In vitro screening of cell bioenergetics to assess mitochondrial dysfunction in drug development.

Drug-induced mitochondrial toxicity is considered as a common cellular mechanism that can induce a variety of organ toxicities. In the present manuscript, 17 in vitro mitochondrial toxic drugs, reported to induce Drug-Induced Liver Injury (DILI) and 6 non-mitochondrial toxic drugs (3 with DILI and 3 without DILI concern), were tested in HepG2 cells using a bioenergetics system. The 17 mitochondrial toxic drugs represent a wide variety of mitochondrial dysfunctions as well as DILI and include 4 pairs of drugs which are structurally related but associated with different DILI concerns in human. Cell bioenergetics were measured using the XF96e analyzer which simultaneous monitor oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), indirect measurements of oxidative phosphorylation and glycolysis, respectively. OCR associated with ATP production, maximal respiration, proton leak and spare respiratory capacity, were also assessed. Duplicate experiments resulted in a sensitivity of 82% (14/17) and specificity of 83% (5/6). The addition of stressors improved specificity considerably. Cut-offs, statistics and rules are clearly discussed to facilitate the use of this assay for screening purposes. Overall, the authors consider that this assay should be part of the battery of safety screening assays at early stages of drug development.

The automated micronucleus assay for early assessment of genotoxicity in drug discovery.

Recent publications on the automated in vitro micronucleus assay show predictive values higher than 85% for the classification of in vitro aneugens, clastogens and non-genotoxic compounds. In the present work, the CHO-k1 micronucleus assay in combination with cellular imaging was further evaluated. Firstly, the effect of a range of S9 concentrations on micronucleus formation and cytotoxicity was investigated. Subsequently, the reproducibility and predictivity of the micronucleus assay on CHO-k1 cells was investigated with a set of four compounds. Then, a larger set of compounds (n=44) was tested on CHO-k1 cells and inter-laboratory correlation was calculated. Finally, cellular imaging was compared with flow cytometry for in vivo assessment of micronucleus formation. The concentration of S9 had a significant impact on micronucleus formation and cytotoxicity. In addition, calculations of relative cell count (RCC) and cytokinesis-block proliferation index (CBPI) showed to be complementary to cytotoxicity assessment. The CHO-k1 micronucleus assay correctly classified the four reference compounds, with a dose-response relationship and low variability. Based on a larger set of compounds, the assay proved to be reliable with a sensitivity of 94% (n=31) and a specificity of 85% (n=13). A correlation coefficient of 97% was obtained when the lowest observable adverse effect levels (LOAELs) from our study were compared with those published by Diaz et al. (2007) [10]. In conclusion, the in vitro CHO-k1 micronucleus assay combined with cellular imaging is a predictive assay appropriate for genotoxicity screening at early stages of drug development. In addition, for in vivo assessment of micronucleus formation, we preferred to use flow cytometry rather than cell imaging.

Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins.

In the pharmaceutical industry, improving the early detection of drug-induced hepatotoxicity is essential as it is one of the most important reasons for attrition of candidate drugs during the later stages of drug development. The first objective of this study was to better characterize different cellular models (i.e., HepG2, HepaRG cells, and fresh primary human hepatocytes) at the gene expression level and analyze their metabolic cytochrome P450 capabilities. The cellular models were exposed to three different CYP450 inducers; beta-naphthoflavone (BNF), phenobarbital (PB), and rifampicin (RIF). HepG2 cells responded very weakly to the different inducers at the gene expression level, and this translated generally into low CYP450 activities in the induced cells compared with the control cells. On the contrary, HepaRG cells and the three human donors were inducible after exposure to BNF, PB, and RIF according to gene expression responses and CYP450 activities. Consequently, HepaRG cells could be used in screening as a substitute and/or in complement to primary hepatocytes for CYP induction studies. The second objective was to investigate the predictivity of the different cellular models to detect hepatotoxins (16 hepatotoxic and 5 nonhepatotoxic compounds). Specificity was 100% with the different cellular models tested. Cryopreserved human hepatocytes gave the highest sensitivity, ranging from 31% to 44% (depending on the donor), followed by lower sensitivity (13%) for HepaRG and HepG2 cells (6.3%). Overall, none of the models under study gave desirable sensitivities (80-100%). Consequently, a high metabolic capacity and CYP inducibility in cell lines does not necessarily correlate with a high sensitivity for the detection of hepatotoxic drugs. Further investigations are necessary to compare different cellular models and determine those that are best suited for the detection of hepatotoxic compounds.

