Two cases of cartilaginous metaplasia in the sclera of Japanese White rabbits.

Spontaneous cartilaginous metaplasia of the sclera has not been reported in rabbits. Herein, we report two cases of spontaneous cartilaginous metaplasia in the sclera of Japanese White (JW) rabbits. Case 1 was noted in a 14-week-old male Kbs:JW rabbit that received a single ocular instillation of 20% isoproterenol (IP) a day before necropsy, and showed no abnormalities in clinical signs, ophthalmological assessments, and necropsy. Case 2 was noted in a 38-week-old male Kbs:JW rabbit that was housed under light-emitting diode (LED) lighting for 26 weeks and showed no effects of LED on clinical signs, ophthalmological assessments, and necropsy. Histological sections of the eyes of both animals were prepared and stained with hematoxylin and eosin (H&E) and Alcian blue, and immunohistochemical staining for vimentin was performed. The H&E-stained specimens showed focal hyaline cartilage-like tissues distributed between the scleral fibers at the posterior pole in both cases. The surrounding scleral fibers were compressed and/or partially destroyed by the cartilage-like tissue. The cartilage-like matrix was stained blue by Alcian blue, and immunohistochemistry showed that chondrocyte-like cells were positive for vimentin. Based on these findings, we diagnosed cartilaginous metaplasia in the sclera of Kbs:JW rabbits. The lesion was farther from the IP administration site in Case 1 and was not accompanied by other ophthalmological or histopathological abnormalities in either of the cases. This implies that the lesions occurred spontaneously owing to the abnormal differentiation of neural crest-derived cells.

Investigation of in vivo unscheduled DNA synthesis in rabbit corneas following instillation of genotoxic agents.

PURPOSE: An unscheduled DNA synthesis (UDS) test is used for in vitro or in vivo genotoxicity evaluation. The UDS test with hepatocytes is well established; however, drug exposure levels at the application site for topically administered drugs (e.g. ophthalmic drugs) often exceed the exposure levels for systemic administration. To establish in vivo genotoxicity on the ocular surface, we performed the UDS test using rabbit corneas from eyes subjected to instillation of genotoxic agents. MATERIALS AND METHODS: Five genotoxic agents - 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat); acridine orange; ethidium bromide; acrylamide; and 4-nitroquinoline 1-oxide (4-NQO) - were instilled once onto both eyes of male Japanese white rabbits. Physiological saline or a general vehicle for ophthalmic solution were instilled as the negative controls. Dimethyl sulfoxide was instilled as the vehicle control. Isolated corneas were incubated with tritium-labelled thymidine and the number of sparsely labelled cells (SLCs, cells undergoing UDS) was counted by autoradiography. RESULTS: Statistically significant increases in the mean appearance rates of SLCs in the corneal epithelium were noted in paraquat-, acridine orange-, ethidium bromide-, and 4-NQO-treated eyes compared with those of the controls. These increases generally appeared in a dose-dependent manner. Acrylamide did not induce an increase in the mean appearance rates of SLCs, presumably because it caused the generation of fewer metabolites in the cornea. CONCLUSIONS: UDS tests revealed DNA damage in the cornea epitheliums treated with well-known genotoxic agents. These results suggest that the UDS test is one of the useful tools for the assessment of in vivo genotoxicity on the ocular surface in the development of ophthalmic drugs.

Orbital malignant schwannoma and embryonal rhabdomyosarcoma in rats caused by leakage of locally injected nickel subsulfide to the orbit.

Nickel subsulfide (Ni3S2) is known to induce intraocular neoplasms when injected intravitreally into the eyes of rats. Here, we found two extraocular orbital neoplasms in two different rat strains, presumably due to the leakage of locally injected Ni3S2 to the extraocular orbital tissues. In the F344/DuCrlCrlj rat, an orbital mass arose at 30 weeks after injection, and invaded into the cranium. Histologically, the orbital mass was composed of areas arranged in parallel bundles formed by densely packed elongated or spindle-shaped cells with indistinct cytoplasmic borders, and of areas of hypocellular arrangement consisting of round cells in eosinophilic myxoid-like substances. Metastases were observed in the right submandibular and cervical lymph nodes. The neoplastic cells were immunopositive for S-100 protein and vimentin. Transmission electron microscopy revealed that the neoplastic cells had cellular processes and pericytoplasmic basal laminae. In the RccHanTM:WIST rat, an orbital mass arose at 36 weeks after injection. Histologically, the mass consisted of rhabdoid-like large round cells with proliferation of small round-to-polygonal cells, and these neoplastic cells infiltrated into the extraocular muscles. Immunohistochemically, the neoplastic cells were positive for desmin and vimentin. Transmission electron microscopy detected immature myofibrils with Z-band structures in the cytoplasm of these neoplastic cells. Consequently, the tumors were diagnosed as an orbital malignant schwannoma in an F344/DuCrlCrlj rat and an orbital embryonal rhabdomyosarcoma in a RccHanTM:WIST rat. The results of this case report suggest that leakage of Ni3S2 to the orbit caused the induction of orbital malignant schwannoma or rhabdomyosarcoma in rats.

