Validation of the 3D reconstructed human skin micronucleus (RSMN) assay: an animal-free alternative for following-up positive results from standard in vitro genotoxicity assays.

In vitro test batteries have become the standard approach to determine the genotoxic potential of substances of interest across industry sectors. While useful for hazard identification, standard in vitro genotoxicity assays in 2D cell cultures have limited capability to predict in vivo outcomes and may trigger unnecessary follow-up animal studies or the loss of promising substances where animal tests are prohibited or not desired. To address this problem, a team of regulatory, academia and industry scientists was established to develop and validate 3D in vitro human skin-based genotoxicity assays for use in testing substances with primarily topical exposure. Validation of the reconstructed human skin micronucleus (RSMN) assay in MatTek Epi-200™ skin models involved testing 43 coded chemicals selected by independent experts, in four US/European laboratories. The results were analysed by an independent statistician according to predefined criteria. The RSMN assay showed a reproducibly low background micronucleus frequency and exhibited sufficient capacity to metabolise pro-mutagens. The overall RSMN accuracy when compared to in vivo genotoxicity outcomes was 80%, with a sensitivity of 75% and a specificity of 84%, and the between- and within-laboratory reproducibility was 77 and 84%, respectively. A protocol involving a 72-h exposure showed increased sensitivity in detecting true positive chemicals compared to a 48-h exposure. An analysis of a test strategy using the RSMN assay as a follow-up test for substances positive in standard in vitro clastogenicity/aneugenicity assays and a reconstructed skin Comet assay for substances with positive results in standard gene mutation assays results in a sensitivity of 89%. Based on these results, the RSMN assay is considered sufficiently validated to establish it as a 'tier 2' assay for dermally exposed compounds and was recently accepted into the OECD's test guideline development program.

Effect of 2-acetylaminofluorene and its genotoxic metabolites on DNA adduct formation and DNA damage in 3D reconstructed human skin tissue models.

In vitro genotoxicity assays utilising human skin models are becoming important tools for the safety assessment of chemicals whose primary exposure is via the dermal route. In order to explore metabolic competency and inducibility of CYP450 activating enzymes, 3D reconstructed human skin tissues were topically treated with 2-acetylaminofluorene (2-AAF) and its genotoxic metabolites, N-hydroxy-2-acetylaminofluorene (N-OH-2-AAF) and N-hydroxy-2-aminofluorene (N-OH-2-AF), which primarily cause DNA damage by forming DNA adducts. 2-AAF did not increase DNA damage measured in the reconstructed skin micronucleus (RSMN) assay when administered in multiple applications at 24 h intervals but was detected in the skin comet assay in the presence of the DNA polymerase inhibitor aphidicolin (APC). Similarly, no increase was found with N-OH-2-AAF in the RSMN assay after multiple treatments whereas a single 3 h exposure to N-OH-2-AAF caused a large dose-related increase in the skin comet assay. A significant increase in the RSMN assay was only obtained with the highly reactive N-OH-2-AF metabolite after multiple treatments over 72 h, whereas N-OH-2-AF caused a strong increase after a single 3 h exposure in the skin comet assay. In support of these results, DNA adduct formation, measured by the 32P-postlabelling assay, was examined. Adduct levels after 2-AAF treatment for 3 h were minimal but increased >10-fold after multiple exposures over 48 h, suggesting that enzyme(s) that metabolise 2-AAF are induced in the skin models. As expected, a single 3 h exposure to N-OH-2-AAF and N-OH-2-AF resulted in adduct levels that were at least 10-fold greater than those after multiple exposures to 2-AAF despite ~100-fold lower tested concentrations. Our results demonstrate that DNA damage caused by 2-AAF metabolites is more efficiently detected in the skin comet assay than the RSMN assay and after multiple exposures and enzyme induction, 2-AAF-induced DNA damage can be detected in the APC-modified comet assay.

Validation of the 3D reconstructed human skin Comet assay, an animal-free alternative for following-up positive results from standard in vitro genotoxicity assays.

