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In the context of a larger testing programme that aimed at assessing the skin sensitisation potential of functional polysiloxanes and silanes, this investigation complements the available in vitro and in vivo data with data in the SENS-IS assay, a human in vitro 3D skin-based model. The SENS-IS assay allowed testing of all functional polysiloxanes and silanes without any solubility issues or limitations related to the multiconstituent nature of the commercial grade test substances. It appeared to encompass skin metabolism, a factor which we considered important for the skin sensitisation hazard assessment particularly of aminofunctionalised siloxanes and silanes. These three technical aspects posed significant challenges in the first part of the in vitro programme with the OECD-validated in vitro assays. The SENS-IS assay delivered promising results for this group of substances. On its own, it was the best performing model, as it did not pose any technical issues with the assay and it matched all in vivo outcomes. Considering its performance and avoidance of any limitations due to lack of solubility or chemical composition aspects, we concluded that the SENS-IS assay to be a suitable starting point for an integrated testing strategy for skin sensitisation for the group of functional polysiloxanes and silanes.

The skin sensitization potential of chemicals has traditionally been evaluated in vivo according to OECD testing guidelines in guinea pigs or the mouse local lymph node assay. There has lately been a great emphasis on establishing in vitro test methods reflecting the key biological events in the adverse outcome pathway (AOP) for skin sensitization as published by the OECD. Against this background, a group of 8 polysiloxanes and silanes, seven of them aminofunctionalised, for which in vivo data were already available, has been tested in vitro in the direct peptide reactivity assay (DPRA), the KeratinoSens™ and the human cell line activation test (h-CLAT) and in the modified myeloid U937 skin sensitization test (mMUSST) as far as technically feasible. The main objective of the programme was to determine the utility of these systems for this heterogeneous group of silicone-based substances, recognizing that some substances are outside the assays applicability domains. The presented data provided some interesting mechanistical insights into the performance of these assays for functionalised siloxanes and silanes. The data also allow for a preliminary evaluation of proposed integrated testing strategies (ITS) to determine the skin sensitization potential of chemicals which were not considered in the training sets of the respective ITS.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of a diverse range of chemicals, including the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Although no endogenous physiological ligand for the AhR has yet been described, numerous studies support the existence of such a ligand(s). Here we have examined the ability of prostaglandins and related chemicals to activate the AhR signaling system. Using two AhR-based bioassay systems we report that relatively high concentrations of several prostaglandins (namely, PGB3, PGD3, PGF3α, PGG2, PGH1, and PGH2) can not only stimulate AhR transformation and DNA binding in vitro, but also induce AhR-dependent reporter gene expression in mouse hepatoma cells in culture. PGG2 also induced AhR-dependent reporter gene expression to a level three-to four fold greater than that observed with a maximal inducing dose of TCDD. Sucrose gradient ligand binding analysis revealed that PGG2 could competitively displace [3H]TCDD from the AhR. Overall, our results demonstrate that selected prostaglandins are weak agonists for the AhR and they represent a structurally distinct and novel class of activator of the AhR signal transduction pathway. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:187–196, 2001

The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biologic and toxicologic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. Here we utilized two AhR-dependent bioassay systems as screening tools to identify novel AhR agonists and to detect the presence of AhR agonists in sample extracts. These assays measure the ability of a chemical to activate AhR DNA binding in vitro (GRAB bioassay) or AhR-dependent (luciferase) gene expression in cultured cells (CALUX bioassay). Known AhR agonists (halogenated and nonhalogenated aromatic hydrocarbons) were positive in both assays, whereas the AhR antagonist alpha-naphthoflavone exhibited agonist activity only in the GRAB assay. In vitro GRAB analysis has identified several imidazoline receptor ligands and beta-carbolines as AhR agonists and also revealed the presence of AhR agonist activity in crude DMSO extracts of commercial newspapers. In contrast to their positive activity in the GRAB assay, the majority of these chemicals/extracts were only weakly active or inactive in the cell-based CALUX assay. Our results not only reveal that the ability of a chemical to activate the AhR in vitro does not necessarily correlate with its ability to induce gene expression in intact cells, but the high level of false positives obtained with the GRAB assay clearly demonstrates its inability to accurately identify AhR agonists or agonist activity. Screening of unknown chemicals, chemical classes, and samples for AhR agonist activity will require the use of intact cell bioassays.