Contribution of anti-inflammatory and anti-virulence effects of azithromycin in the treatment of experimental Staphylococcus aureus keratitis.

BACKGROUND: We aimed to demonstrate the contribution of anti-inflammatory and anti-virulence effects of azithromycin (AZM) in ocular surface infection treatment. METHODS: Staphylococcus aureus was injected into the corneal stroma of rabbits to induce keratitis. AZM at concentrations of 0.01, 0.1, and 1% was instilled into the eye twice daily. The eyes were examined using a slit lamp and scored. The viable bacteria in the cornea were counted at 48 h post infection. To evaluate the anti-inflammatory efficacy of AZM, S. aureus culture supernatant-induced anterior ocular inflammation in rabbit was examined using a slit lamp and scored. To evaluate the inhibitory effect of AZM on bacterial toxin production, S. aureus was cultured with AZM and hemolytic reaction in the culture supernatant was determined. RESULTS: In the bacterial keratitis model, AZM dose-dependently inhibited the increase in the clinical score. The viable bacterial count in the cornea treated with 1% AZM significantly decreased compared with that of the vehicle, whereas bacterial count in 0.01 and 0.1% AZM-treated corneas was similar to that of the vehicle. In the anterior ocular inflammation model, 0.1 and 1% AZM inhibited the increase in the clinical score. AZM inhibited hemolytic reaction at concentrations that did not inhibit bacterial growth. CONCLUSIONS: The results demonstrated that AZM has not only anti-bacterial, but also anti-inflammatory effects, and inhibits bacterial toxin production leading to ocular surface damage in bacterial infection. Thus, the therapeutic effect of AZM against ocular infections is expected to be higher than that which could be assumed if it only had anti-bacterial activity.

Characteristics of corneal phospholipidosis induced by topical ocular application of chloroquine and amiodarone in rabbits.

Several cationic-amphiphilic drugs such as chloroquine and amiodarone are known to induce phospholipidosis in the cornea by systemic administration. However, the characteristics of ophthalmological and pathological changes when phospholipidosis-inducing drugs are topically applied have not been well studied. This study was conducted to investigate the characteristics of corneal changes caused by topical application of chloroquine and amiodarone to Japanese white rabbits. The changes were evaluated by ophthalmological, histopathological, and ultrastructural examinations. An in vivo confocal microscopy was also applied to the chloroquine-treated corneas. In both chloroquine- and amiodarone-treated corneas, diffuse cloudiness was observed by slit-lamp biomicroscopy, and its transparency increased with duration of dosing. Confocal microscopy showed punctate dots in the corneal epithelium. Histopathologically, cytoplasmic vacuolation was found in the corneal epithelium and keratocytes in both chloroquine- and amiodarone-treated eyes. Furthermore, foamy cytoplasm of the corneal endothelium was observed in the chloroquine-treated eyes. Ultrastructural examination showed multi-lamellar inclusion bodies or membrane-like debris in the lysosome-like vacuoles in the cytoplasm of corneal cells, which is a characteristic of the lesions of phospholipidosis. These changes disappeared after a withdrawal period. Continuous dosing of chloroquine resulted in corneal erosion and focal corneal opacity as shown by gross observation and slit-lamp biomicroscopy. Confocal microscopy could detect the corneal changes prior to the appearance of these ophthalmological changes. The present study showed that phospholipidosis caused by ocular administration of chloroquine and amiodarone first induces reversible diffuse corneal cloudiness. Confocal microscopy is a useful method for monitoring induction of corneal phospholipidosis.

Time-course changes in DNA damage of corneal epithelial cells in rabbits following ocular instillation with genotoxic compounds.

BACKGROUND: In eye-drop drug development, the additional genotoxicity tests in some cases might be necessary to assess genotoxicity in the ocular surface since the ocular surface is exposed directly to high drug concentrations. Recently, an in vivo comet assay using corneal epithelial cells in rabbits following single ocular instillation was developed as an assay to evaluate genotoxicity in ocular tissues. In this study, we investigated the time-course changes in DNA damage after ocular instillation of genotoxic compounds to evaluate the optimal sampling timing for in vivo comet assay of the ocular surface tissue. Ethidium bromide (EtBr), methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4-NQO) were administered to the eyes of the rabbits. Corneas were collected at 0.5, 2, 4, 6, and 24 h after administration, and the comet assay was performed. In addition, the in vitro comet assay was performed to assess the time-course changes in DNA damage induced by short-time exposure to the genotoxic compounds. RESULTS: The mean % tail DNA, which is an indicator for DNA damage, in the corneal epithelial cells treated with all compounds exhibited statistically significant increases compared with those in the negative controls of saline at 0.5, 2, 4, and 6 h. There was a difference in the DNA damage response between EtBr and the other two compounds. In the 3% MMS- and 1% 4-NQO-treated eyes, the values of the % tail DNA were the highest at 0.5 h and then decreased gradually. In contrast, in the 1% EtBr-treated eyes, the highest value was noted at 4 h. The results of the in vitro comet assay showed that the % tail DNA increased in all groups. A further increase in the % tail DNA occurred in the EtBr-treated cells even after removing the compound but not in the MMS- and 4-NQO-treated cells. CONCLUSION: Relatively high values of the % tail DNA were maintained from 0.5 to 6 h after administration, suggesting that the optimal sampling time is any one point from 0.5 to 6 h in the comet assay of the corneal surface.

