Leucine-rich repeat kinase 2 (LRRK2)-deficient rats exhibit renal tubule injury and perturbations in metabolic and immunological homeostasis.

Genetic evidence links mutations in the LRRK2 gene with an increased risk of Parkinson's disease, for which no neuroprotective or neurorestorative therapies currently exist. While the role of LRRK2 in normal cellular function has yet to be fully described, evidence suggests involvement with immune and kidney functions. A comparative study of LRRK2-deficient and wild type rats investigated the influence that this gene has on the phenotype of these rats. Significant weight gain in the LRRK2 null rats was observed and was accompanied by significant increases in insulin and insulin-like growth factors. Additionally, LRRK2-deficient rats displayed kidney morphological and histopathological alterations in the renal tubule epithelial cells of all animals assessed. These perturbations in renal morphology were accompanied by significant decreases of lipocalin-2, in both the urine and plasma of knockout animals. Significant alterations in the cellular composition of the spleen between LRRK2 knockout and wild type animals were identified by immunophenotyping and were associated with subtle differences in response to dual infection with rat-adapted influenza virus (RAIV) and Streptococcus pneumoniae. Ontological pathway analysis of LRRK2 across metabolic and kidney processes and pathological categories suggested that the thioredoxin network may play a role in perturbing these organ systems. The phenotype of the LRRK2 null rat is suggestive of a complex biology influencing metabolism, immune function and kidney homeostasis. These data need to be extended to better understand the role of the kinase domain or other biological functions of the gene to better inform the development of pharmacological inhibitors.

GADD45α induction in the GreenScreen HC indicator assay does not occur independently of cytotoxicity.

Mammalian chromosomal integrity assays are influenced by cytotoxicity, a phenomenon which impacts data interpretation, assay specificity, and regulatory testing guidelines. Concordance of the GADD45α GreenScreen HC indicator assay to established in vitro and in vivo genetic toxicological assays has previously been described, yet a detailed description in the manner by which cytotoxicity influences its performance has not. Here we present a post-hoc analysis of a previously tested set of 91 proprietary and nonproprietary compounds investigating the influence of cytotoxicity on GADD45α induction and how varying assay cutoff criteria impacts assay performance. Significant cytotoxicity was identified to accompany the majority (72%) of compounds classified as genotoxic by GADD45α induction. Decreasing the GADD45α genotoxic induction criteria (from a 50 to a 30% increase over solvent controls) resulted in an increased assay sensitivity (from 30 to 68%) and concordance (from 55 to 68%), though a concomitant decrease in specificity was also observed (from 97 to 68%). We conclude that GADD45α induction in the GreenScreen HC indicator assay is influenced by cytotoxicity and that assay performance can be improved if different cutoff criteria are implemented.

Modeling bone marrow toxicity using kinase structural motifs and the inhibition profiles of small molecular kinase inhibitors.

The cellular function of kinases combined with the difficulty of designing selective small molecule kinase inhibitors (SMKIs) poses a challenge for drug development. The late-stage attrition of SMKIs could be lessened by integrating safety information of kinases into the lead optimization stage of drug development. Herein, a mathematical model to predict bone marrow toxicity (BMT) is presented which enables the rational design of SMKIs away from this safety liability. A specific example highlights how this model identifies critical structural modifications to avoid BMT. The model was built using a novel algorithm, which selects 19 representative kinases from a panel of 277 based upon their ATP-binding pocket sequences and ability to predict BMT in vivo for 48 SMKIs. A support vector machine classifier was trained on the selected kinases and accurately predicts BMT with 74% accuracy. The model provides an efficient method for understanding SMKI-induced in vivo BMT earlier in drug discovery.

Identification of a kinase profile that predicts chromosome damage induced by small molecule kinase inhibitors.

Kinases are heavily pursued pharmaceutical targets because of their mechanistic role in many diseases. Small molecule kinase inhibitors (SMKIs) are a compound class that includes marketed drugs and compounds in various stages of drug development. While effective, many SMKIs have been associated with toxicity including chromosomal damage. Screening for kinase-mediated toxicity as early as possible is crucial, as is a better understanding of how off-target kinase inhibition may give rise to chromosomal damage. To that end, we employed a competitive binding assay and an analytical method to predict the toxicity of SMKIs. Specifically, we developed a model based on the binding affinity of SMKIs to a panel of kinases to predict whether a compound tests positive for chromosome damage. As training data, we used the binding affinity of 113 SMKIs against a representative subset of all kinases (290 kinases), yielding a 113x290 data matrix. Additionally, these 113 SMKIs were tested for genotoxicity in an in vitro micronucleus test (MNT). Among a variety of models from our analytical toolbox, we selected using cross-validation a combination of feature selection and pattern recognition techniques: Kolmogorov-Smirnov/T-test hybrid as a univariate filter, followed by Random Forests for feature selection and Support Vector Machines (SVM) for pattern recognition. Feature selection identified 21 kinases predictive of MNT. Using the corresponding binding affinities, the SVM could accurately predict MNT results with 85% accuracy (68% sensitivity, 91% specificity). This indicates that kinase inhibition profiles are predictive of SMKI genotoxicity. While in vitro testing is required for regulatory review, our analysis identified a fast and cost-efficient method for screening out compounds earlier in drug development. Equally important, by identifying a panel of kinases predictive of genotoxicity, we provide medicinal chemists a set of kinases to avoid when designing compounds, thereby providing a basis for rational drug design away from genotoxicity.