Advances in high throughput and high content (HT/HC) methods such as those used in the fields of toxicogenomics, bioinformatics, and computational toxicology have the potential to improve both the efficiency and effectiveness of toxicity evaluations and risk assessments. However, prior to use, scientific confidence in these methods should be formally established. Traditional validation approaches that define relevance, reliability, sensitivity and specificity may not be readily applicable. HT/HC methods are not exact replacements for in vivo testing, and although run individually, these assays are likely to be used as a group or battery for decision making and use robotics, which may be unique in each laboratory setting. Building on the frameworks developed in the 2010 Institute of Medicine Report on Biomarkers and the OECD 2007 Report on (Q)SAR Validation, we present constructs that can be adapted to address the validation challenges of HT/HC methods. These are flexible, transparent, and require explicit specification of context and purpose of use such that scientific confidence (validation) can be defined to meet different regulatory applications. Using these constructs, we discuss how anchoring the assays and their prediction models to Adverse Outcome Pathways (AOPs) could facilitate the interpretation of results and support scientifically defensible fit-for-purpose applications.

In order to gain better insight into factors (strain, gender, and diet) influencing background variability in kidney gene expression, we examined the transcriptomes of male and female Crl: CD(SD)IGSBR (Sprague-Dawley [SD]) and CDF(Fischer 344)/ CrlBR rats maintained for 19 days on three different diets (ad libitum [AL], diet restriction-75% of AL, and casein-based phytoestrogen-free diet). Kidney RNA was analyzed using Agilent Rat oligo microarrays (approximately 20,000 genes). Principal component analysis demonstrated that strain and gender have the most impact on the variability in gene expression, while diet had a lesser effect. The majority of the affected genes differed by a magnitude of four-fold or less between strains/gender, with some previously known to be sex-hormone regulated (SLC22A7 and SLC21A1). One gene of particular interest was ornithine decarboxylase, a significant marker of cell proliferation and tumor promotion, which was expressed at an 18-fold greater level in SD rats. Further analysis revealed that the difference in expression was due to the use of an alternate polyadenylation signal resulting in the production of two different sizes of transcripts. These results demonstrate that gender and strain have significant influence on gene expression which could be a confounder when comparing results, especially when it involves predictive fingerprint/patterns.

Tumorigenic mechanisms due to chemical exposure are broadly classified as either genotoxic or nongenotoxic. Genotoxic mechanisms are generally well defined; however nongenotoxic modes of tumorgenesis are less straightforward. This study was undertaken to help elucidate dose-response changes in gene expression (transcriptome) in the liver of rats in response to administration of known genotoxic or nongenotoxic liver carcinogens. Male Big Blue Fischer 344 rats were treated for 28-days with 0, 0.1, 0.3, 1.0, or 3.0 mg/kg/day of the genotoxin 2-acetylaminofluorene (AAF) or 0, 10, 30, 60, or 100 mg/kg/day of the nongenotoxin phenobarbital (PB). Transcriptome analysis was performed using the relatively focused Clontech Rat Toxicology II microarray (465 genes) and hybridized with 32P-labeled cDNA target. The analysis indicated that after 28 days of treatment, AAF altered the expression of 14 genes (9 up-and 5 down-regulated) and PB altered the expression of 18 genes (10 up- and 8 down-regulated). Of the limited genes whose expression was altered by AAF and PB, four were altered in common, two up-regulated, and two down-regulated. Several of the genes that show modulation of transcriptional activity following AAF and PB treatment display an atypical dose-response relationship such that the expression at the higher doses tended to be similar to that of control. This high-dose effect could potentially be caused by adaptation, toxicity, or tissue remodeling. These results suggest that the transcriptional response of the cells to higher doses of a toxic agent is likely to be different from that of a low-dose exposure.

Treatment of cells with genotoxic chemicals is expected to set into motion a series of events including gene expression changes to cope with the damage. We have investigated gene expression changes in L5178Y TK(+/-) mouse lymphoma cells in culture following treatment with methyl methanesulfonate (MMS), a direct acting genotoxin, and sodium chloride (NaCl), which induces mutations in these cells through indirect mechanisms at high concentrations. The mouse lymphoma cells were treated for 4 or 24 h and the cells were harvested for RNA isolation at the end of the treatment. Analysis of the transcriptome was performed using Clontech Mouse 1.2K cDNA microarrays (1185 genes) and hybridized using 32P-labeled cDNA. The microwell methodology was used to quantify the mutagenic response. Of the genes examined, MMS altered the expression (1.5-fold or more) of only five (four at 4 h and one after 24 h treatment). NaCl altered two genes after 4 h treatment, but after 24 h it altered 19 genes (13 down- and six up-regulated). Both compounds altered the expression of several genes associated with apoptosis and NaCl altered genes involved in DNA damage/response and GTP-related proteins. This, along with other data, indicates that the widely used L5178Y TK(+/-) mouse lymphoma cells in culture are relatively recalcitrant in terms of modulating gene expression to deal with genotoxic insult.

