Cell size sensing in animal cells coordinates anabolic growth rates and cell cycle progression to maintain cell size uniformity.

Cell size uniformity in healthy tissues suggests that control mechanisms might coordinate cell growth and division. We derived a method to assay whether cellular growth rates depend on cell size, by monitoring how variance in size changes as cells grow. Our data revealed that, twice during the cell cycle, growth rates are selectively increased in small cells and reduced in large cells, ensuring cell size uniformity. This regulation was also observed directly by monitoring nuclear growth in live cells. We also detected cell-size-dependent adjustments of G1 length, which further reduce variability. Combining our assays with chemical/genetic perturbations confirmed that cells employ two strategies, adjusting both cell cycle length and growth rate, to maintain the appropriate size. Additionally, although Rb signaling is not required for these regulatory behaviors, perturbing Cdk4 activity still influences cell size, suggesting that the Cdk4 pathway may play a role in designating the cell's target size.

Classification, Source, and Effect of Environmental Pollutants and Their Biodegradation.

Any foreign chemical substance that is unusually present within an organism or is unexpectedly found in the environment at a higher concentration than the permissible limits can be termed a xenobiotic or a pollutant. Such substances include carcinogens, drugs, food additives, hydrocarbons, dioxins, polychlorinated biphenyls, pesticides or even some natural compounds. Pollutants are known for their higher persistence and pervasiveness, and along with their transformed products, they can remain in and interact with the environment for prolonged periods. In this article, the classification of such substances based on their nature, use, physical state, pathophysiological effects, and sources is discussed. The effects of pollutants on the environment, their biotransformation in terms of bioaccumulation, and the different types of remediation such as in situ and ex situ remediation, are also presented.

Discovery of Novel Anti-prion Compounds Using In Silico and In Vitro Approaches.

Prion diseases are associated with the conformational conversion of the physiological form of cellular prion protein (PrP(C)) to the pathogenic form, PrP(Sc). Compounds that inhibit this process by blocking conversion to the PrP(Sc) could provide useful anti-prion therapies. However, no suitable drugs have been identified to date. To identify novel anti-prion compounds, we developed a combined structure- and ligand-based virtual screening system in silico. Virtual screening of a 700,000-compound database, followed by cluster analysis, identified 37 compounds with strong interactions with essential hotspot PrP residues identified in a previous study of PrP(C) interaction with a known anti-prion compound (GN8). These compounds were tested in vitro using a multimer detection system, cell-based assays, and surface plasmon resonance. Some compounds effectively reduced PrP(Sc) levels and one of these compounds also showed a high binding affinity for PrP(C). These results provide a promising starting point for the development of anti-prion compounds.

Human pluripotent stem cell-derived neural constructs for predicting neural toxicity.

Human pluripotent stem cell-based in vitro models that reflect human physiology have the potential to reduce the number of drug failures in clinical trials and offer a cost-effective approach for assessing chemical safety. Here, human embryonic stem (ES) cell-derived neural progenitor cells, endothelial cells, mesenchymal stem cells, and microglia/macrophage precursors were combined on chemically defined polyethylene glycol hydrogels and cultured in serum-free medium to model cellular interactions within the developing brain. The precursors self-assembled into 3D neural constructs with diverse neuronal and glial populations, interconnected vascular networks, and ramified microglia. Replicate constructs were reproducible by RNA sequencing (RNA-Seq) and expressed neurogenesis, vasculature development, and microglia genes. Linear support vector machines were used to construct a predictive model from RNA-Seq data for 240 neural constructs treated with 34 toxic and 26 nontoxic chemicals. The predictive model was evaluated using two standard hold-out testing methods: a nearly unbiased leave-one-out cross-validation for the 60 training compounds and an unbiased blinded trial using a single hold-out set of 10 additional chemicals. The linear support vector produced an estimate for future data of 0.91 in the cross-validation experiment and correctly classified 9 of 10 chemicals in the blinded trial.

Development of a panel of high-throughput reporter-gene assays to detect genotoxicity and oxidative stress.

