Heinz Hildebrand

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.

Chondrotoxicity of quinolones in vivo and in vitro

Chondrotoxicity is a rare toxicological finding which is observed in dogs after administration of quinolone antibacterials. To study this effect chondrocytes from articular cartilage of dogs were isolated, and incubated with quinolone derivatives. The effects on cell viability, mitochondrial dehydrogenase, and proteoglycan synthesis were determined. These results were compared with in vivo findings in dogs treated with these quinolones. It was concluded that inhibition of mitochondrial dehydrogenase activity and of proteoglycan synthesis are major reasons for cartilage damage. Therefore this in vitro model is capable of identifying strongly arthropathogenic quinolones without the need of performing animal studies.

Urinary antigens as markers of papillary toxicity. I. Identification and characterization of rat kidney papillary antigens with monoclonal antibodies.

Abstract Monoclonal antibodies were prepared in an attempt to develop diagnostic tools for the identification of toxic damage to the rat renal papilla. One IgG and five IgM monoclonal antibodies, reacting with antigens localized in the papilla were obtained. Three of the IgM class and the IgG class monoclonal antibodies were found to be specific for antigens localized in collecting ducts, two of them staining papillary collecting ducts more intensely than cortical collecting ducts. The IgG class antibody, termed Pap X 5C10, recognizes an antigen located at high density on the luminal side of papillary collecting duct epithelial cells and at lower density in cortical collecting duct cells. One of the IgM class monoclonal antibodies reacts with an antigen localized in epithelial cells of ascending and descending loops of Henle and of connecting tubules. Another of the IgM class monoclonal antibodies reacts with an antigen localized in the interstices of the inner medulla. All these monoclonal antibodies react with their antigens in native frozen as well as in Bouin-fixed and paraffin-embedded tissue slices. Molecular properties of the Pap X 5C10 antigen have been investigated by gel permeation chromatography, SDS-PAGE, Western blotting, and isoelectric focusing. The results indicate that the antigen in both its tissue-derived and urinary form is of large (150–200 kDa) molecular size and can be separated into two molecular species with isoelectric points of pH 7.2 and 7.3 respectively. In the urine the antigens recognized by the monoclonal antibodies form large complexes with Tamm-Horsfall protein. The antigen-containing complexes can be extracted from urine by adsorption to diatomaceous earth and elution with SDS-containing buffer. Using sandwich ELISA-type assays it is possible to determine the concentration of the antigens. In preliminary experiments we were able to show that at least three of the antigens are detected in the urine following toxic insults to the kidney. The monoclonal antibodies prepared and the tests developed thus may provide direct diagnostic access to the renal papilla and allow, for the first time, early detection of papillary damage.

Serotonin (5-HT1A-Receptor) Agonist-Induced Collecting Duct Vacuolation and Renal Papillary Necrosis in the Rat

General anxiety in humans is treated with azaspirodecanedions, which act through a reduction of serotonin transmission. Ipsapirone also represents a serotonin (5-HT1A) receptor agonist and was under development as an anxiolytic drug. Histopathologic evaluation of animal experiments revealed cellular swelling and/or vacuolation of renal papillary and medullary collecting duct (MCD) epithelium in rats but not in dogs or mice. The changes ensued already after 1 wk of dosing and were first localized in the inner MCDs. Longer treatment periods showed that these changes proceeded from proximal to distal, approaching the papillary collecting ducts. The changes were most likely the result of altered hemodynamics in the papillary tip. Swelling resulted in partial or total papillary necrosis in some cases. Furthermore, rats treated with ipsapirone showed a sharp and transient rise in urinary endothelin excretion. Concomitantly, urinary PGE2 levels were elevated. In contrast, no elevated levels of endothelin were detected in urine samples of patients from a volunteer study, leading to the conclusion that the human kidney is not susceptible to the ipsapirone-induced alterations seen in the collecting ducts of rats.

Preparation and characterization of a mouse monoclonal antibody against a rat kidney papillary antigen

Monoclonal antibodies were prepared in an attempt to develop diagnostic tools for the identification of toxic damage to the renal papilla. One of these antibodies, termed PAP X 5C10, recognizes an antigen that is located on the luminal side of collecting duct epithelial cells. As shown by Western blotting experiments, this antigen can be extracted from papillary tissue and visualized as a broad band of high molecular weight. Enzyme-linked immunosorbent assays have shown that increased amounts of this antigen can be detected in urine of rats treated with bromoethaneamine. This procedure thus enables this antigen to be detected also in supernatants of cultured ductal cells.

Determination of apoptosis in primary rat hepatocytes by real-time quantitative PCR (TaqMan PCR)

Primary hepatocytes in collagen gel sandwich culture are a well suited model for studying effects like xenobiotic metabolism, hepatotoxicity, apoptosis, and others. Apoptosis is programmed cell death. It plays an essential role in embryonal development as well as in adult tissue for the elimination of undesired cells. The physiologic balance between growth and death can be disturbed by xenobiotics which may induce apoptosis. For the characterization of apoptosis, the expression of two genes, namely bax and p53 was analysed by quantitative real-time PCR. Following incubation of rat primary hepatocytes with camptothecin (CPT), a time- and concentration-dependent increase of mRNA expression was measured for bax at approximately 350% and p53 at approximately 600%. Further, camptothecin and topotecan (TPT) were compared for their effect on bax mRNA expression. Whereas camptothecin induced a continuous increase in bax transcription from 0.1 mum to 10 mum, only the highest concentration of topotecan (10 mum) led to a comparable increase of bax transcription. Our results demonstrate that apoptosis can be rapidly and simply quantitated in hepatocytes by TaqMan PCR.