As part of the safety assessment process, all industrial sectors employ genotoxicity test batteries, starting with well-established in vitro assays. However, these batteries have limited predictive capacity for the in vivo situation, which may result in unnecessary follow-up in vivo testing or the loss of promising substances where animal tests are prohibited or not desired. To address this, a project involving regulators, academia and industry was established to develop and validate in vitro human skin-based genotoxicity assays for topically exposed substances, such as cosmetics ingredients. Here, we describe the validation of the 3D reconstructed skin (RS) Comet assay. In this multicenter study, chemicals were applied topically three times to the skin over 48 h. Isolated keratinocytes and fibroblasts were transferred to slides before electrophoresis and the resulting comet formation was recorded as % tail DNA. Before decoding, results of the validation exercise for 32 substances were evaluated by an independent statistician. There was a high predictive capacity of this assay when compared to in vivo outcomes, with a sensitivity of 77 (80)%, a specificity of 88 (97)% and an overall accuracy of 83 (92)%. The numbers reflect the calls of the performing laboratories in the coded phase, whereas those in parenthesis reflect calls according to the agreed evaluation criteria. Intra- and inter-laboratory reproducibility was also very good, with a concordance of 93 and 88%, respectively. These results generated with the Phenion® Full-Thickness skin model demonstrate its suitability for this assay, with reproducibly low background DNA damage and sufficient metabolic capacity to activate pro-mutagens. The validation outcome supports the use of the RS Comet assay to follow up positive results from standard in vitro genotoxicity assays when the expected route of exposure is dermal. Based on the available data, the assay was accepted recently into the OECD test guideline development program.

Validation of the 3D Skin Comet assay using full thickness skin models: Transferability and reproducibility.

Recently revised OECD Testing Guidelines highlight the importance of considering the first site-of-contact when investigating the genotoxic hazard. Thus far, only in vivo approaches are available to address the dermal route of exposure. The 3D Skin Comet and Reconstructed Skin Micronucleus (RSMN) assays intend to close this gap in the in vitro genotoxicity toolbox by investigating DNA damage after topical application. This represents the most relevant route of exposure for a variety of compounds found in household products, cosmetics, and industrial chemicals. The comet assay methodology is able to detect both chromosomal damage and DNA lesions that may give rise to gene mutations, thereby complementing the RSMN which detects only chromosomal damage. Here, the comet assay was adapted to two reconstructed full thickness human skin models: the EpiDerm™- and Phenion® Full-Thickness Skin Models. First, tissue-specific protocols for the isolation of single cells and the general comet assay were transferred to European and US-American laboratories. After establishment of the assay, the protocol was then further optimized with appropriate cytotoxicity measurements and the use of aphidicolin, a DNA repair inhibitor, to improve the assay's sensitivity. In the first phase of an ongoing validation study eight chemicals were tested in three laboratories each using the Phenion® Full-Thickness Skin Model, informing several validation modules. Ultimately, the 3D Skin Comet assay demonstrated a high predictive capacity and good intra- and inter-laboratory reproducibility with four laboratories reaching a 100% predictivity and the fifth yielding 70%. The data are intended to demonstrate the use of the 3D Skin Comet assay as a new in vitro tool for following up on positive findings from the standard in vitro genotoxicity test battery for dermally applied chemicals, ultimately helping to drive the regulatory acceptance of the assay. To expand the database, the validation will continue by testing an additional 22 chemicals.

Weak silica nanomaterial-induced genotoxicity can be explained by indirect DNA damage as shown by the OGG1-modified comet assay and genomic analysis.

In a previous study, 15-nm silica nanoparticles (NPs) caused small increases in DNA damage in liver as measured in the in vivo comet and micronucleus assays after intravenous administration to rats at their maximum tolerated dose, a worst-case exposure scenario. Histopathological examination supported a particle-induced, tissue damage-mediated inflammatory response. This study used a targeted approach to provide insight into the mode of action (MoA) by examining transcriptional regulation of genes in liver in a time and dose-dependent manner at 1, 2, 4, 8 and 24 h after intravenous administration of 15-nm silica NPs. DNA damage was assessed using the standard comet assay and hOGG1 glycosylase-modified comet assay that also measures oxidative DNA damage. Potassium bromate, an IARC Class 2B carcinogen that specifically operates via an oxidative stress MoA, was used as a positive control for the hOGG1 comet assay and gave a strong signal in its main target organ, the kidney, while showing less activity in liver. Treatment of rats with silica NPs at 50 mg/kg body weight (bw) caused small, statistically insignificant increases in DNA damage in liver measured by the standard comet assay, while a statistically significant increase was observed at 4 h with the hOGG1 comet assay, consistent with a MoA involving reactive oxygen species. Histopathology showed liver damage and neutrophil involvement while genomic analysis and response pattern of key genes involved in inflammation and oxidative stress supported a tissue damage-mediated inflammatory response involving the complement system for removing/phagocytising damaged cells. No changes were observed for histopathology or gene array for the low-dose (5 mg/kg bw) silica NPs. The results of this study confirm our hypothesis that the weak DNA damage observed by silica NPs occurs secondary to inflammation/immune response, indicating that a threshold can be applied in the risk assessment of these materials.

