Evaluation of the skin phototoxicity of systemically administered pharmaceuticals in Sprague-Dawley rats.

In vivo phototoxicity testing is important for predicting drug-induced phototoxicity in humans. Currently, there is no internationally validated in vivo test method for the photosafety evaluation of pharmaceuticals. In this study, we evaluated the phototoxicity of systemically administered drugs using SD rats. We first determined the appropriate ultraviolet A (UVA) dose using 8-methoxypsoralen, a well-known phototoxic drug. Compared to lower and higher UVA doses, we found that a UVA dose of 10 J/cm2 allowed for the detection of phototoxic responses in both a dose- and time-dependent manner. We next performed a phototoxicity study using seven pharmaceutical drugs which included known phototoxic and non-phototoxic drugs using a UVA dose of 10 J/cm2. In order to improve the accuracy of our assessment, we evaluated both gross skin findings as well as histopathological findings. Using gross skin findings alone resulted in an accuracy of 85.7% which could be increased to 100% accuracy when the gross skin findings were combined with histopathological findings. This study suggests that the inclusion of histopathological findings increases the accuracy of the phototoxicity evaluation of systemically administered drugs in SD rats. In conclusion, we found that for studying drug-induced phytotoxicity, a 10 J/cm2 UVA dose serves as the optimal radiation dose, and that the inclusion of histopathological findings increases the accuracy of the phototoxicity evaluation of the drugs.

Evaluation of skin phototoxicity of transdermally administered pharmaceuticals in Sprague-Dawley rats.

Some drugs cause phototoxicity in humans when exposed to light, thus there is a need for an in vivo phototoxicity test to evaluate them. However, an in vivo phototoxicity test method to evaluate this has not been established. This study aimed to establish an in vivo phototoxicity test method for transdermally administered drugs. For this, we evaluated the phototoxicity using Sprague-Dawley (SD) rats for transdermal administered drugs and we studied the appropriate UVA dose using 8-methoxypsalen, which is a well-known phototoxic drug. We found that a UVA dose of 15 J/cm2 was dose and time dependent response compared to other UVA doses. We performed the Minimum Erythema Dose (MED) test because UVB can cause skin irritation by itself and selected 0.01 J/cm2 as an appropriate dose of UVB. Using the selected UVA and UVB doses, we performed a phototoxicity study of 6 pharmaceutical drugs, which included phototoxic and non-phototoxic drugs. As a result of the phototoxicity test, 100% accuracy was obtained when compared with previous studies. In addition, we performed histopathology to confirm the new findings. We found that histopathology can be used as an additional indicator of phototoxicity test for transdermally administered drugs.

Two tiered approaches combining alternative test methods and minimizing the use of reconstructed human cornea-like epithelium tests for the evaluation of eye irritation potency of test chemicals.

In order to overcome the limitations of single in vitro eye irritation tests, Integrated Approaches to Testing Assessment strategies have been suggested for evaluating eye irritation. This study developed two tiered approaches combining alternative test methods. They were designed in consideration of the solubility property of test chemicals and to use the RhCE tests at final steps. The tiered approach A is composed of the STE, BCOP, HET-CAM or RhCE tests, whereas the tiered approach B is designed to perform simultaneously two in vitro test methods at the first stage and the RhCE test at the final stage. The predictive capacity of the two tiered approaches was estimated using 47 chemicals. The accuracy, sensitivity, and specificity value of the tiered approach A were 95.7% (45/47), 100% (34/34), and 84.6% (11/13), respectively, whereas those of the tiered approach B were 95.7% (45/47), 97.1% (33/34), and 92.3% (12/13), respectively. The approach A and B were considered to be available methods for distinguishing test chemicals of Category 1 (all 73.3%) and No Category (84.6% and 92.3%), respectively. Especially, the approach B was considered as an efficient method as the Bottom-Up approach, because it predicted correctly test chemicals classified as No Category.

Evaluation of skin sensitization potential of chemicals by local lymph node assay using 5-bromo-2-deoxyuridine with flow cytometry.

