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.

Screening for (anti)androgenic properties using a standard operation protocol based on the human stably transfected androgen sensitive PALM cell line. First steps towards validation.

Despite more than a decade of research in the field of endocrine active compounds targeting the androgen receptor (AR), and although suitable cell lines can be obtained, no validated human stably transfected androgen sensitive transactivation assay is available. Bayer Schering Pharma (BSP) and the Flemish Institute for Technological Research (VITO), partners within the EU-sponsored 6th framework project ReProTect, made first steps towards such a validation. A standard operation protocol (SOP) developed at BSP based on the androgen sensitive PALM cell line was transferred to VITO and its performance and transferability were thoroughly studied. The investigation followed a generic protocol prepared for all reporter gene assays evaluated within ReProTect, and in both laboratories at least three independent experiments were performed. The highest concentration to be tested was limited to 10 microM, if needed. A few compounds, 17alpha-methyltestosterone (17alpha-MT), vinclozolin and linuron, were studied using a real world scenario, i.e., assuming that their interaction with the AR was not known: A prescreening for agonism and true, competitive antagonism was used to select conditions such as the appropriate mode of action, and the working range excluding cytotoxicity for the final screening. All other compounds were tested according to the generic protocol: Compounds screened for agonism were the reference androgen 17alpha-methyldihydrotestosterone (MDHT), levonorgestrel, norethynodrel, progesterone, o,p'-DDT, and dibutylphthalate (DBP), while compounds screened for antagonism were the reference anti-androgen flutamide, prochloraz, o,p'-DDT, progesterone, norethynodrel, and DBP. Cytotoxicity was assessed in parallel as lactate dehydrogenase release. The prescreen classified 17alpha-MT as androgenic, vinclozolin and linuron as anti-androgenic and compounds were tested accordingly. In the absence of cytotoxicity, appropriate androgenic properties of reference and test compounds were detected by both laboratories, o,p'-DDT and DBP had no androgenic activity. Across the two laboratories EC(50)-values for MDHT, 17alpha-MT, and levonorgestrel varied by not more than a factor of 3.4, for norethynodrel by a factor of 9.7. Progesterone effects could not fully be evaluated, as frequently concentration response curves were incomplete. In the absence of cytotoxicity anti-androgenic properties of reference and test compounds were also detected in both laboratories. DBP, the putative negative reference compound, was inactive, norethynodrel rather showed agonistic properties. Progesterone was an antagonist at low concentrations, but agonistic properties were observed in one laboratory at high concentrations. Since the highest test concentration was limited to 10 microM, for some compounds no complete concentration response curves were obtained and estimation of EC(50)-values was less robust. Our data demonstrated that the SOP was transferable, and that the assay was able to rank compounds with strong, weak, and without affinity for the AR and to discriminate agonists and antagonists. The sensitivity of the assay could be improved further, if the limit of solubility or beginning cytotoxicity was chosen as the highest test concentration. The assay avoids the use of tissues from laboratory animals, and thus contributes to the 3R concept. Furthermore, it could be adjusted to an intermediate/high throughput format. On the whole, this PALM assay is a promising candidate for further validation.

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.

In vitro differentiation of skin sensitizers by cell signaling pathways.

Animal testing causes ethical problems and in view of EU regulations (e.g. EU-Guideline (76/768/EEC, February 2003)) or REACH the development of reliable in vitro assays has become even more important. Up to now, we use the modified local lymph node assay (IMDS) for toxicological hazard identification of sensitizing and irritant properties of chemicals in accordance with OECD Guideline 429. In this study, we investigated whether analyses of cell signaling pathways can provide a methodology for the detection of sensitizing compounds in vitro. Murine and human skin explants as well as reconstituted skin models (epidermal model EST-1000 and full-thickness model AST-2000) were exposed to sensitizing (oxazolone and DNFB) or irritant compounds (SDS and TritonX-100). Phosphorylation of MAP-kinases (p38, ERK1/2 and JNK1/2), STAT1 and PLCgamma were determined by cytometric bead array (CBA). In skin explants, all three MAP-kinases were exclusively activated after exposure to sensitizing compounds. For the reconstituted skin models phosphorylations of p38 and JNK1/2 were obtained after stimulation with allergens, whereas treatments with irritant compounds led to ERK1/2 activation. Activation of PLCgamma and STAT1 were never detected. In conclusion, MAP-kinase activation provides a promising in vitro tool for the discrimination between sensitizers and irritants.