Comet assay in reconstructed 3D human epidermal skin models--investigation of intra- and inter-laboratory reproducibility with coded chemicals.

Reconstructed 3D human epidermal skin models are being used increasingly for safety testing of chemicals. Based on EpiDerm™ tissues, an assay was developed in which the tissues were topically exposed to test chemicals for 3h followed by cell isolation and assessment of DNA damage using the comet assay. Inter-laboratory reproducibility of the 3D skin comet assay was initially demonstrated using two model genotoxic carcinogens, methyl methane sulfonate (MMS) and 4-nitroquinoline-n-oxide, and the results showed good concordance among three different laboratories and with in vivo data. In Phase 2 of the project, intra- and inter-laboratory reproducibility was investigated with five coded compounds with different genotoxicity liability tested at three different laboratories. For the genotoxic carcinogens MMS and N-ethyl-N-nitrosourea, all laboratories reported a dose-related and statistically significant increase (P < 0.05) in DNA damage in every experiment. For the genotoxic carcinogen, 2,4-diaminotoluene, the overall result from all laboratories showed a smaller, but significant genotoxic response (P < 0.05). For cyclohexanone (CHN) (non-genotoxic in vitro and in vivo, and non-carcinogenic), an increase compared to the solvent control acetone was observed only in one laboratory. However, the response was not dose related and CHN was judged negative overall, as was p-nitrophenol (p-NP) (genotoxic in vitro but not in vivo and non-carcinogenic), which was the only compound showing clear cytotoxic effects. For p-NP, significant DNA damage generally occurred only at doses that were substantially cytotoxic (>30% cell loss), and the overall response was comparable in all laboratories despite some differences in doses tested. The results of the collaborative study for the coded compounds were generally reproducible among the laboratories involved and intra-laboratory reproducibility was also good. These data indicate that the comet assay in EpiDerm™ skin models is a promising model for the safety assessment of compounds with a dermal route of exposure.

Silica nanoparticles administered at the maximum tolerated dose induce genotoxic effects through an inflammatory reaction while gold nanoparticles do not.

While the collection of genotoxicity data and insights into potential mechanisms of action for nano-sized particulate materials (NPs) are steadily increasing, there is great uncertainty whether current standard assays are suitable to appropriately characterize potential risks. We investigated the effects of NPs in an in vivo Comet/micronucleus (MN) combination assay and in an in vitro MN assay performed with human blood. We also incorporated additional endpoints into the in vivo study in an effort to delineate primary from secondary mechanisms. Amorphous silica NPs (15 and 55 nm) were chosen for their known reactivity, while gold nano/microparticles (2, 20, and 200 nm) were selected for their wide size range and lower reactivity. DNA damage in liver, lung and blood cells and micronuclei in circulating reticulocytes were measured after 3 consecutive intravenous injections to male Wistar rats at 48, 24 and 4h before sacrifice. Gold nano/microparticles were negative for MN induction in vitro and in vivo, and for the induction of DNA damage in all tissues. Silica particles, however, caused a small but reproducible increase in DNA damage and micronucleated reticulocytes when tested at their maximum tolerated dose (MTD). No genotoxic effects were observed at lower doses, and the in vitro MN assay was also negative. We hypothesize that silica NPs initiate secondary genotoxic effects through release of inflammatory cell-derived oxidants, similar to that described for crystalline silica (quartz). Such a mechanism is supported by the occurrence of increased neutrophilic infiltration, necrosis, and apoptotic cells in the liver, and induction of inflammatory markers TNF-α and IL-6 in plasma at the MTDs. These results were fairly consistent between silica NPs and the quartz control, thereby strengthening the argument that silica NPs may act in a similar, thresholded manner. The observed profile is supportive of a secondary genotoxicity mechanism that is driven by inflammation.