It is believed that some aspects of genotoxicity are associated with changes in the transcription levels of certain genes, especially those involved in DNA repair and cell cycle control. Additionally, it is hypothesized that chemicals sharing a common mode of genotoxicity should exhibit similar changes in gene expression. We have evaluated these hypotheses by analyzing transcriptome profiles of mouse lymphoma L5178Y/TK(+/-) cells treated with bleomycin and hydrogen peroxide, two mutagens that produce genotoxicity by generating reactive free radicals. The cells were treated for 4 hr and RNA was isolated at the end of the treatment and after a 20 hr recovery. Transcriptome analyses were performed using the Clontech Mouse 1.2K cDNA microarray (1,185 genes) and hybridization with a (32)[P]-labeled probe. Of the genes examined, each mutagen altered the expression (1.5-fold or greater) of only two genes after the 4 hr treatment. In cells allowed to recover for 20 hr after treatment, bleomycin and hydrogen peroxide altered the expression of 8 and 5 genes, respectively. Many of the altered genes have some association with apoptosis. Of these genes, three (the genes encoding granzyme A, integrin beta 7, and 45 kDa calcium-binding protein precursor) were in common between chemical treatments. The expression of DNA repair and cell cycle controlling genes present on the array was not affected by the treatments. These results show that bleomycin and hydrogen peroxide both have unique and commonly regulated genes that have the potential to serve as biomarkers of exposure to agents causing DNA damage by free radical mechanisms.

The aryl hydrocarbon nuclear translocator (Arnt) is a basic helix-loop-helix (per/Arnt/Ahr/sim) PAS-containing protein that can heterodimerize with the aryl hydrocarbon receptor (AhR), the hypoxia-inducible factor-1 alpha, and other PAS-containing proteins to form transcriptionally active complexes. To identify the genes whose expression is modulated by Arnt, we used the technique of differential display to compare the expression of genes in wild-type and Arnt-defective (BPRc1) mouse hepatoma (Hepa1c1c7) cells. Here we report two gene products whose expression was reduced in BPRc1 cells (a WW domain-binding, protein-like factor and one unknown gene product) when compared to wild-type cells, and two that were elevated (Steel factor and a serpin-like protein). Comparison of the relative expression of these gene products between two independently-derived, Arnt-defective cell lines, as well as in BPRc1 cells in which Arnt expression was restored by a stably integrated Arnt-expression plasmid, revealed that each gene was expressed in an Arnt-independent manner. Our results clearly demonstrate that gene expression in the variant cell clones is distinctly different from that of the parental wild-type Hepa1c1c7 cells from which they were derived and involves genes in addition to, and unrelated to, that of Arnt. The identification of these differentially expressed gene products suggests that caution should be exercised when using these variant cell lines to confirm the role of the AhR/Arnt-signaling pathway in a given cellular response.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of a variety of hydrophobic natural and synthetic chemicals, including the environmental contaminant 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). A variety of indole-containing chemicals, such as indole-3-carbinol, indolo[3, 2-b]carbazole, and UV photoproducts of tryptophan (TRP), have previously been identified as ligands for AhR. Here we have examined the ability of endogenous metabolites of tryptophan (TRP) to bind to and activate AhR in vitro and in cells in culture. Although hydroxylated TRP metabolites were inactive, two metabolites, namely tryptamine (TA) and indole acetic acid (IAA), were shown to be AhR agonists. Not only do TA and IAA bind competitively to AhR, but they also can stimulate AhR transformation and DNA binding and induce expression of an AhR-dependent reporter gene in cells. In addition to being an AhR ligand, TA is also a competitive substrate for cytochrome P4501A1, a well-characterized AhR- and TCDD-inducible gene product. Although these compounds are relatively weak ligands, compared to TCDD, they represent some of the first endogenous hydrophilic AhR agonists identified to date.