Characterization of primary rat proximal tubular cells by gene expression analysis.

The kidney plays a major role in excretory and reabsorptive processes. The kidney cortex consists primarily of proximal tubular cells, which are epithelial cells that are often involved in the induction and progression of various kidney diseases. Therefore primary proximal tubular cells are widely used as a renal cell model. To further characterize this kidney in vitro model different time points in culture after isolation of the cells were compared to the cortex in vivo using gene expression analysis based on microarrays. This study revealed that many metabolic pathways and some kidney-specific functions are lacking in the in vitro model. Furthermore genes involved in RNA and protein synthesis, intracellular transport, extracellular matrix and cytoskeletal organization were upregulated in culture compared to in vivo, indicating proliferation of the cells and differentiation into a cell culture phenotype. The data represented here may help to evaluate the in vivo relevance of results obtained with this in vitro model.

Epidermal-skin-test 1,000 (EST-1,000)--a new reconstructed epidermis for in vitro skin corrosivity testing.

The determination of a possible corrosive or irritative potential of certain products and ingredients is necessary for their classification and labeling requirements. Reconstructed skin as a model system provides fundamental advantages to single cell culture testing and leads to promising results as shown by different validation studies (for review: Fentem, J.H., Botham, P.A., 2002. ECVAM's activities in validating alternative tests for skin corrosion and irritation. ATLA 30(Suppl. 2), 61-67). In this study we introduce our new reconstructed epidermis "Epidermal-Skin-Test" (EST-1,000). This fully grown epidermis consists of proliferating as well as differentiating keratinocytes. EST-1,000 shows a high comparability to normal human skin as shown by histological and immunohistochemical data. Characteristic markers (KI-67, CK 1/10/5/14, transglutaminase, collagen IV, involucrin, beta 1 integrin) can be identified easily. The main focus of this work was to characterize EST-1,000 especially with respect to its barrier function by testing several substances of known corrosive potential. Skin corrosion was detected by the cytotoxic effect of the substances on a reconstructed epidermis after short-term application to the stratum corneum. The effect was determined by standard MTT assay and accompanying histological analysis. Hence EST-1,000 shows a very high predictive potential and closes the gap between animal testing and the established full-thickness model Advanced-Skin-Test 2,000 (AST-2,000) (Noll, M., Merkle, M.-L., Kandsberger, M., Matthes, T., Fuchs, H., Graeve, T., 1999. Reconstructed human skin (AST-2,000) as a tool for pharmaco-toxicology. ATLA 27, 302).

Molecular multiple endpoint embryonic stem cell test--a possible approach to test for the teratogenic potential of compounds.

The embryonic stem cell test (EST) examines the cytotoxicity of chemical compounds on embryonic stem (ES) cells and 3T3.A31 fibroblasts. Additionally, the EST measures the ability of ES cells to differentiate into contracting cardiomyocytes following drug exposure. In this study, we introduce new endpoints to obtain a molecular multiple endpoint EST (mme-EST), enabling the identification of potential chemical effects on osteogenic, chondrogenic and neural differentiation in addition to the traditional endpoint of cardiomyocyte differentiation. Six compounds in three classes with known teratogenic in vivo potential were assayed with the mme-EST in a pilot study: penicillin G (non-teratogenic), 5-fluorouracil and retinoic acid (strongly teratogenic), diphenylhydantoin, valproic acid and thalidomide (moderately teratogenic). While the traditional EST measures a morphological endpoint, we included molecular markers of differentiation as endpoints. With the mme-EST, every compound could be classified correctly according to its known teratogenic potential in vivo. Penicillin G, 5-fluorouracil and diphenylhydantoin inhibited differentiation of all endpoints equally. Interestingly, valproic acid showed the strongest inhibition of neural differentiation, while thalidomide specifically inhibited osteogenic development. Retinoic acid, on the other hand, supported neural but inhibited chondrogenic and osteogenic differentiation concentration-dependently. Valproic acid and thalidomide, classified incorrectly with the established EST model, were classified correctly with the mme-EST according to their effects on specific endpoints. This pilot study indicates that the predictive value of the EST may be enhanced by including further differentiation endpoints.