The lack of toxicological information on many of the compounds that humans use or are exposed to, intentionally or unintentionally, poses a big problem in risk assessment. To fill this data gap, more emphasis is given to fast in vitro screening tools that can add toxicologically relevant information regarding the mode(s) of action via which compounds can elicit adverse effects, including genotoxic effects. By use of bioassays that can monitor the activation of specific cellular signalling pathways, many compounds can be screened in a high-throughput manner. We have developed two new specific reporter-gene assays that can monitor the effects of compounds on two pathways of interest: the p53 pathway (p53 CALUX) for genotoxicity and the Nrf2 pathway (Nrf2 CALUX) for oxidative stress. To exclude non-specific effects by compounds influencing the luciferase reporter-gene expression non-specifically, a third assay was developed to monitor changes in luciferase expression by compounds in general (Cytotox CALUX). To facilitate interpretation of the data and to avoid artefacts, all three reporter-gene assays used simple and defined reporter genes and a similar cellular basis, the human U2OS cell line. The three cell lines were validated with a range of reference compounds including genotoxic and non-genotoxic agents. The sensitivity (95%) and specificity (85%) of the p53 CALUX was high, showing that the assay is able to identify various types of genotoxic compound, while avoiding the detection of false positives. The Nrf2 CALUX showed specific responses to oxidants only, enabling the identification of compounds that elicit part of their genotoxicity via oxidative stress. All reporter-gene assays can be used in a high-throughput screening format and can be supplemented with other U2OS-based reporter-gene assays that can profile nuclear receptor activity, and several other signalling pathways.

Evaluation of the national toxicology program report on carcinogens.

This study evaluates the National Toxicology Program's Report on Carcinogens program (RoCP) and compares it with the International Agency for Research on Cancer Monographs Program (IMP). We tracked agents classified in the RoCP since 1983 as known human carcinogens (A-List), or as reasonably anticipated to be human carcinogens (B-List). The first A-list included 24 agents, and twenty-four unique agents were added in the following 28years; twenty were listed by IMP as Group 1 (carcinogenic to humans) 7years before their A-list appearance. Group 1 also includes 30 or more agents eligible for, but not on, the A-list. The first B-list included 98 agents, and this increased to 185. Of these, 39 are in Group 2A (probably carcinogenic), and 122 are in Group 2B (possibly carcinogenic). Only 5% of the 204 agents ever on the B-list have been upgraded to the A-list. The RoCP is severely limited because it evaluates few agents and because its B-list does not distinguish between probable and possible human carcinogens. Further, it mislabels likely non-carcinogens as reasonably anticipated to be carcinogens. If the RoCP were terminated there would be no loss or delay of information available to scientific, public health and regulatory communities.

Statistical evaluation of mortality in long-term carcinogenicity bioassays using a Williams-type procedure.

Several doses and a control group can be compared under order restriction using the Williams procedure for normally distributed endpoints assuming variance homogeneity. Comparison of the survival functions represents a secondary endpoint in long-term in vivo bioassays of carcinogenicity. Therefore, a Williams-type procedure for the comparison of survival functions is proposed for the assumption of the Cox proportional hazards model or the general frailty Cox model to allow a joint analysis over sex and strains. Interpretation according to both statistical significance and biological relevance is possible with simultaneous confidence intervals for hazard ratios. Related survival data can be analyzed using the R packages survival, coxme, and multcomp. Together with the R packages MCPAN and nparcomp, Dunnett- or Williams-type procedures are now available for the statistical analysis of the following endpoint types in toxicology: (i) normally distributed, (ii) non-normally distributed, (iii) score (ordered categorical) data, (iv) crude proportions, (v) survival functions, and (vi) time-to-tumor data with and without cause-of-death information.

Development of a multiparametric cell-based protocol to screen and classify the hepatotoxicity potential of drugs.