Comparison of a genomic and a multiplex cell imaging approach for the detection of phospholipidosis.

Phospholipidosis (PLD) is a topic of concern in drug development because it may be associated with toxicological consequences. The aim of the study was to determine the best method to screen proprietary compounds with regard to their potential to induce PLD. Two in vitro approaches, a genomic method previously evaluated in our laboratory and a fluorescent cell based approach to detect PLD were compared using HepG2 cells. The same set of reference compounds (15 PLD inducing, 7 non-PLD inducing and 4 additional compounds) were used to compare both approaches. The same sensitivity (15/15) and similar specificity (7/7 versus, 6/7 for the genomic approach) were obtained. In addition, 11 proprietary compounds were tested in 4-day exploratory rat toxicity studies as well as in both in vitro approaches. Two of the 11 compounds induced alveolar foamy macrophages and lung vacuolization in vivo and were considered as PLD inducers. Sensitivity (2/2) and specificity (7/9) were better with the fluorescent method than with the genomic approach (1/2 and 3/9, respectively). In conclusion, compared to the genomic approach, the fluorescent method is the test of choice for screening compounds at an early stage of drug development. Indeed, the fluorescent method is more adapted to medium throughput, detects the positive reference compounds at lower (8/15) or equal (7/15) concentrations, allows multiplexing and is associated with higher sensitivity and specificity to predict lung foamy macrophages and vacuolization in vivo. Nevertheless, to confirm our conclusion, it would be necessary to compare the predictivity of both in vitro approaches by using a wide range of reference and proprietary compounds, with information on their potential to induce PLD under in vivo conditions.

p53 expression and apoptosis in melanomas of dogs and cats.

Twenty-seven melanocytic tumours from 20 dogs and four cats were examined for p53 expression and apoptosis. They included tumours that were histologically classified as benign (BM), primary malignant (PMM) and metastatic malignant melanomas (MMM). For all cases clinical follow-up was available. p53 expression was examined immunohistochemically using different monoclonal and polyclonal antibodies. Apoptosis was detected using the TUNEL technique. The tissue sections were analysed using a quantitative image analysing system. A p53 index (p53I) and an apoptotic index (AI) were determined. p53 over-expression was found infrequently in these canine and feline melanocytic tumours. Apoptosis was observed in some of the malignant tumours. In one feline case of malignant melanoma, p53 accumulation together with apoptosis was seen in three metastases but not in the primary tumour. p53I and AI were not significantly correlated with survival. These results are similar to those reported for human cutaneous melanomas.

Proliferation, DNA ploidy, p53 overexpression and nuclear DNA fragmentation in six equine melanocytic tumours.

Melanocytic tumours are a well-known clinical and pathological entity in horses, but further phenotypic characterization of these tumours is lacking. Six melanocytic tumours from five horses (two metastatic and four benign) were examined by Ki67, PCNA and p53 immunostaining, DNA nick end labelling (Tunel) and Feulgen staining. The stainings were evaluated using quantitative image analysis. The resulting parameters of growth fraction (Ki67), S-phase index (PCNA), p53 index, apoptotic index, DNA index, nuclear diameter, ploidy balance, proliferation index (Feulgen) and hyperploidy were analysed. The metastatic melanomas showed overexpression of p53 in a large portion of the cells. Apoptosis was also found in the metastatic melanomas. No differences were found in growth fraction, S-phase index (PCNA) nor in DNA configuration between the metastatic and the benign tumours. No immunohistochemical evidence of mutant p53 could be found in the tumours. In conclusion, melanocytic tumours in horses seem to have different phenotypic characteristics in comparison with melanocytic tumours in dogs, cats and humans, especially with respect to proliferative activity of the benign tumours. Therefore, markers put forward in these other species for predicting the clinical behaviour of the melanomas seem to be of no value in the horse. Moreover, quantitative DNA changes or p53 mutations do not seem to be involved in tumourogenesis in these cases.

Detection of "Candidatus Helicobacter suis" in gastric samples of pigs by PCR: comparison with other invasive diagnostic techniques.