In vitro to in vivo concordance of a high throughput assay of bone marrow toxicity across a diverse set of drug candidates.

The development of predictive toxicology assays is necessary to optimize the drug candidate selection process. The colony forming assay (CFA) is used routinely to assess bone marrow toxicity and represents a viable tool for the discovery toxicologist, but the assay is not widely accepted as a standard screening tool due to technical challenges. A higher throughput and standardized version of the assay recently was developed such that the proliferative capacity of a cell lineage is measured indirectly via ATP levels, replacing the cumbersome identification and enumeration of specific colonies. In this study, a high-throughput assay of bone marrow toxicity prediction using the granulocyte, erythrocyte, monocyte, and macrophage (GEMM) progenitor cell lineage was evaluated using a training set of 56 structurally diverse compounds with known in vivo bone marrow effects. In general, compounds identified as toxic in vivo had lower IC(50) values, whereas those identified as non-toxic had higher IC(50) values. Concordance (i.e., predictive accuracy) to in vivo bone marrow toxicity results was 82% when an in vitro toxicity threshold of 20 microM was used. Additional experiments in other hematopoietic lineages were conducted to determine if predictivity of several false positive and negative compounds in the GEMM lineage could be improved; however an increase in sensitivity or specificity was not observed. The high-throughput GEMM assay has good concordance to in vivo bone marrow toxicity results and, with the high-throughput and standardized format, can be incorporated readily into the pharmaceutical toxicological screening paradigm, aiding in the early identification of compounds that eventually may fail due to bone marrow toxicity.

Interlaboratory evaluation of genomic signatures for predicting carcinogenicity in the rat.

The Critical Path Institute recently established the Predictive Safety Testing Consortium, a collaboration between several companies and the U.S. Food and Drug Administration, aimed at evaluating and qualifying biomarkers for a variety of toxicological endpoints. The Carcinogenicity Working Group of the Predictive Safety Testing Consortium has concentrated on sharing data to test the predictivity of two published hepatic gene expression signatures, including the signature by Fielden et al. (2007, Toxicol. Sci. 99, 90-100) for predicting nongenotoxic hepatocarcinogens, and the signature by Nie et al. (2006, Mol. Carcinog. 45, 914-933) for predicting nongenotoxic carcinogens. Although not a rigorous prospective validation exercise, the consortium approach created an opportunity to perform a meta-analysis to evaluate microarray data from short-term rat studies on over 150 compounds. Despite significant differences in study designs and microarray platforms between laboratories, the signatures proved to be relatively robust and more accurate than expected by chance. The accuracy of the Fielden et al. signature was between 63 and 69%, whereas the accuracy of the Nie et al. signature was between 55 and 64%. As expected, the predictivity was reduced relative to internal validation estimates reported under identical test conditions. Although the signatures were not deemed suitable for use in regulatory decision making, they were deemed worthwhile in the early assessment of drugs to aid decision making in drug development. These results have prompted additional efforts to rederive and evaluate a QPCR-based signature using these samples. When combined with a standardized test procedure and prospective interlaboratory validation, the accuracy and potential utility in preclinical applications can be ascertained.

The histone deacetylase inhibitor trichostatin a has genotoxic effects in human lymphoblasts in vitro.

Histone deacetylase inhibitors (HDACi) are a class of putative chemotherapeutic agents for which the mechanism of toxicity has not been fully identified. To explore the possibility that HDACi are genotoxic, human TK6 lymphoblastoid cells were exposed to trichostatin A (TSA) and genetic damage was measured. TSA caused a dose-dependent increase of G1-arrested cells at 24 h that correlated with increasing levels of p21 and apoptosis. Significantly elevated frequencies of structural chromosomal aberrations in cells exposed to TSA were observed using both the kinetochore-antibody micronucleus assay and nonbanding metaphase chromosome analysis. Increased tail intensities, indicative of elevated levels of DNA damage, were observed using the alkaline comet assay. Elevated levels of phosphorylated histone gammaH2AX protein were observed as early as 3 h following TSA exposure and peaked at 12 h for 200nM TSA. Significant levels of aneuploidy at the 200nM TSA dose were observed using metaphase analysis, but interestingly, kinetochore-positive micronuclei were not detected at any dose using the kinetochore micronucleus assay, suggesting that TSA induces aneuploidy via a nondisjunction event rather than chromosome lagging. Increases in chromosomal loss and breakage were observed using simultaneous FISH metaphase analysis of chromosomes 5, 7, 8, and 21, consistent with data obtained from the micronucleus and metaphase chromosome analyses. We conclude that TSA is both a clastogen and aneugen in the TK6 cell line and propose that the observed cytostatic and apoptotic properties of TSA may partially be due to this genotoxicity.

