Hans Jürgen Ahr

Molecular markers in embryonic stem cells.

Embryonic stem cells are pluripotent cells derived from mouse blastocysts, which have the capacity to differentiate in vitro into a wide variety of cell types. Based on this potential the embryonic stem cell test (EST) has been developed, which represents an assay system for the classification of compounds for their teratogenic potential, based on the morphological evaluation of contracting myocard cells compared to the cytotoxic effects on undifferentiated stem cells and adult 3T3 fibroblasts. To expand the EST, the quantitative expression of the alpha- and beta-myosin heavy chain (MHC) genes under the influence of test compounds was studied employing real-time TaqMan PCR analysis. The molecular evaluation of the MHC genes allows a higher sensitivity for the classification of substances and the transfer of the EST to the molecular level allows to start experimental procedures at day 9 of culture. Thus, the modulated EST holds promise as a new easily quantifiable in vitro screening assay in teratology.

Meeting report: Validation of toxicogenomics-based test systems: ECVAM-ICCVAM/NICEATM considerations for regulatory use

This is the report of the first workshop “Validation of Toxicogenomics-Based Test Systems” held 11–12 December 2003 in Ispra, Italy. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and organized jointly by ECVAM, the U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). The primary aim of the workshop was for participants to discuss and define principles applicable to the validation of toxicogenomics platforms as well as validation of specific toxicologic test methods that incorporate toxicogenomics technologies. The workshop was viewed as an opportunity for initiating a dialogue between technologic experts, regulators, and the principal validation bodies and for identifying those factors to which the validation process would be applicable. It was felt that to do so now, as the technology is evolving and associated challenges are identified, would be a basis for the future validation of the technology when it reaches the appropriate stage. Because of the complexity of the issue, different aspects of the validation of toxicogenomics-based test methods were covered. The three focus areas include a) biologic validation of toxicogenomics-based test methods for regulatory decision making, b) technical and bioinformatics aspects related to validation, and c) validation issues as they relate to regulatory acceptance and use of toxicogenomics-based test methods. In this report we summarize the discussions and describe in detail the recommendations for future direction and priorities.

Ciprofloxacin: in vivo genotoxicity studies.

The fluoroquinolone ciprofloxacin is widely used in antimicrobial therapy. It inhibits the bacterial gyrase and in high concentrations in vitro also the functionally related eukaryotic topoisomerase-II, which resulted in genotoxic effects in several in vitro tests. In order to evaluate the relevance of these findings, ciprofloxacin was tested in vivo for genotoxic activity using the following test systems: micronucleus test in bone marrow of mice, cytogenetic chromosome analysis in Chinese hamster, dominant lethal assay in male mice and UDS tests in primary rat and mouse hepatocytes in vivo. These results are compared with already published in vitro and in vivo studies with ciprofloxacin. All in vivo genotoxicity revealed no genotoxic effect for ciprofloxacin. In addition, ciprofloxacin was found to be non-carcinogenic in two rodent long-term bioassays. Therefore, ciprofloxacin is considered to be safe for therapeutic use.

Improved in vitro method for screening organophosphate-induced delayed polyneuropathy

Organophosphate-induced delayed polyneuropathy (OPIDP) has been tested mainly byin vivo methods in the chicken and by examination of the inhibition of neuropathy target esterase in their neuronal tissue. An alternative method, using permanent neuronal cell lines and detecting the growth of neurite-like sprouts by morphometric means, has meanwhile been validated to substitute thesein vivo investigations, at least in part. This paper reports some modifications of this in vitro method in order to optimize it for a routine laboratory screening test of organophosphates. By determination of cytoskeletal elements with cell ELISA, it was demonstrated that seven OPIDP-inducing compounds [tri-o-cresyl phosphate (TOCP) and its metabolite cresyl-saligeninphosphate, haloxon, mipafox, leptophos, EPN and chlorpyrifos] and nine negative control substances [paraoxon, methylazinphos, ethylazinphos, dimefox, dimethoate, phenylmethanesulfonylfluoride (PMSF) and three organophosphate metabolite-cresol, phenol and p-nitrophenol] resulted in the same effects as seen in the original test method. The cell ELISA technique thus represents an alternative method that is much easier to perform than the morphometric method.