Comparison of the effects of oestrogen deficiency and old age on primary bladder afferent activity and voiding behaviour in the ageing female rat.

OBJECTIVE: To determine if a decrease in oestrogen (E) levels increases activity in bladder afferent nerves, by investigating the effects of E loss on afferent nerve activity in ovariectomized (OVX) rats, and making comparisons with findings in ageing female rats. MATERIALS AND METHODS: Female Sprague-Dawley rats were divided into four groups: ageing; OVX+ E; OVX+ vehicle (V); and sham-operated (SHAM) + V. The E (250 µg/kg) or V (cottonseed oil) was injected s.c. once a week for 8 weeks. Frequency-volume recordings were performed with the rats in metabolic cages, and single Aδ- or C-fibre activities from the bladder were measured. After measurements, the blood serum was collected and the estradiol (E2) level was measured. RESULTS: The C-fibre activity of OVX + V rats was significantly lower than in the other groups. Aδ-fibre activity was unchanged. Despite low E2 levels, ageing rats showed high afferent activity and voiding frequency. CONCLUSION: Although a low E level may affect storage function, it is unlikely that it is the main cause of the high afferent activity and voiding frequency observed in the ageing female rat.

Cannabinor, a selective cannabinoid-2 receptor agonist, improves bladder emptying in rats with partial urethral obstruction.

PURPOSE: We studied the effects of chronic treatment with the novel selective cannabinoid 2 receptor agonist cannabinor (Procter & Gamble Pharmaceuticals, Cincinnati, Ohio) on bladder function in conscious rats with partial urethral obstruction and on the functional properties of isolated detrusor muscle. MATERIALS AND METHODS: A total of 24 female Sprague-Dawley® rats with surgically created partial urethral obstruction received daily intraperitoneal injections of 3 mg/kg cannabinor (12) or saline as controls (12) for 2 weeks. Cystometry was done, the rats were sacrificed and the bladders were prepared for in vitro studies. RESULTS: Mean ± SEM bladder weight was 0.97 ± 0.15 gm in controls and 0.53 ± 0.08 gm in cannabinor treated rats (p <0.05). There was no difference between the groups in the mean micturition interval, or mean baseline, threshold, flow or maximum pressure. In controls and cannabinor treated rats mean post-void residual volume was 0.28 ± 0.07 and 0.06 ± 0.02 ml, mean micturition compliance was 0.032 ± 0.006 and 0.069 ± 0.016 ml/cm H(2)O, and mean bladder wall force at the start of flow was 950 ± 280 and 1,647 ± 325 mN/gm, respectively (each p <0.05). Nonvoiding contractions were significantly less frequent in cannabinor treated rats than in controls. We noted no difference in carbachol (Sigma®) half maximum concentration between the groups but the carbachol maximum response in detrusor strips from cannabinor treated rats was significantly higher than that in control strips. CONCLUSIONS: In rats with partial urethral obstruction treated daily for 14 days with cannabinor bladder weight was lower, the ability to empty the bladder was preserved and nonvoiding contraction frequency was low compared to those in controls. Detrusor preparations from cannabinor treated rats showed a higher response to nerve stimulation than those from controls. Selective cannabinoid 2 receptor activation may be a novel principle to enable improved bladder function after partial urethral obstruction.

Urothelial beta-3 adrenergic receptors in the rat bladder.