The local lymph node assay using 5-bromo-2-deoxyuridine with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA used to identify skin sensitizers. This assay measures the proliferation of auricular lymph node cells (LNCs) during the induction phase of skin sensitization and the number of BrdU-positive LNCs using flow cytometry. We determined if LLNA: BrdU-FCM can evaluate the skin sensitization potential of 20 substances, including 16 sensitizers and 4 non-sensitizers, that were tested using LLNA: DA and LLNA: BrdU-ELISA but not listed in OECD TG 429. After selecting appropriate vehicles and conducting pre-screen tests in 2 phases, solvents and test concentrations for the main test were determined. In the main study, we measured changes in LN weight, the number of LNCs, and the proportion of BrdU incorporated into LNCs to calculate stimulation indexes (SI). SI was calculated based on the total number of LNCs and BrdU incorporation in LNCs. We found that all substances were correctly classified as sensitizers or non-sensitizers. Overall, we confirmed that the LLNA: BrdU-FCM can evaluate skin sensitization potential of the 20 substances. Additionally, our results of combining 22 reference substances listed in OECD TG 429 and 20 additional substances showed that concordance of LLNA: BrdU-FCM with the LLNA was higher than before.

Assessment of the predictive capacity of the optimized in vitro comet assay using HepG2 cells.

Evaluation of DNA damage is critical during the development of new drugs because it is closely associated with genotoxicity and carcinogenicity. The in vivo comet assay to assess DNA damage is globally harmonized as OECD TG 489. However, a comet test guideline that evaluates DNA damage without sacrificing animals does not yet exist. The goal of this study was to select an appropriate cell line for optimization of the in vitro comet assay to assess DNA damage. We then evaluated the predictivity of the in vitro comet assay using the selected cell line. In addition, the effect of adding S9 was evaluated using 12 test chemicals. For cell line selection, HepG2, Chinese hamster lung (CHL/IU), and TK6 cell lines were evaluated. We employed a method for the in vitro comet assay based on that for the in vivo comet assay. The most appropriate cell line was determined by% tail DNA increase after performing in vitro comet assays with 6 test chemicals. The predictivity of the in vitro comet assay using the selected cell line was measured with 10 test chemicals (8 genotoxins and 2 non-genotoxic chemicals). The HepG2 cell line was found to be the most appropriate, and in vitro comet assays using HepG2 cells exhibited a high accuracy of 90% (9/10). This study suggests that HepG2 is an optimal cell line for the in vitro comet assay to assess DNA damage.

Ezrin as a complementary marker in ocular toxicity assessment using a three-dimensional reconstructed human corneal-like epithelium model, EpiOcular™.

INTRODUCTION: A variety of in vitro tests to replace the Draize test have been developed; however, there is no available method for assessing the full spectrum of Globally Harmonized System (GHS) categories. Human cornea-like three-dimensional (3D) reconstructed tissue models are the most promising in vitro systems. The objective of this study was to evaluate the ocular toxicity of 11 test substances using the EpiOcular™ model after performing proficiency tests. We further evaluated the effectiveness of ezrin staining as a complementary marker in histological analysis to overcome the limitation of eye irritation tests using 3D reconstructed human corneal epithelium models. METHODS: The assessment of ocular toxicity was performed by the suggested OECD TG 492 procedure. After treatment with proficiency test chemicals and 10 test substances, EpiOcular™ tissue models were stained with hematoxylin and eosin and ezrin, and the histological changes were observed by immunofluorescence microscopy. RESULTS: The ocular toxicity assessment of 10 test chemicals using the EpiOcular™ eye irritation test were in accordance with the UN GHS classification of test chemicals. Histological analysis of ezrin staining showed that the cell membranes of models treated with 10 out of 11 non-irritant chemicals were maintained, whereas those of models treated with 14 eye irritant substances resulted in the apparent translocation of ezrins from the cell membrane to the cytoplasm or nucleus by destruction of cell membrane. DISCUSSION: Ezrin may be used as a complementary marker to more accurately assess ocular toxicity using 3D reconstructed human cornea-like epithelium models.

Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells.

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration (IC50) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity.

Phototoxicity Evaluation of Pharmaceutical Substances with a Reactive Oxygen Species Assay Using Ultraviolet A.

With ultraviolet and visible light exposure, some pharmaceutical substances applied systemically or topically may cause phototoxic skin irritation. The major factor in phototoxicity is the generation of reactive oxygen species (ROS) such as singlet oxygen and superoxide anion that cause oxidative damage to DNA, lipids and proteins. Thus, measuring the generation of ROS can predict the phototoxic potential of a given substance indirectly. For this reason, a standard ROS assay (ROS assay) was developed and validated and provides an alternative method for phototoxicity evaluation. However, negative substances are over-predicted by the assay. Except for ultraviolet A (UVA), other UV ranges are not a major factor in causing phototoxicity and may lead to incorrect labeling of some non-phototoxic substances as being phototoxic in the ROS assay when using a solar simulator. A UVA stimulator is also widely used to evaluate phototoxicity in various test substances. Consequently, we identified the applicability of a UVA simulator to the ROS assay for photoreactivity. In this study, we tested 60 pharmaceutical substances including 50 phototoxins and 10 non-phototoxins to predict their phototoxic potential via the ROS assay with a UVA simulator. Following the ROS protocol, all test substances were dissolved in dimethyl sulfoxide or sodium phosphate buffer. The final concentration of the test solutions in the reaction mixture was 20 to 200 µM. The exposure was with 2.0~2.2 mW/cm2 irradiance and optimization for a relevant dose of UVA was performed. The generation of ROS was compared before and after UVA exposure and was measured by a microplate spectrophotometer. Sensitivity and specificity values were 85.7% and 100.0% respectively, and the accuracy was 88.1%. From this analysis, the ROS assay with a UVA simulator is suitable for testing the photoreactivity and estimating the phototoxic potential of various test pharmaceutical substances.