Hepatotoxicity is a major reason for drug nonapprovals and withdrawals. The multiparametric analysis of xenobiotic toxicity at the single cells level using flow cytometry and cellular imaging-based approaches, such as high-content screening (HCS) technology, could play a key role in the detection of toxicity and the classification of compounds based on patterns of cellular injury. This study aimed to develop and validate a practical, reproducible, in vitro multiparametric cell-based protocol to assess those drugs that are potentially hepatotoxic to humans and to suggest their mechanisms of action. The assay was applied to HepG2 human cell line cultured in 96-well plates and exposed to 78 different compounds for 3 and 24 h at a range of concentrations (1-1000µM). After treatments, cells were simultaneously loaded with five fluorescent dyes showing optical compatibility and were then analyzed with the High-Content Screening Station Scan^R (Olympus). By using the new technology of HCS cell parameters associated with nuclear morphology, plasma membrane integrity, mitochondrial function, intracellular calcium concentration, and oxidative stress, indicative of prelethal cytotoxic effects and representative of different mechanisms of toxicity, were measured at the single cells level, which allows high-throughput screening. This strategy appears to identify early and late events in the hepatotoxic process and also suggests the mechanism(s) implicated in the toxicity of compounds to thereby classify them according to their degree of injury (no injury, low, moderate, and high injury).

A high content screening assay for identifying lysosomotropic compounds.

Lysosomes are acidic organelles that are essential for the degradation of old organelles and engulfed microbes. Furthermore, lysosomes play a key role in cell death. Lipophilic or amphiphilic compounds with a basic moiety can become protonated and trapped within lysosomes, causing lysosomal dysfunction. Therefore, high-throughput screens to detect lysosomotropism, the accumulation of compounds in lysosomes, are desirable. Hence, we developed a 96-well format, high content screening assay that measures lysosomotropism and cytotoxicity by quantitative image analysis. Forty drugs, including antidepressants, antipsychotics, antiarrhythmics and anticancer agents, were tested for their effects on lysosomotropism and cytotoxicity in H9c2 cells. The assay correctly identified drugs known to cause lysosomotropism and revealed novel information showing that the anticancer drugs, gefitinib, lapatinib, and dasatinib, caused lysosomotropism. Although structurally and pharmacologically diverse, drugs that were lysosomotropic shared certain physicochemical properties, possessing a ClogP>2 and a basic pKa between 6.5 and 11. In contrast, drugs which did not lie in this physicochemical property space were not lysosomotropic. The assay is a robust, rapid screen that can be used to identify lysosomotropic, as well as, cytotoxic compounds, and can be positioned within a screening paradigm to understand the role of lysosomotropism as a contributor to drug-induced toxicity.

ABC multidrug transporters: target for modulation of drug pharmacokinetics and drug-drug interactions.

Nine proteins of the ABC superfamily (P-glycoprotein, 7 MRPs and BCRP) are involved in multidrug transport. Being localised at the surface of endothelial or epithelial cells, they expel drugs back to the external medium (if located at the apical side [P-glycoprotein, BCRP, MRP2, MRP4 in the kidney]) or to the blood (if located at the basolateral side [MRP1, MRP3, MRP4, MRP5]), modulating thereby their absorption, distribution, and elimination. In the CNS, most transporters are oriented to expel drugs to the blood. Transporters also cooperate with Phase I/Phase II metabolism enzymes by eliminating drug metabolites. Their major features are (i) their capacity to recognize drugs belonging to unrelated pharmacological classes, and (ii) their redundancy, a single molecule being possibly substrate for different transporters. This ensures an efficient protection of the body against invasion by xenobiotics. Competition for transport is now characterized as a mechanism of interaction between co-administered drugs, one molecule limiting the transport of the other, potentially affecting bioavailability, distribution, and/or elimination. Again, this mechanism reinforces drug interactions mediated by cytochrome P450 inhibition, as many substrates of P-glycoprotein and CYP3A4 are common. Induction of the expression of genes coding for MDR transporters is another mechanism of drug interaction, which could affect all drug substrates of the up-regulated transporter. Overexpression of MDR transporters confers resistance to anticancer agents and other therapies. All together, these data justify why studying drug active transport should be part of the evaluation of new drugs, as recently recommended by the FDA.

A novel framework for predicting in vivo toxicities from in vitro data using optimal methods for dense and sparse matrix reordering and logistic regression.

