Transcriptomic alterations induced by Ochratoxin A in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model.

Ochratoxin A (OTA) is a widely studied compound due to its role in renal toxicity and carcinogenicity. However, there is still no consensus on the exact mechanisms of toxicity or carcinogenicity. In the current study, we analysed the effect of OTA on three human renal proximal tubular models (human primary, RPTEC/TERT1 and HK-2 cells) and two rat renal proximal tubular models (rat primary and NRK-52E cells). Global transcriptomics analysis at two exposure times was performed to generate a set of 756 OTA sensitive genes. This gene set was then compared in more detail across all models and additionally to a rat in vivo renal cortex model. The results demonstrate a well-conserved response across all models. OTA resulted in deregulation of a number of pathways including cytoskeleton, nucleosome regulation, translation, transcription, ubiquitination and cell cycle pathways. Interestingly, the oxidative stress activated Nrf2 pathway was not enriched. These results point to an epigenetic action of OTA, perhaps initiated by actin binding as the actin remodelling gene, advillin was the highest up-regulated in all models. The largest model differences were observed between the human and the rat in vitro models. However, since the human in vitro models were more similar to the rat in vivo model, it is more likely that these differences are model-specific rather than species-specific per se. This study demonstrates the usefulness of in vitro cell culture models combined with transcriptomic analysis for the investigation of mechanisms of toxicity and carcinogenicity. In addition, these results provide further evidence supporting a non-genotoxic mechanism of OTA-induced carcinogenicity.

Establishment of a protocol for the gene expression analysis of laser microdissected rat kidney samples with affymetrix genechips.

Laser microdissection in conjunction with microarray technology allows selective isolation and analysis of specific cell populations, e.g., preneoplastic renal lesions. To date, only limited information is available on sample preparation and preservation techniques that result in both optimal histomorphological preservation of sections and high-quality RNA for microarray analysis. Furthermore, amplification of minute amounts of RNA from microdissected renal samples allowing analysis with genechips has only scantily been addressed to date. The objective of this study was therefore to establish a reliable and reproducible protocol for laser microdissection in conjunction with microarray technology using kidney tissue from Eker rats p.o. treated for 7 days and 6 months with 10 and 1mg Aristolochic acid/kg bw, respectively. Kidney tissues were preserved in RNAlater or snap frozen. Cryosections were cut and stained with either H&E or cresyl violet for subsequent morphological and RNA quality assessment and laser microdissection. RNA quality was comparable in snap frozen and RNAlater-preserved samples, however, the histomorphological preservation of renal sections was much better following cryopreservation. Moreover, the different staining techniques in combination with sample processing time at room temperature can have an influence on RNA quality. Different RNA amplification protocols were shown to have an impact on gene expression profiles as demonstrated with Affymetrix Rat Genome 230_2.0 arrays. Considering all the parameters analyzed in this study, a protocol for RNA isolation from laser microdissected samples with subsequent Affymetrix chip hybridization was established that was also successfully applied to preneoplastic lesions laser microdissected from Aristolochic acid-treated rats.

In vitro differentiation of embryonic stem cells into mineralized osteoblasts.

Embryonic stem cells are pluripotent cells derived from the inner cell mass of mouse blastocysts that have been shown to differentiate spontaneously into cell types representing all three germ layers. This study shows that ES cells were induced to differentiate in vitro into mineralized osteoblasts under the influence of ascorbic acid, beta-glycerophosphate and 1alpha,25-OH vitamin D3. The activity of alkaline phosphatase, an early osteoblast marker, was found to be increased around day 12 of culture. Mineralized cells were clearly identified by histochemical staining, which detects mineralized calcium. The major noncollagenous component of bone matrix, osteocalcin, was localized to the mineralized cells by immunofluorescence. The expression of bone-specific genes was analyzed by real-time quantitative PCR. Osteocalcin and bone sialoprotein (BSP) were identified as early as in the fourth week of embryonic stem cell culture, both being characteristic for late stages of osteoblastic differentiation, indicating that at this time of culture the identified cells represent "mature" osteoblasts. The osteoblast-specific transcription factor Cbfa1 was induced a few days earlier. The expression of osteopontin and osteonectin, both being involved in binding calcium ions and hydroxyapatite during mineralization processes, as well as of collagen type I, representing by far the most predominant collagen in vertebrate organisms, is enhanced at the beginning of the second culture week upon addition of supplements. In the third week of culture, treated cells showed a second peak of osteopontin, osteonectin and collagen type I expression, osteopontin and osteonectin being stimulated 3-4-fold and collagen type I being induced 6-fold over control values. Alkaline phosphatase (ALP) expression was enhanced at the beginning of the third week of culture and was found to be increased again at later stages of culture at days 27-34. The in vitro differentiation of mouse embryonic stem cells into osteoblasts may provide a suitable model for studying the molecular processes of osteoblastic development in vivo.

Application of toxicogenomics to drug development.

The application of advanced modern biomedical and chemical research technologies in the pharmaceutical industry has led to a significant increase in the number of potential drug targets and lead candidates. Whereas the drug discovery process is enhanced significantly, the failure rate of new compounds due to toxicity remains very high. The pharmaceutical industry is setting high hopes on the new discipline of toxicogenomics to revolutionize the process of drug toxicity assessment by reducing the bottleneck of new drug candidates and minimizing late-stage developmental failures. Toxicogenomics is expected to facilitate the efficient screening of new compounds at an early stage, resulting in significant savings of time and cost associated with new drug development. In this review, a general description of the new discipline of toxicogenomics and its potential impact on the safety assessment of new drugs in the pharmaceutical industry is provided. An overview of the key issues and questions that are confronting investigators in this field today is also given as well as a prospective view of the future of this new discipline.