AIMS: To investigate the distribution of beta-3 adrenergic receptors (ß(3)ARs) in the rat bladder and to examine the contribution of urothelial ß(3)ARs to agonist-induced suppression of bladder reflexes and relaxation of smooth muscle. METHODS: Bladder tissue was collected from 8- to 10-month old female SD rats. In some samples, the urothelium was surgically separated from the smooth muscle. The expression and localization of ßAR mRNA and ß(3)AR protein were determined using RT-PCR and immunohistochemistry. Contractile responses to the specific ß(3)AR agonists TAK-677 and BRL37344 were measured in bladder strips with or without the urothelium. The contribution of urothelial ß(3)ARs to the micturition reflex was assessed in continuous cystometry in urethane anesthetized rats using intravesical delivery of ß(3)AR agonists. RESULTS: RT-PCR detected mRNA of all ßARs in urothelium and smooth muscle. Immunostaining detected ß(3)ARs throughout the urothelium, in the smooth muscle, myofibroblast-like cells, and in the peripheral nerves. Ovariectomy did not change the distribution of ß(3)ARs in any bladder structure. Intravesical administration of TAK-677 and BRL37344 (1-5 × 10(-4) M) decreased voiding frequency and amplitude of bladder contractions. In bladder strips in vitro both ß(3)AR agonists (10(-12) to 10(-4) M) relaxed the smooth muscle in a concentration-dependent manner to the same extent in strips with and without the urothelium. CONCLUSIONS: In addition to their presence in bladder smooth muscle, ß(3)ARs are present in the urothelium where their activation may alter reflex voiding via release of factor(s) that act on non-myocyte structures including the afferent and/or efferent nerves to influence bladder contractility.

Effects of cannabinor, a novel selective cannabinoid 2 receptor agonist, on bladder function in normal rats.

BACKGROUND: Cannabinoid (CB) receptors may be involved in the control of bladder function; the role of CB receptor subtypes in micturition has not been established. OBJECTIVES: Our aim was to evaluate the effects of cannabinor, a novel CB2 receptor agonist, on rat bladder function. DESIGN, SETTING, AND PARTICIPANTS: Sprague Dawley rats were used. Distribution of CB2 receptors in sensory and cholinergic nerves of the detrusor was studied. Selectivity of cannabinor for human and rat CB receptors was evaluated. Effects of cannabinor on rat detrusor and micturition were investigated. MEASUREMENTS: Immunohistochemistry, radioligand binding, tritium outflow assays, organ bath studies of isolated bladder tissue, and cystometry in awake rats were used. RESULTS AND LIMITATIONS: CB2 receptor immunoreactivity was expressed in the urothelium and in sensory and cholinergic bladder nerves. Cannabinor exhibited similar binding at human and rat CB2 receptors and a 321-fold functional selectivity for the CB2 receptor versus the CB1 receptor. Cannabinor had no effect on isolated detrusor muscle function. In vivo, cannabinor 3.0mg/kg increased micturition intervals and volumes by 52% (p<0.05) and 96% (p<0.01), respectively, and increased threshold and flow pressures by 73% (p<0.01) and 49% (p<0.001), respectively. Cannabinor 0.3 or 1.0mg/kg or vehicle did not affect urodynamic parameters. CONCLUSIONS: Considering that CB2 receptors are localized on sensory nerves and on the urothelium and that cannabinor had effects on "afferent" urodynamic parameters, peripheral CB2 receptors may be involved in sensory functions of rat micturition. Effects of cannabinor on cholinergic nerve activity in normal bladder tissue appear to be limited.

Effects of beta3-adrenergic receptor activation on rat urinary bladder hyperactivity induced by ovariectomy.

Voiding dysfunctions, including increased voiding frequency, urgency, or incontinence, are prevalent in the postmenopausal population. Beta(3)-adrenergic receptor (beta(3)AR) agonists, which relax bladder smooth muscle, are being developed to treat these conditions. We utilized the rat ovariectomy (OVX) model to investigate the effect of ovarian hormone depletion on bladder function and the potential for beta(3)AR agonists to treat bladder hyperactivity in this setting. OVX increased voiding frequency and decreased bladder capacity by approximately 25% in awake rats and induced irregular cystometrograms in urethane-anesthetized rats. Reverse transcription-polymerase chain reaction revealed three betaARs subtypes (beta(1,2,3)) in bladder tissue, and immunostaining indicated beta(3)AR localization in urothelium and detrusor. Receptor expression was not different in OVX and SHAM rats. The beta(3)AR agonist selectivity of BRL37344 [(+/-)-(R(*),R(*))-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid sodium hydrate], TAK-677 [(3-((2R)-(((2R)-(3-chlorophenyl)-2-hydroxyethyl)amino)propyl)-1H-indol-7-yloxy)acetic acid], and FK175 [acetic acid, 2-[[(8S)-8-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-yl]oxy], ethyl ester, hydrochloride] was confirmed by examining the relative potency for elevation of cAMP in CHOK1 cells overexpressing the various rat betaARs. Intravenous injection of each of the beta(3)AR agonists (0.1-500 microg/kg) in anesthetized rats decreased voiding frequency, bladder pressure, and amplitude of bladder contractions. In bladder strips, beta(3)AR agonists (10(-12)-10(-4) M) decreased baseline tone and reduced spontaneous contractions. BRL37344 (5 mg/kg) and TAK-677 (5 mg/kg) injected intraperitoneally in awake rats decreased voiding frequency by 40 to 70%. These effects were not altered by OVX. The results indicate that OVX-induced bladder dysfunction, including decreased bladder capacity and increased voiding frequency, is not associated with changes in beta(3)AR expression or the bladder inhibitory effects of beta(3)AR agonists. This suggests that beta(3)AR agonists should prove effective for the treatment of overactive bladder symptoms in the postmenopausal population.