Tetraploidy and chromosomal instability are early events during cervical carcinogenesis.

Chromosomal instability as manifested by increases in aneuploidy and structural chromosome aberrations is believed to play a critical role in the intermediate to late stages in the development of cervical malignancies. The current study was designed to determine the role of tetraploidy in the formation of aneuploidy and ascertain the occurrence of these alterations during the earlier stages of cervical carcinogenesis. Cervical cell samples, with diagnoses ranging from Normal to high-grade lesions, (HSIL) were obtained from 143 women and were evaluated for chromosomal alterations using dual-probe fluorescence in situ hybridization. Cervical cells from a subset of the group were also evaluated for chromosomal instability in the form of micronuclei. The frequencies of cells exhibiting either tetrasomy or aneusomy for Chromosomes 3 and 17 increased significantly with disease progression and displayed distinctive patterns where aneusomy was rarely present in the absence of tetrasomy. The frequencies of micronuclei that formed through either chromosomal loss or breakage increased significantly in both the low-grade and high-grade diagnostic categories and were highly correlated with both the number of tetrasomic and aneusomic cervical cells. In addition, a unique chromosomal alteration involving a significant non-random loss of Chromosome 17 specific to near-tetraploid aneusomic cells (trisomy 17 and tetrasomy 3) was observed. We conclude that tetraploidy and chromosomal instability are related events occurring during the early stages of cervical carcinogenesis that predispose cervical cells to the formation of aneuploidy frequently involving the loss of Chromosome 17.

The flavoring agent dihydrocoumarin reverses epigenetic silencing and inhibits sirtuin deacetylases.

Sirtuins are a family of phylogenetically conserved nicotinamide adenine dinucleotide-dependent deacetylases that have a firmly established role in aging. Using a simple Saccharomyces cerevisiae yeast heterochromatic derepression assay, we tested a number of environmental chemicals to address the possibility that humans are exposed to sirtuin inhibitors. Here we show that dihydrocoumarin (DHC), a compound found in Melilotus officinalis (sweet clover) that is commonly added to food and cosmetics, disrupted heterochromatic silencing and inhibited yeast Sir2p as well as human SIRT1 deacetylase activity. DHC exposure in the human TK6 lymphoblastoid cell line also caused concentration-dependent increases in p53 acetylation and cytotoxicity. Flow cytometric analysis to detect annexin V binding to phosphatidylserine demonstrated that DHC increased apoptosis more than 3-fold over controls. Thus, DHC inhibits both yeast Sir2p and human SIRT1 deacetylases and increases p53 acetylation and apoptosis, a phenotype associated with senescence and aging. These findings demonstrate that humans are potentially exposed to epigenetic toxicants that inhibit sirtuin deacetylases.

Elevated levels of tetraploid cervical cells in human papillomavirus-positive Papanicolaou smears diagnosed as atypical squamous cells of undetermined significance.

BACKGROUND: Recommendations for the proper treatment of women diagnosed with an equivocal atypical squamous cells of undetermined significance (ASCUS) Papanicolaou (Pap) smear are controversial. To the authors' knowledge, there currently are no methods available that can identify accurately ASCUS/human papillomavirus (HPV)-positive women who have an increased risk of developing progressive cervical lesions without the use of invasive procedures. An additional diagnostic tool is needed to triage women properly who are diagnosed with ASCUS. Numerical chromosomal abnormalities, such as tetraploidy and aneuploidy, frequently accompany cervical carcinoma development and are believed to represent early and important genetic alterations during cervical carcinogenesis. The identification of elevated levels of numerical chromosomal aberrations in women diagnosed with ASCUS Pap smears, therefore, may be of prognostic value. METHODS: Multiple-probe fluorescence in situ hybridization was used to analyze 1000 cervical epithelial cells from each of 257 women for the presence of numerical chromosomal aberrations. RESULTS: A statistically significant proportion of women diagnosed with HPV-positive ASCUS had elevated levels of tetraploid cervical cells (5 of 69 women) compared with normal/HPV-negative women (0 of 75 women) (P = 0.02). CONCLUSIONS: The frequency of numerical chromosomal aberrations in cervical cells obtained from the majority of women diagnosed with an ASCUS Pap smear did not differ significantly from the frequency in women with smears that were diagnosed as normal. However, a modest but statistically significant proportion of women diagnosed as HPV-positive ASCUS did have elevated levels of tetraploid cervical cells, a genetic abnormality often associated with cervical carcinogenesis, suggesting that these women may be at an increased risk of developing more advanced cervical lesions. Given these results, the authors recommend performing additional studies with histologic follow-up.