of toxicodynamic endpoints.

Identification of an enzymatically formed glutathione conjugate of the cyanobacterial hepatotoxin microcystin-LR: the first step of detoxication.

Cyanobacterial toxins have adverse effects on mammals, birds and fish and are being increasingly recognised as a potent stress factor and health hazard factor in aquatic ecosystems. Microcystins, cyclic heptapeptides and a main group of the cyanotoxins are mainly retained within the producer cells during cyanobacterial bloom development. However, these toxins are released into the surrounding medium by senescence and lysis of the blooms. Any toxin present could then come into contact with a wide range of aquatic organisms including phytoplankton grazers, invertebrates, fish and aquatic plants. Recent studies showed the conversion of microcystin in animal liver to a more polar compound in correlation with a depletion of the glutathione pool of the cell. The present study shows the existence of a microcystin-LR glutathione conjugate formed enzymatically via soluble glutathione S-transferase in various aquatic organisms ranging from plants (Ceratophyllum demersum), invertebrates (Dreissena polymorpha, Daphnia magna) up to fish eggs and fish (Danio rerio). The main derived conjugate was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry yielding a mass of m/z 1302, which is equivalent to the mass assumed for a glutathione microcystin-LR conjugate. This conjugate appears to be the first step in the detoxication of a cyanobacterial toxin in aquatic organisms.

Mueggelone, a novel inhibitor of fish development from the fresh water cyanobacterium Aphanizomenon flos-aquae.

A novel C18 lipid, containing a 10-membered lactone, mueggelone (1), was isolated from a field-collected sample of Aphanizomenon flos-aquae, together with the known compound lupenyl acetate (3). Both structures were secured using extensive spectroscopic analysis (1D and 2D NMR, MS, IR). Biological activity assessment of both compounds indicated them to have significant inhibitory effects on fish embryo larval development.

Valproic acid induced abnormal development of the central nervous system of three species of amphibians: implications for neural tube defects and alternative experimental systems.

Embryos of Ambystoma mexicanum, Xenopus laevis, and Hyperolius viridiflavus taeniatus were exposed to various concentrations of valproic acid (VPA: 0.1, 1.5, 10 mM) from blastula stage (S) 9 on up to advanced gastrulation of control embryos (S 11 1/2-12). At 10 and 5 mM VPA early development was affected in all species tested. However, the most pronounced effects occurred in Ambystoma: the neural folds appeared delayed and showed a flattened and wavy shape; the neural tube was not formed and embryos successively died. In Xenopus and Hyperolius (10, 5 mM VPA) the beginning of gastrulation was delayed up to neurulation of control embryos. In Xenopus many of the embryos completed neurulation, whereas some embryos exposed to 10 mM VPA showed neural tube defects (NTDs) of different type and degree (open neural tube at different regions of the dorsum). In Hyperolius neural folds arose around the blastoporus and fused later on (earlier in embryos treated with 5 mM VPA), but the shape of these embryos was abnormal and the development was not continued (pronounced effect at 10 mM VPA). Comparing the three species, Xenopus proved to be the least sensitive species (at 5 mM VPA 14.2% NTDs of total malformations compared to 100% in the other species). The most sensitive species, Ambystoma, developed head-oedema at 1 mM VPA, whereas the anurans were not affected. Our results suggest a similar mechanism of VPA-induced NTDs in mammals and amphibians.

Demonstration of sensory-motor innervation of postmetamorphic forelimb regenerates of Triturus alpestris (Urodela) on cryotome serial sections using horseradish peroxidase.

A method has been developed to obtain horseradish peroxidase-treated serial sections containing spinal cord as well as bilateral ventral and dorsal roots, dorsal root ganglia and spinal nerves. Young postmetamorphic newts (Triturus alpestris) served as experimental animals. After cryotome cross sectioning the forelimb region of the trunk, slices 80 microns in thickness were mounted serially with up to 15 sections per slide. This facilitated subsequent staining manipulations and made partial loss of sections less likely.