Comparison of BALB/c and CBA/J mice for the local lymph node assay using bromodeoxyuridine with flow cytometry (LLNA: BrdU-FCM).

The local lymph node assay using 5-bromo-2-deoxyuridine (BrdU) with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA that is used to identify skin sensitizers by counting BrdU-incorporated lymph node cells (LNCs) with flow cytometry. Unlike other LLNA methods (OECD TG 429, 442A and 442B) in which the CBA/J mouse strain is used, LLNA: BrdU-FCM was originally designed to be compatible with BALB/c, a mouse strain that is more widely used in many countries. To justify the substitution of CBA/J for BALB/c, the equivalence of the test results between two strains shall be established prior to the official implementation of LLNA: BrdU-FCM. This study aims to compare the test results of LLNA: BrdU-FCM produced in BALB/c mice with those in CBA/J mice for 18 reference substances, including 13 sensitizers and 5 non-sensitizers, listed in OECD Test Guideline 429. Based on the LLNA: BrdU-FCM test procedure, we selected an appropriate solvent and then performed preliminary tests to determine the non-irritating dose ranges for the main study, which revealed the difference in the irritation responses to 8 of the 18 chemicals between the two strains. In the main study, we measured the changes in the number of total LNCs, which indicated differences in the responses to test chemicals between the two strains. However, the stimulation index obtained with the counts of BrdU-incorporated LNCs with 7-AAD using flow cytometry yielded comparable results and 100% concordance between the BALB/c and CBA/J mouse strains was achieved, suggesting that the performance of LLNA: BrdU-FCM using BALB/c mice was equivalent to that with CBA/J mice.

Performance standard-based validation study for local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method.

Local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method (LLNA: BrdU-FCM) is a modified non-radioisotopic technique with the additional advantages of accommodating multiple endpoints with the introduction of FCM, and refinement and reduction of animal use by using a sophisticated prescreening scheme. Reliability and accuracy of the LLNA: BrdU-FCM was determined according to OECD Test Guideline (TG) No. 429 (Skin Sensitization: Local Lymph Node Assay) performance standards (PS), with the participation of four laboratories. Transferability was demonstrated through successfully producing stimulation index (SI) values for 25% hexyl cinnamic aldehyde (HCA) consistently greater than 3, a predetermined threshold, by all participating laboratories. Within- and between-laboratory reproducibility was shown using HCA and 2,4-dinitrochlorobenzene, in which EC2.7 values (the estimated concentrations eliciting an SI of 2.7, the threshold for LLNA: BrdU-FCM) fell consistently within the acceptance ranges, 0.025-0.1% and 5-20%, respectively. Predictive capacity was tested using the final protocol version 1.3 for the 18 reference chemicals listed in OECD TG 429, of which results showed 84.6% sensitivity, 100% specificity, and 88.9% accuracy compared with the original LLNA. The data presented are considered to meet the performance criteria for the PS, and its predictive capacity was also sufficiently validated.

Pyruvate protects against kainate-induced epileptic brain damage in rats.

Although the majority of epileptic seizures can be effectively controlled with antiepileptic drugs and/or surgery, a significant number progress to status epilepticus of sufficient duration to cause permanent brain damage. Combined treatment with antiepileptic drugs and neuroprotective agents, however, may help protect these individuals from permanent brain damage. Since toxicity induced by endogenous zinc contributes to epileptic brain injury, and since pyruvate is effective in reducing zinc-triggered neuronal death in cortical culture as well as ischemic neuronal death in vivo, we examined whether systemic pyruvate administration reduces seizure-induced brain damage. Na pyruvate (500 mg/kg) or osmolarity-matched saline (265 mg/kg NaCl, i.p.) were given to adult SD rats 30 or 150 min after 10 mg/kg kainite injection (i.p.), and there was no significant difference in the time course or severity of seizures between these groups. Zinc accumulation in neuronal cell bodies in the hippocampus, however, was much lower in the pyruvate than in the saline group. There was a close correlation between zinc accumulation and cell death, as assessed by acid-fuchsin and TUNEL staining. Pyruvate treatment markedly reduced neuronal death in the hippocampus, neocortex and thalamus. Pyruvate increased HSP-70 expression in hippocampal neurons. These results suggest that pyruvate, a natural glucose metabolite, may be useful as adjunct treatment in status epilepticus to reduce permanent brain damage.