The SEURAT-1 Approach towards Animal Free Human Safety Assessment

SEURAT-1 is a European public-private research consortium that is working towards animal-free testing of chemical compounds and the highest level of consumer protection. A research strategy was formulated based on the guiding principle to adopt a toxicological mode-of-action framework to describe how any substance may adversely affect human health.The proof of the initiative will be in demonstrating the applicability of the concepts on which SEURAT-1 is built on three levels:(i) Theoretical prototypes for adverse outcome pathways are formulated based on knowledge already available in the scientific literature on investigating the toxicological mode-of-actions leading to adverse outcomes (addressing mainly liver toxicity);(ii)adverse outcome pathway descriptions are used as a guide for the formulation of case studies to further elucidate the theoretical model and to develop integrated testing strategies for the prediction of certain toxicological effects (i.e., those related to the adverse outcome pathway descriptions);(iii) further case studies target the application of knowledge gained within SEURAT-1 in the context of safety assessment. The ultimate goal would be to perform ab initio predictions based on a complete understanding of toxicological mechanisms. In the near-term, it is more realistic that data from innovative testing methods will support read-across arguments. Both scenarios are addressed with case studies for improved safety assessment. A conceptual framework for a rational integrated assessment strategy emerged from designing the case studies and is discussed in the context of international developments focusing on alternative approaches for evaluating chemicals using the new 21st century tools for toxicity testing.

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.

Calcium channel blockers and cancer : Is there preclinical evidence for an association?

The preclinical evidence for a potential influence of calcium channel blockers (CCB) on carcinogenesis is discussed in the light of rodent carcinogenicity studies as well as mechanistic data. In the bioassays performed in rats and mice on the dihydropyridine CCB nifedipine, nimodipine, nisoldipine and nitrendipine, no evidence was found for a carcinogenic potential of these compounds. Moreover, the mechanistic knowledge on the influence of CCB on the fundamental processes of cell proliferation and apoptosis is not in favor of a tumor-promoting activity of these compounds. It is, therefore, concluded that there is no preclinical evidence that the therapeutic use of CCB of the dihydropyridine class is associated with an increased risk of cancer.

Effects of fluoroquinolones and glucocorticoids on cultivated tendon cells in vitro

Achilles tendon rupture is known to occur after administration of various drugs to patients. It is also reported to occur after treatment with fluoroquinolone and glucocorticoids. To study potential cytotoxic effects of fluoroquinolones (ciprofloxacin, pefloxacin, sparfloxacin) and triamcinolonacetonide on tendons an in vitro model of cultivated tendon cells (human, dog, mini-pig, rat, marmoset) was established. The cells were characterized by their morphological appearance and by the synthesis of proteoglycans and collagen type I. The cytotoxicity of the tested drugs was determined by measurement of mitochondrial dehydrogenase activity (MTT assay) and other parameters (viability, proteoglycan content, proliferation). These investigations revealed the following: (1) no marked differences were found between various species after ciprofloxacin treatment, only rat tendon cells reacted slightly less sensitively at the highest concentration (100 mug/ml); (2) no age-dependent effects of ciprofloxacin and triamcinolonacetonide were found in cultivated human tendon cells from patients of different ages; (3) the simultaneous administration of fluoroquinolones and triamcinolonacetonide resulted in a significantly greater reduction of cell viability than when they were administered alone. This could indicate that the combined administration of fluoroquinolones and glucocorticoids can favour the occurrence of tendon rupture.