In this work, we combine the strengths of mixed-integer linear optimization (MILP) and logistic regression for predicting the in vivo toxicity of chemicals using only their measured in vitro assay data. The proposed approach utilizes a biclustering method based on iterative optimal reordering (DiMaggio, P. A., McAllister, S. R., Floudas, C. A., Feng, X. J., Rabinowitz, J. D., and Rabitz, H. A. (2008). Biclustering via optimal re-ordering of data matrices in systems biology: rigorous methods and comparative studies. BMC Bioinformatics 9, 458-474.; DiMaggio, P. A., McAllister, S. R., Floudas, C. A., Feng, X. J., Rabinowitz, J. D., and Rabitz, H. A. (2010b). A network flow model for biclustering via optimal re-ordering of data matrices. J. Global. Optim. 47, 343-354.) to identify biclusters corresponding to subsets of chemicals that have similar responses over distinct subsets of the in vitro assays. The biclustering of the in vitro assays is shown to result in significant clustering based on assay target (e.g., cytochrome P450 [CYP] and nuclear receptors) and type (e.g., downregulated BioMAP and biochemical high-throughput screening protein kinase activity assays). An optimal method based on mixed-integer linear optimization for reordering sparse data matrices (DiMaggio, P. A., McAllister, S. R., Floudas, C. A., Feng, X. J., Li, G. Y., Rabinowitz, J. D., and Rabitz, H. A. (2010a). Enhancing molecular discovery using descriptor-free rearrangement clustering techniques for sparse data sets. AIChE J. 56, 405-418.; McAllister, S. R., DiMaggio, P. A., and Floudas, C. A. (2009). Mathematical modeling and efficient optimization methods for the distance-dependent rearrangement clustering problem. J. Global. Optim. 45, 111-129) is then applied to the in vivo data set (21.7% sparse) in order to cluster end points that have similar lowest effect level (LEL) values, where it is observed that the end points are effectively clustered according to (1) animal species (i.e., the chronic mouse and chronic rat end points were clearly separated) and (2) similar physiological attributes (i.e., liver- and reproductive-related end points were found to separately cluster together). As the liver and reproductive end points exhibited the largest degree of correlation, we further analyzed them using regularized logistic regression in a rank-and-drop framework to identify which subset of in vitro features could be utilized for in vivo toxicity prediction. It was observed that the in vivo end points that had similar LEL responses over the 309 chemicals (as determined by the sparse clustering results) also shared a significant subset of selected in vitro descriptors. Comparing the significant descriptors between the two different categories of end points revealed a specificity of the CYP assays for the liver end points and preferential selection of the estrogen/androgen nuclear receptors by the reproductive end points.

Prediction of the contact sensitizing potential of chemicals using analysis of gene expression changes in human THP-1 monocytes.

The aim of this study was to find differentially regulated genes in THP-1 monocytic cells exposed to sensitizers and nonsensitizers and to investigate if such genes could be reliable markers for an in vitro predictive method for the identification of skin sensitizing chemicals. Changes in expression of 35 genes in the THP-1 cell line following treatment with chemicals of different sensitizing potential (from nonsensitizers to extreme sensitizers) were assessed using real-time PCR. Verification of 13 candidate genes by testing a large number of chemicals (an additional 22 sensitizers and 8 nonsensitizers) revealed that prediction of contact sensitization potential was possible based on evaluation of changes in three genes: IL8, HMOX1 and PAIMP1. In total, changes in expression of these genes allowed correct detection of sensitization potential of 21 out of 27 (78%) test sensitizers. The gene expression levels inside potency groups varied and did not allow estimation of sensitization potency of test chemicals. Results of this study indicate that evaluation of changes in expression of proposed biomarkers in THP-1 cells could be a valuable model for preliminary screening of chemicals to discriminate an appreciable majority of sensitizers from nonsensitizers.

Inhibition of in vitro cytokine production by human peripheral blood mononuclear cells treated with xenobiotics: implications for the prediction of general toxicity and immunotoxicity.