Respiratory hypersensitivity to trimellitic anhydride in Brown Norway rats: evidence for different activation pattern of immune cells following topical and respiratory induction.

The elicitation of respiratory allergy in animal models is exquisitely complex and interpretation of results from different laboratories cannot readily be compared due to variability in testing protocols, biomarkers and techniques used to identify 'positive' responses. On the one hand, guinea-pigs have been proposed as a good model with which to study allergic and irritant bronchial hyperresponsiveness. On the other hand, considerable efforts have been made to develop animal models that take the immunological mechanisms into account to reduce the complexity as well as duration of the guinea-pig assays. In principle, local skin reactions can easily be determined by the local lymph node assay (LLNA) introduced by Kimber and Weisenberger. In contrast to lung sensitization there are already simplified and reliable models available to test for and discriminate contact sensitizers from skin irritants, i.e. the modified local lymph node assay IMDS (integrated model for the differentiation of skin reactions). Modifications of this assay verified that methods other than radioactive labelling may be comparably sensitive, and that it is possible to eliminate 'false positive' results induced by irritants (IMDS). Thus, we asked whether there could be a similar simplified model like the modified LLNA or IMDS for investigations of respiratory allergens. Therefore, we analysed immune reactions induced by the dermal and respiratory route, respectively. Analyses of the draining lymph nodes of the lung and the ear were carried out before and after challenge via the pulmonary tract. The results clearly support that (1) the reactions in the lung draining lymph nodes could be used as early indicators of respiratory sensitization, and (2) the specificity of the immune competent cells seem to be dependent of the route of administration during induction.

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.

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.

Rat cortical neuron cultures: an in vitro model for differentiating mechanisms of chemically induced neurotoxicity.

Various structurally unrelated chemicals [2,5 hexandione, acrylamide, organophosphates like mipafox, beta,beta iminodipropionnitrile (IDPN), 3-nitropropionic acid (3-NP), potassium cyanide (KCN), paraquat, and NMDA (N-methyl-D-apartic acid)] are known to cause degenerative damage of the peripheral or central nervous system. Differentiated neuronal cell cultures obtained from fetal rats have been used to differentiate the mechanisms underlying this type of neurotoxicity. Cytotoxicity as measured by a viability assay was not sensitive enough and had to be supplemented by further endpoints covering effects on cytoskeleton and on the energy state of the cells [glucose consumption, mitochondrial membrane potential and adenosine 5'-triphosphate (ATP) concentration]. Compounds like the delayed neurotoxic organophosphates, exert a selective direct effect on cytoskeleton elements in this model at concentrations distinctly below cytotoxic concentrations. Other compounds, like KCN, paraquat, and 3-NP selectively disrupt the balance between energy supply and demand of the neurons either by interacting with mitochondrial respiration or glycolysis. For these compounds cytoskeletal damage seemed to be secondary to the energy depletion. For NMDA, 2,5 hexandione and acrylamide, both mechanisms may contribute to the neuronal damage. In conclusion, primary cortical neuronal cultures of the rat are well suited to detect a neurotoxic potential and to differentiate its underlying mechanisms. Damage of the cytoskeleton may be considered as an endpoint mechanistically related to degenerative neuropathic effects.

An intra-laboratory validation of the Integrated Model for the Differentiation of Skin Reactions (IMDS): discrimination between (photo)allergic and (photo)irritant skin reactions in mice.