Analyses of cutaneous fluoroquinolones photoreactivity using the integrated model for the differentiation of skin reactions

Currently available test models for the differentiation of photoallergic and photoirritant reactions are extremely time consuming and the protocols are very heterogeneous. In vitro tests are of proven value in predicting irritant or toxic effects, but these tests fail to predict chemical-induced allergic side effects. We developed test systems for this endpoint which is not easily detected by existing assays. In a previous publication we were able to discriminate between a contact sensitizer and a skin irritant with a combination of primary ear swelling analysis and cell counting of the ear-draining lymph nodes [Toxicol. Appl. Pharm. 153 (1998) 83; Arch. Toxicol. 73 (2000) 501]. This combination of tests was called the Integrated Model for the Differentiation of chemical-induced allergic and irritant Skin reactions (IMDS). In addition, it had been shown before that inclusion of UV irradiation in the local lymph node assay enables discrimination of photoallergic from photoirritant reactions after dermal application [Photodermatol. Photoimmunol. Photomed. 10 (1994) 57]. Because of the fact that fluoroquinolones are known to induce photoreactions after oral but not dermal treatment, the aim of the present study was to apply the IMDS for the fast and reliable differentiation of photoreactions due to fluoroquinolones after oral treatment. Enoxacin, lomefloxacin, ofloxacin, sparfloxacin and BAY y 3118 were tested in this system. We found a good correlation between the results of UV light-irradiated IMDS and a guinea pig model with the quinolones as far as photoirritancy was concerned. This holds true also for the photoallergic standard olaquindox and the photoirritant standard 8-methoxypsoralen. However, in contrast to the guinea pig assays the IMDS is fast and extremely predictive for the risk of both photosensitization and photoirritancy depending on the route of exposure. Thus, the UV light-irradiated IMDS turned out to be a good tool for the preclinical risk assessment procedure in terms of discriminating photoreactions. In addition, flow cytometric analyses were used to underline the fact that antigen-independent activation occurred after the induction of photoirritant reactions.

Damage to mitochondrial DNA induced by the quinolone Bay y 3118 in embryonic turkey liver.

Quinolones are a class of antibiotics that induce damage to and loss of DNA from bacteria. The structural organization of bacterial DNA is more similar to eukaryotic mitochondrial DNA (mtDNA) than to eukaryotic chromosomal or nuclear DNA (nDNA). Antibiotics affecting the bacterial genome may therefore preferentially damage mtDNA rather than nDNA. We investigated the effect of a quinolone on mtDNA in avian embryonic hepatocytes in ovo. The quinolone Bay y 3118 (1-cyclopropyl-7-(2,8-diazabicyclo[4.3.0]non-8-yl) 6-fluoro-8-chloro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid hydrochloride, chemical structure see Bremm et al. [K.D. Bremm, U. Petersen, K.G. Metzger, R. Endermann, In vitro evaluation of Bay-y 3118, a new full-spectrum fluoroquinolone, Chemotherapy 38 (1992) 376-387] was injected into fertilized turkey eggs 8 days before hatching at doses of 1, 3, 10 and 30 mg per egg. The embryos were removed from the eggs after 4 days and liver samples were shock frozen. Mitochondrial DNA was purified from samples of the embryonic liver. The integrity of mtDNA was investigated by electrophoresis on agarose gels with native mtDNA and with ribonuclease-treated mtDNA. Fluorescent staining of the electrophoresis gels allows the densitometric quantification of the mtDNA of the regular band at 16 kilobases (kb) and the amount of DNA fragments of irregular size (smear). The genotoxic nitrosamine nitrosodiethylamine (NDEA) has previously been shown to reduce the content of mtDNA of the regular size of 16 kb and to induce the occurrence of smaller fragments of mtDNA [H. Enzmann, C. Kühlem, E. Löser, P. Bannasch, Damage to mitochondrial DNA induced by the hepatocarcinogen, diethylnitrosamine in ovo, Mutation Res. 329 (1995) 113-120]. After exposure to 10 and 30 mg Bay y 3118, a dose-dependent induction of damage to the mtDNA was found, whereas exposure to 3 and 1 mg showed no effect. NDEA (25 mg) was used as positive control. Testing chemical compounds in the in ovo model is a simple and rapid approach for investigations on chemically induced alterations of mtDNA.