The use of human peripheral blood mononuclear cells (PBMC) as an in vitro system to predict in vivo toxicity was investigated. For 58 chemicals, the effect on cytokine secretion (IL-5, IFNgamma and TNFalpha) by phytohaemagglutinin-activated PBMC was measured, IC50 values were calculated and correlations of these endpoints with human LC50 values were determined. The best result was obtained with IFNgamma as an endpoint for which the calculated R(2) value was 0.58 which is comparable with the R(2) values for the classical neutral red uptake (NRU) assays using murine 3T3 cells and normal human keratinocytes (R(2)=0.56 and 0.59, respectively). When for each chemical the lowest IC50 value of the three endpoints was correlated with LC50 the calculated R(2) increased slightly to 0.63. A specific strength of our test is that it corrects several outliers (diazepam, digoxin, malathion and verapamil hydrochloride) which do not fit in the linear regression analysis for IC50 values obtained with the classical 3T3 NRU assay. Furthermore, 2,4-dichlorophenoxyacetic acid, cyclosporine A and pentachlorophenol had a 10 times lower IC50 value than the estimated human LC50 value and were identified as immunotoxic alerts. In conclusion, new endpoints investigated in this study contribute to the prediction of immunotoxic effects and correct outliers of classical cytotoxicity assays.

Simian retroviruses: infection and disease--implications for immunotoxicology research in primates.

Non-human primates have assumed an important role in preclinical safety assessment studies, particularly in the evaluation of biopharmaceutical and immunomodulatory therapies. Naturally occurring simian retrovirus infections may adversely affect the suitability of primates for use in such studies. Various species of non-human primates are the natural hosts for six exogenous retroviruses, representing five genera within the family Retroviridae. Retroviruses establish persistent infections with a broad spectrum of pathogenic potential, ranging from nonpathogenic to highly pathogenic, depending on the variety of the host, virus, and environmental factors. In the context of immunotoxicology, in which the research objective is to specifically evaluate the effect of drugs or biologics on the immune system, the immune modulatory effects of simian retroviruses, which may be subtle or profound, may introduce significant confounding into the studies of immunotoxic effects utilizing non-human primates. Latent or subclinical retrovirus infections are common and research-related procedures may lead to virus reactivation or overt disease. Adverse effects of undetected retrovirus infections on preclinical research include the loss of experimental subjects (and potentially of statistical power) due to increased morbidity and mortality, virus-induced clinical abnormalities, histologic lesions, alteration of physiologic parameters and biologic responses, and interference with in vitro assays and/or cytolytic destruction of primary cell cultures. The aim of this review is to provide an overview of the key biological, clinical, and pathological features of several important simian retroviruses, with emphasis on viruses infecting macaques and other primate species commonly used in preclinical research, and a discussion of the implications of these infections for immunotoxicology and other preclinical research in primates. Adequate pre-study retrovirus screening is essential to exclude retrovirus-infected primates from research protocols.

New strategy for alerting central nervous system toxicity: Integration of blood-brain barrier toxicity and permeability in neurotoxicity assessment.

The combination of an in vitro BBB model (4d/24w) with a neuronal cell line (SH-SY5Y) provides a convenient approach to explore the importance of BBB permeability in neurotoxicity assessment of compounds. The toxicity of 16 compounds on SH-SY5Y cells was evaluated after 24h incubation with each compound and compared to their toxicity on SH-SY5Y after passage through the BBB model. Nine out of 16 compounds were found toxic after direct exposure at 100muM while only three still induced toxicity on SH-SY5Y cells after BBB transport. The BBB permeability values of each compound revealed that in the case of compounds that did not induce toxicity, the amount that crossed the BBB was not enough to exert a toxic effect on the neuronal cells. Since disrupting the BBB may also cause unwanted effect on brain cells, the BBB toxicity of these compounds have been assessed. Our results prompted the importance of BBB permeability assessment in neurotoxicity evaluation, as it allows a better estimation of the actual concentration at the target site.

Identification of tumor promotion marker genes for predicting tumor promoting potential of chemicals in BALB/c 3T3 cells.