We recently presented a modified local lymph node test which made it possible to quickly and reliably differentiate between irritative and allergic skin reactions with extremely simple parameters. The Integrated Model for the Differentiation of Skin Reactions (IMDS) test combines measurement of cell proliferation in draining lymph nodes with measurement of primary ear swelling after topical application of the test substance on three consecutive days. In contrast to the 'classic' skin sensitisation test in guinea-pigs the IMDS test is considerably faster and is based on objective measured data, not subjective skin evaluations. Like the Local Lymph Node Assay (LLNA), measurement of allergic potential in the IMDS test is based on the underlying immunological mechanisms, but also considers the behaviour of immune competent cells following non-specific activation by irritants. In addition, the IMDS test can employ UV radiation after application of the substance and, therefore, make differentiation possible between different types of skin photoreaction (photoallergy and photoirritation) after both topical and systemic administration. Attempts to achieve this kind of discrimination with the LLNA necessitate considerably greater expenditure, as proliferation in the draining lymph nodes can also be induced by moderate to extreme (photo)irritants. In a previous paper in which we presented the IMDS test, we examined each type of reaction in reference to one single standard; the next logical step was therefore a broad-based intra-laboratory validation. An important factor in the validation was the use of standards that had been thoroughly examined in both guinea pig and mouse systems and were also relevant with regard to estimation of the risk for humans. The data presented here show that the IMDS is a simple and reliable tool for obtaining fast and reproducible assessments of potential (photo)allergic and (photo)irritant skin reactions to substances.

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.

Primary immune response in skin and skin-associated lymphoid tissue of interleukin-4 transgenic mice.

The interleukin-4 transgenic mice investigated here exhibit a ubiquitous expression of interleukin-4 in all organs, including the skin. In this study, the induction phase of oxazolone-induced local primary contact hypersensitivity and croton oil-induced irritant contact dermatitis in transgenic and wild-type mice was analysed. Compared to wild-type mice, the transgenic mice showed a decreased activation of the skin-draining lymph nodes but a strong hyperreactivity in the skin after topical sensitisation. In contrast to this, both the transgenic and the wild-type mice developed a strong and comparable inflammatory skin reaction after topical irritation. A striking increased expression level of tumour necrosis factor-alpha and macrophage inflammatory protein-2 genes were found in the skin of the transgenic mice during primary local contact hypersensitivity, while both the transgenic and the wild-type mice developed comparable expression levels of these cytokines during irritant contact dermatitis. Compared to wild-type mice, a strongly enhanced expression level of interleukin-6 transcripts derived from epidermal antigen presenting cells were detected in the skin of IL-4 transgenic mice, whereas in the skin-draining lymph nodes of transgenic mice significantly lower levels were detected. We conclude that the migration of epidermal antigen-presenting cells towards the skin-draining lymph nodes is reduced in transgenic mice, which could be due to the different cytokine balance in these mice strains. The atypical irritant-like reaction observed in transgenic mice after topical sensitisation is a phenomenon comparable to atopic diseases and therefore this transgenic strain might be a helpful model for investigating the immunopathophysiological features of these diseases.

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.

An in vitro model for peroxisome proliferation utilizing primary hepatocytes in sandwich culture.

Peroxisome proliferators comprise a group of structurally diverse chemicals which share as a common biologic effect the induction of peroxisomal fatty acid degrading enzymes. Concomitantly, the number and size of peroxisomes within hepatocytes increases. Following chronic administration some peroxisome proliferators act as non-genotoxic hepatocarcinogens in susceptible species such as rodents. To establish an in vitro model for the toxicological investigation of peroxisome proliferation, primary hepatocytes of rats, dogs and humans were cultivated in an organotypic cell culture model (sandwich model). By employing a panel of diverse compounds in this model a graded response was observed in the induction of carnitine acetyl transferase (CAT), the activity of which was determined as an endpoint. The following results were obtained in the order of decreasing inducing potential for rat hepatocytes: FOE 3798>nafenopin>fenofibrate (ciprofibrate>bezafibrate DEHP approximately ETYA>DEHA. Induction of CAT activity was generally higher than reported in earlier cell culture systems, probably reflecting the effect of the extracellular matrix provided by the collagen gel sandwich. In parallel, transcription of the rat CYP4A1 gene was induced by a similar order of magnitude as measured by TaqMan RT-PCR. In accordance with literature data, human and dog hepatocytes did not display such a strong and graded response but rather were not susceptible to this effect. In addition, (3)H-thymidine incorporation data demonstrated that nafenopin was able to induce DNA synthesis in rat hepatocytes whereas it did not in human hepatocytes.