Systemic pyruvate administration markedly reduces infarcts and motor deficits in rat models of transient and permanent focal cerebral ischemia.

Pyruvate markedly reduces neuronal death following transient global ischemia. In the present study, we investigated the possible neuroprotective effect of pyruvate in focal ischemia. Pyruvate (62.5-250 mg/kg) treatment, regardless of whether given intraperitoneally (ip) or intravenously (iv), decreased infarct volume by more than 50% in both transient (1 h) and permanent occlusion models. The infarct-reducing effects of pyruvate were maintained 14 days (d) after MCAO. Interestingly, higher doses failed to reduce the infarct size. Pyruvate administration also reduced motor deficits. Magnetic resonance (MR) spectroscopy revealed that protective doses of pyruvate, but not the non-protective doses, were associated with a reduction in the level of lactate compared with saline controls. Diffusion-weighted MR images further confirmed infarct reduction in pyruvate-treated rats. Pyruvate is an endogenous metabolite of glycolysis, and hence is unlikely to have serious side effects. Considering its substantial neuroprotective capacity in focal cerebral ischemia, a clinical trial is warranted.

Infarct reduction in rats following intraventricular administration of either tissue plasminogen activator (tPA) or its non-protease mutant S478A-tPA.

In addition to its thrombolytic effect, human recombinant tissue plasminogen activator (tPA) may have parenchymal effects such as protease-dependent neurotoxic and protease-independent neuroprotective effects. The purpose of this study was to examine parenchymal effects of tPA and its non-protease mutant S478A-tPA in permanent focal cerebral ischemia in rats. However, before doing in vivo experiments, effects of tPA and S478A-tPA on zinc or NMDA toxicity were first studied in cortical cultures. Like tPA, which has protease-independent cytoprotective effects, the non-protease mutant S478A-tPA blocked zinc toxicity in cortical cell cultures, but did not affect calcium-mediated NMDA toxicity. Then, effects of tPA and S478A-tPA on infarcts induced by permanent occlusion of middle cerebral artery (MCA) were investigated. tPA and S478A-tPA were administered into the cerebral ventricle 15 min or 1 h after MCA occlusion. Both tPA and its non-protease mutant S478A-tPA, when given 15 min after ischemia, substantially reduced infarcts and ameliorated motor deficits in the MCA occlusion model of focal cerebral ischemia. However, when administered 1 h after MCA occlusion, neither showed protective effects. The protective effects of tPA or S478A-tPA remained unchanged at 7 days after MCA occlusion. Indicating that the native protein conformation is necessary for the protective effect of tPA and S478A-tPA, heat-denatured tPA did not exhibit any protective effect. Since S478A-tPA lacks protease activity, which has been implicated in causing cerebral hemorrhage or aggravating excitotoxicity, its parenchymal neuroprotective effect may be useful in treatment of ischemic stroke.

Co-induction of p75(NTR) and the associated death executor NADE in degenerating hippocampal neurons after kainate-induced seizures in the rat.

Zinc induces in cultured cortical neurons both p75(NTR) and p75(NTR)-associated death executor (NADE), which together contribute to caspase-dependent neuronal apoptosis. Since zinc neurotoxicity may contribute to neuronal death following seizures, we examined whether p75(NTR) and NADE are co-induced also in rat hippocampal neurons degenerating after seizures. Staining of brain sections with a zinc-specific fluorescent dye (N-(6-methoxy-8-quinolyl)-p-carboxybenzoylsulphonamide) and acid fuchsin revealed zinc accumulation in degenerating neuronal cell bodies in CA1 and CA3 of hippocampus 24 h after kainate injection. Both anti-p75(NTR) and anti-NADE immunoreactivities appeared in zinc-accumulating/degenerating neurons in both areas. Intraventricular injection of CaEDTA, without altering the severity or time course of kainate-induced seizures, markedly attenuated the induction of p75(NTR)/NADE in hippocampus, which correlated with the decrease of caspase-3 activation and zinc accumulation/cell death. The present study has demonstrated that p75(NTR) and NADE are co-induced in neurons degenerating after kainate-induced seizures in rats, likely in a zinc-dependent manner.