Recently, a new 16S ribosomal DNA-based PCR assay was developed for the specific detection of "Candidatus Helicobacter suis" (former "Gastrospirillum suis") in porcine gastric samples. In the present study, this PCR assay was compared to three other invasive diagnostic methods (rapid urease test, immunohistochemistry, histologic analysis by Giemsa staining). Antral stomach samples from 200 slaughterhouse pigs from Belgium and The Netherlands were examined. Bacterial presence was determined in 77% (154 of 200) of the samples by PCR in combination with Southern blot hybridization, 56% (111 of 200) of the samples by immunohistochemistry, 61% (122 of 200) of the samples by urease testing (20 h postinoculation [p.i.]), 36% (71 of 200) of the samples by urease testing (3 h p.i.), and 33% (65 of 200) of the samples by Giemsa staining. The intrinsic specificity of the PCR assay was assessed by Southern blot analysis with an "Candidatus H. suis"-specific probe and sequencing of PCR products. Interassay sensitivity and specificity values were assessed for each test by pairwise comparisons between tests. Agreement between tests was evaluated by calculating Cohen's kappa coefficient. From that analysis, the PCR assay was considered the most reliable benchmark. Microscopic detection of immunohistochemically labeled or Giemsa-stained "Candidatus H. suis" cells in stomach sections proved to be highly specific (100%) but relatively insensitive (72 and 42%, respectively) compared to the PCR assay. A longer incubation time of the urease test improved its sensitivity considerably (74 versus 55%) but was accompanied by a loss of specificity (72 versus 93%). In conclusion, we found the "Candidatus H. suis"-specific PCR assay to be a sensitive and reliable diagnostic method for the detection of "Candidatus H. suis" in the stomachs of pigs and could prove to be a valuable tool for further epidemiological studies both for "Candidatus H. suis"- and for "Helicobacter heilmannii" type 1-related research.

Phylogenetic characterization of 'Candidatus Helicobacter bovis', a new gastric helicobacter in cattle.

Recently helicobacter-like organisms have been reported in the pyloric part of the abomasum of calves and adult cattle. Cultivation of these spiral bacteria has not been successful to date. In the present study, comparative 16S rDNA sequence analysis was used to determine the taxonomic position of these bacteria. Seven abomasal biopsies of adult cattle were sampled from different Belgian and Dutch farms. In all samples the presence of helicobacter-like organisms was demonstrated by biochemical, immunohistochemical and electron microscopical data. Bacterial 16S rDNA was amplified by PCR and sequences were determined either by direct or indirect sequence analysis. Pairwise comparisons revealed all sequences to be more than 99% homologous. Phylogenetic analysis placed the organism, corresponding to the reference sequence R2XA, within the genus Helicobacter. A diagnostic PCR assay was designed, differentiating all of the bovine 16S rDNA sequences from Helicobacter and Wolinella species. The low similarity level towards Helicobacter bilis (92.8%), its closest validly named neighbour, indicates that this novel taxon is indeed a novel Helicobacter species. An in situ hybridization procedure associated the bovine sequences to the helicobacter-like organisms in the abomasum. The name 'Candidatus Helicobacter bovis' is proposed for this new abomasal helicobacter from cattle.

PCNA and Ki67 proliferation markers as criteria for prediction of clinical behaviour of melanocytic tumours in cats and dogs.

Melanocytic tumours in domestic animals vary from benign to highly malignant. Diagnosis and prognosis are mainly based on the somewhat uncertain interpretation of cytological signs of malignancy in histological sections. The purpose of the present retrospective study was to compare the accuracy of prognosis based on the classical morphological criteria with that provided by quantitative computerized analysis of proliferation-associated antigens. Twenty-seven melanocytic tumours (20 canine and seven feline) from 24 animals (20 dogs and four cats) were examined. These comprised eight tumours histologically classified as benign melanoma (BM) (seven canine, one feline), 16 classified as primary malignant melanoma (PMM) (13 canine, three feline) and three classified as metastatic melanoma (MMM) (three feline). Tumour size, predominant histological cell type, invasive growth and clinical outcome were recorded for each case. In addition, the proliferation (phase) index and growth fraction were measured, after bleaching, by means of quantitative image analysis of tissue sections immunolabelled for proliferating cell nuclear antigen (PCNA) and MIB-1 (Ki67). Lesions classified histologically as benign or malignant, differed significantly (Copyright P<0.001) in respect of Ki67 and PCNA positivity. No such difference could be shown between PMM and MMM. High growth fraction (measured by the Ki67 positive cells) and macroscopic invasive growth were associated with decreased survival time (P=0.027 and P=0.024, respectively). Moreover, the established cytological classification also proved to be associated with the survival time (P<0.001). The results suggest that Ki67 is a potentially important prognostic factor in melanomas of the cat and dog. Other criteria, including tumour size, predominant cell type and intensity of PCNA expression, were not of significant prognostic value (P>0.05).