1,2,3,4-Tetrahydroisoquinolinyl sulfamic acids as phosphatase PTP1B inhibitors.

High-throughput screening of the P&GP corporate repository against several protein tyrosine phosphatases identified the sulfamic acid moiety as potential phosphotyrosine mimetic. Incorporation of the sulfamic acid onto a 1,2,3,4-tetrahydroisoquinoline scaffold provided a promising starting point for PTP1B inhibitor design.

A nonspecific phosphotyrosine phosphatase inhibitor, bis(maltolato)oxovanadium(IV), improves glucose tolerance and prevents diabetes in Zucker diabetic fatty rats.

The molecular basis of insulin resistance, a major risk factor for development of Type II diabetes, involves defective insulin signaling. Insulin-mediated signal transduction is negatively regulated by the phosphotyrosine phosphatase, PTP1B, and numerous studies have demonstrated that organo-vanadium compounds, which are nonselective phosphotyrosine phosphatase inhibitors, have insulin-mimetic properties. However, whether or not vanadium compounds can prevent the transition from insulin resistance to overt diabetes is unknown. We compared the ability of bis(maltolato)oxovanadium(IV) (BMOV), an orally bioavailable organo-vanadium compound, and rosiglitazone maleate (RSG), a known insulin sensitizer, to prevent development of diabetes in Zucker diabetic fatty (ZDF) rats. Treatment began at 6 weeks of age when animals are insulin resistant and hyperinsulinemic, but not yet hyperglycemic, and ended at 12 weeks of age, which is 4 weeks after ZDF rats typically develop overt diabetes. BMOV-treated ZDF rats did not develop hyperglycemia, showed significant improvement in insulin sensitivity, and retained normal pancreatic islet morphology and endocrine cell distribution, similar to RSG-treated animals. BMOV and RSG treatment also prevented the hyper-phagia and polydipsia present in untreated ZDF rats; however, BMOV-treated ZDF rats gained much less weight than did RSG-treated animals. Circulating levels of adiponectin decreased in untreated ZDF rats compared to lean controls, but these levels remained normal in BMOV-treated ZDF rats. In contrast, in RSG-treated ZDF rats, plasma adiponectin levels were nearly 4-fold higher than in lean control rats, primarily as a result of a large increase in the amount of low-molecular weight forms of adiponectin in circulation. These data demonstrate that phosphatase inhibition offers a new approach to diabetes prevention, one that may have advantages over current approaches.

Comparison of high throughput screening technologies for luminescence cell-based reporter screens.

As higher density formats become more and more common in HTS labs, the expectations for maintaining faster, lower cost screens puts great pressure on traditional 96-well screens. In some cases higher density formats are not compatible with the assay. This seems especially true in cell-based assays. In our case, the nature of the cells' response forced us to remain in 96-well plates. In this paper, we describe the development of a luminescence reporter assay and its performance in two detection modes, flash and glow. The advantages in cost and throughput for each technique are explored, along with automation considerations. An additional new technology, the use of pins for low-volume transfers, is also briefly described because of its dramatic effect on our screen's throughput. However, it will be more thoroughly presented in a future publication. Comparing the technologies available for HTS aids in designing automated systems that meet the unique needs of each assay.