Tumor promoters can cause development of tumors in initiated cells and the majority of them are non-genotoxic carcinogens. The detection of tumor promoters is important for the prevention of cancer. The in vitro two-stage transformation assay, using BALB/c 3T3 cells, is a useful system, and benefits from a convenient protocol and high predictability of mammalian carcinogenicity. But these assays are time-consuming and often require expertise for microscopic observation. To construct an in vitro tumor promoting activity test system, we performed large-scale gene expression analyses, using DNA microarrays, of BALB/c 3T3 cells following treatment with nine chemicals that are known to induce tumor promotion: TPA, zinc chloride, sodium orthovanadate, okadaic acid, insulin, lithocolic acid, phenobarbital sodium, sodium saccharide, sodium arsenite. As a result of DNA microarray and real time PCR analyses, 22 marker genes were identified. These consisted of genes related to cell cycle, regulation of transcription, anti-apoptosis, and positive regulation of cell proliferation. There was a correlation between these 22 marker genes and the cell transformation assay results in BALB/c 3T3 cells. These results suggest that this tumor promoting activity test system, based on 22 marker genes, can become a valuable tool for screening potential tumor promoters.

Selection of cytotoxicity markers for the screening of new chemical entities in a pharmaceutical context: a preliminary study using a multiplexing approach.

The present study was undertaken to validate a battery of cytotoxicity assays performed in a multiplex format to screen pharmaceutical compounds at an early stage of drug development. Two experiments were performed on HepG2 cells and the parameters were measured in 96-well plates. Biological and technical triplicates were performed to evaluate the reproducibility of the assay. In the first experiment, HepG2 cells were exposed to tamoxifen, staurosporine, phenobarbital and triton X-100 for 2 and 24h. The following nine cytotoxicity parameters were analyzed, cell viability, lactate dehydrogenase (LDH), adenosine triphosphate (ATP), caspase-3/7, aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and alpha-glutathione-S-transferase (alpha-GST). In the second experiment, HepG2 cells were exposed to doxorubicin, t-butyl hydroperoxide, ferrous sulfate and sulfamoxole for 2 and 24h. Based on the results of the first experiment, six cytotoxicity parameters were selected for further evaluation (cell viability, ATP, LDH, caspase, AST and GLDH). ALT (activity always below detection limit), ALP (no response to drug treatment) and alpha-GST (too labor intensive and not possible to multiplex) were eliminated. The analysis of the data revealed that the reproducibility of the assays was accurate according to principal component analysis. Our data also clearly indicated that the potential of this battery of selected assays measured in a multiplex format not only made it possible to rank and select the most promising drug candidates based on their cytotoxic potential, but also to gather information that may help to understand some of the toxic events occurring in the cells.

A stable and sensitive testing system for potential carcinogens based on DNA damage-induced gene expression in human HepG2 cell.

In order to analyze potential carcinogenic and genotoxic responses caused by exposure to pollutants existing in environment, a screening method has been established in our laboratory that uses a stably transfected HepG2 cell lines containing gadd153 promoter regions which drive a luciferase reporter gene. Activation of the exogenous gadd153 promoter was quantified using the luciferase activity following drug exposure. Twenty four agents were used to evaluate this screening assay. We selected the agents, ranging from DNA alkylating agents, oxidative agent, radiation, DNA cross-linking agent, nongenotoxic carcinogens, precarcinogenic agents, which included cadmium chloride, chromium trichloride, mercuric chloride, lead nitrate, dichloro-diphenyl-trichloroethane, deltamethrin, biphenylamine, 2-aminofluorene, benzo[a]pyrene, 2,3,7,8,-tetracblorodibenzo-p-dioxin, diethyl-stilbestrol, carbon tetrachloride, mitomycin C, hydroxycamptothecin, UV, sodium fluoride, acrylamide, hydrogen peroxide. In addition, two complex genotoxic agents (water samples) existing in the environment were selected. The results showed that all 20 tested known carcinogenic and genotoxic agents were able to induce gadd153-Luc expression at a sublethal dose. In contrast, four tested non-carcinogens, included 4-acetylaminofluorene, pyrene, benzylpenicillin sodium and vitamin C, were unable to induce gadd153-Luc expression. In conclusion, this reporter system can facilitate in vitro screening for potential carcinogens. Therefore, the gadd153-Luc test system we have developed appears to be a useful and complementary system to existing genotoxic and mutagenic tests.

Application of a high-content multiparameter cytotoxicity assay to prioritize compounds based on toxicity potential in humans.