Short- and intermediate-term carcinogenicity testing--a review. Part 1: the prototypes mouse skin tumour assay and rat liver focus assay.

Carcinogenicity testing is by far the most expensive and time-consuming study type of toxicology. For many years, the lifetime exposure with the maximum tolerated dose in two rodent species has been the gold standard of carcinogenicity testing of pharmaceuticals. Major change was introduced by the Fourth International Conference on Harmonization in July 1997; a chronic rodent bioassay in one species and a short-term carcinogenicity assay are regarded as sufficient for registration. Such requirements provide the opportunity to redirect the vast resources previously spent on the lifetime study in the second species. Numerous experimental protocols for short- and intermediate-term carcinogenicity testing in many target tissues have been available for years. The first part of this review describes the basic principles of short- and intermediate-term carcinogenicity testing using the examples of the widely used mouse skin tumour assay and the rat liver foci assay. In the context of these experimental models, the discrimination and quantification of initiating and promoting activity and the use of preneoplastic lesions as endpoints in carcinogenicity testing are described. The review includes the limitations of the models with regard to the extrapolation from effects observed in animal experiments to a potential exposure of humans.

Short- and intermediate-term carcinogenicity testing--a review. Part 2: available experimental models.

Numerous experimental protocols for short- and intermediate-term carcinogenicity assays have been available for many years. This paper surveys various of these test systems in rodents, fish species, non-vertebrates and avian embryos in ovo. The mouse skin tumour assay and the rat liver foci assay were used to introduce the basic concepts of short- and intermediate-term carcinogenicity testing in the previous part of the review. The focus of this second part of the review is on rodent assays for carcinogenicity testing in the lung, kidney, urinary bladder, pancreas, stomach, oral cavity, small intestine, colon, and on the possibility to combine several target organs in multi-organ models. The potential use of various fish species, non-vertebrates and hatching eggs for carcinogenicity testing is outlined and the advantages and limitations are discussed. This review also presents the problem of validation of any carcinogenicity test system and proposes a strategy for contemporary safety assessment of chemicals with regard to the detection and evaluation of carcinogenicity.

Calcium channel blockers and the risk of cancer: a preclinical assessment.

The preclinical evidence for a potential influence of calcium channel blockers (CCBs) on carcinogenesis is discussed in the light of a broad database from rodent carcinogenicity studies as well as literature data. In all bioassays performed in rats and mice on the dihydropyridine CCBs--nifedipine, nimodipine, nisoldipine, and nitrendipine--no evidence was found for a carcinogenic potential of these compounds. Calcium is an essential intracellular signal for cell proliferation and apoptosis. The crucial role of increased cell proliferation in all stages of carcinogenesis is well documented. Some indirect experimental evidence also points to a role of defective apoptosis in tumor promotion. CCBs uniformly inhibit cell proliferation, whereas the influence of CCBs on apoptosis is inconsistent, resulting in an inhibition or increase in apoptosis dependent on cell type. Accordingly, antitumorigenic effects of CCBs have been reported based on their antiproliferative action. A tumor-promoting effect of CCBs based on inhibition of apoptosis, however, remains purely speculative and, in fact, can be denied based on the results of in vivo bioassays. It is therefore concluded that there is no preclinical evidence that should give rise to concern over the carcinogenic potential of dihydropyridine-type CCBs.