Prioritization of compounds based on human hepatotoxicity potential is currently a key unmet need in drug discovery, as it can become a major problem for several lead compounds in later stages of the drug discovery pipeline. The authors report the validation and implementation of a high-content multiparametric cytotoxicity assay based on simultaneous measurement of 8 key cell health indicators associated with nuclear morphology, plasma membrane integrity, mitochondrial function, and cell proliferation. Compounds are prioritized by (a) computing an in vitro safety margin using the minimum cytotoxic concentration (IC(20)) across all 8 indicators and cell-based efficacy data and (b) using the minimal cytotoxic concentration alone to take into account concentration of drug in tissues. Feasibility data using selected compounds, including quinolone antibiotics, thiazolidinediones, and statins, suggest the viability of this approach. To increase overall throughput of compound prioritization, the authors have identified the higher throughput, plate reader-based CyQUANT assay that is similar to the high-content screening (HCS) assay in sensitivity of measuring inhibition of cell proliferation. It is expected that the phenotypic output from the multiparametric HCS assay in combination with other highly sensitive approaches, such as microarray-based expression analysis of toxic signatures, will contribute to a better understanding and predictivity of human hepatotoxicity potential.

Evaluation of the rodent Hershberger bioassay: testing of coded chemicals and supplementary molecular-biological and biochemical investigations.

Under the auspices of the Organization for Economic Cooperation and Development (OECD) the Hershberger assay is being validated as an in vivo screen for compounds with (anti)androgenic potential. We participated in the final activity, the testing of coded chemicals. Test compounds included trenbolone (TREN; 1.5, 40 mg/kg), testosterone propionate (TP; 0.4 mg/kg), flutamide (FLUT; 3mg/kg), linuron (LIN; 10, 100mg/kg), 1,1-bis-(4-chlorophenyl)-2,2-dichloroethylene (p,p'-DDE; 16, 160 mg/kg), and two negative reference substances, i.e., compounds not considered to affect androgen-sensitive tissue weights (ASTWs) in the Hershberger assay, namely 4-nonylphenol (NP; 160 mg/kg) and 2,4-dinitrophenol (DNP; 10mg/kg); TREN, LIN, p,p'-DDE, NP, and DNP being used under code. Compounds were administered for 10 days by oral intubation or subcutaneous injection (TP). Additional investigations not mandatorily requested by OECD included organ gravimetry of the liver, gene expression analysis in prostate using quantitative RT PCR for prostate specific binding protein polypeptide C3 (PBPC3) and ornithine decarboxylase 1 (ODC1) and determination of testosterone metabolizing and phase II conjugating enzymes in the liver. After submission of all study reports to OECD by participants uncoding revealed the following results: (A) When assessing androgenic potential in castrated rats, administration of TREN increased the weights of ventral prostate (VP), seminal vesicles (SV), glans penis, levator ani and bulbocavernosus muscles, and Cowper's glands at the high dose. A similar or stronger (VP, SV) increase of ASTWs was observed for TP; NP and DNP were ineffective. TREN dose-dependently increased gene expression of ODC1 and PBPC3, TP induced expression of these genes even more strongly (almost) to the level of untreated intact animals, whereas NP and DNP were inactive. Liver enzyme activities depending on physiological androgen levels were lower in castrated than in intact rats and could not be restored by androgen treatment. (B) When assessing antiandrogenic potential in TP-supplemented castrated rats, administration of LIN and p,p'-DDE decreased ASTWs only at the high dose. FLUT even more effectively decreased ASTWs, NP and DNP were again without effect. Decreases in androgen-responsive gene expression in the prostate corresponding to the organ weight changes were only observed for p,p'-DDE (high dose) and flutamide (PBPC3 only). p,p'-DDE dose-dependently induced liver weights and most liver enzyme activities including androgen-dependent ones. Our study accurately reproduced ASTW changes obtained in previous studies also under code suggesting that the Hershberger assay is a robust tool to screen for an (anti)androgenic potential. Assessment of ODC1 and PBPC3 gene expression in prostate, however, may only represent a sensitive tool for the detection of an androgenic potential. Finally, p,p'-DDE may affect ASTWs by several mechanisms including enhanced testosterone metabolism.