Presumed congenital nictitating membrane dysplasia in a Japanese white rabbit.

OBJECTIVE: The present report characterises a spontaneous nictitating membrane abnormality in a Japanese white rabbit. ANIMAL STUDIED: The animal was a male Japanese white rabbit (Oryctolagus cuniculus, Kbs:JW, 10 weeks old at the time of purchase) that had not received any treatment. A morphological abnormality of the nictitating membrane in the animal's right eye was detected. PROCEDURES: Ophthalmological examinations, including slit-lamp biomicroscopy, corneal and conjunctival staining with fluorescein and lissamine green, fundoscopy, blinking rate measurement, Schirmer's tear test, and tonometry were performed. The animal was euthanised at 15 weeks of age, and histopathological examinations of the abnormal nictitating membrane, palpebral conjunctiva, eyelid and eyeball were performed. RESULTS: The tip of the nictitating membrane adhered to the palpebral conjunctiva of the medial canthus. The eye and periocular tissues showed no abnormalities in the ophthalmological examinations, except for the structure of the nictitating membrane. Histopathological examination revealed that the adhered site of the nictitating membrane and the palpebral conjunctiva consisted of dense fibrous connective tissue that was consecutive to the conjunctiva adjacent to the eyelid margin and lamina propria of the nictitating membrane. The fibrous connective tissue was covered with stratified squamous epithelium. CONCLUSION: Based on these results, we diagnosed this abnormal finding as congenital nictitating membrane dysplasia. This paper is the first case report describing a congenital structural abnormality of the nictitating membrane in a Japanese white rabbit.

In vivo comet assay in rabbit corneal epithelial cells following ocular instillation with genotoxic compounds.

BACKGROUND: The in vivo comet assay is used to evaluate the genotoxic potential of compounds by detecting DNA strand breaks in cells isolated from animal tissue. The comet assay of hepatocytes is well established; however, the levels of systemic drug exposure following systemic administration are often insufficient to evaluate the genotoxic potential of compounds on the ocular surface following ocular instillation. To investigate the possibility of using the comet assay as a genotoxic evaluation tool for the ocular surface, we performed this assay on the corneal epithelial cells of rabbit eyes 2 h after the single ocular instillation of five genotoxic compounds, namely ethidium bromide, 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat), methyl methanesulfonate (MMS), acrylamide, and 4-nitroquinoline 1-oxide (4-NQO). RESULTS: The mean % tail DNA, as an indicator of DNA damage, in the corneal epithelial cells treated with ethidium bromide, MMS, and 4-NQO exhibited statistically significant increases compared with those in the negative controls (saline or 5 % dimethyl sulfoxide in saline). However, paraquat and acrylamide did not increase the mean % tail DNA, presumably because of the high antioxidant levels and low cytochrome P450 levels present in the corneal epithelium, respectively. CONCLUSIONS: The comet assay was able to detect genotoxic potential on the ocular surface following ocular instillation with genotoxic compounds. The study findings indicate that the in vivo comet assay may provide a useful tool for assessing the genotoxicity of compounds topically administrated on the ocular surface under mimicking clinical condition.

A reaction mechanism-based prediction of mutagenicity: α-halo carbonyl compounds adduct with DNA by SN2 reaction.

Most of the α-halo carbonyl (AHC) compounds tend to be predicted as mutagenic by structure-activity relationship based on structural category only, because they have an alkyl halide structure as a structural alert of mutagenicity. However, some AHC compounds are not mutagenic. We hypothesized that AHC reacts with DNA by SN2 reaction, and the reactivity relates to mutagenicity. As an index of SN2 reactivity, we focused on molecular orbitals (MOs), as the direction and position of two molecules in collision are important in the SN2 reaction. The MOs suitable for SN2 reaction (SN2MOs) were selected by chemical-visual inspection based on the shape of the MO. We used the level gap and the energy gap between SN2MO and the lowest unoccupied molecular orbital as the descriptors of SN2 reactivity. As the results, SN2 reactivity related to mutagenicity and we were able to predict mutagenicity of 20 AHC compounds with 95.0% concordance. It was suggested that SN2 reaction is a reaction mechanism of AHC compounds and DNA in the mutagenic process. The method allows for discrimination among structurally similar compounds by combination with quantitative structure-activity relationships. The combination approach is expected to be useful for the mutagenic assessment of pharmaceutical impurities.

Establishment of an in silico phospholipidosis prediction method using descriptors related to molecular interactions causing phospholipid-compound complex formation.

Although phospholipidosis (PLD) often affects drug development, there is no convenient in vitro or in vivo test system for PLD detection. In this study, we developed an in silico PLD prediction method based on the PLD-inducing mechanism. We focused on phospholipid (PL)-compound complex formation, which inhibits PL degradation by phospholipase. Thus, we used some molecular interactions, such as electrostatic interactions, hydrophobic interactions, and intermolecular forces, between PL and compounds as descriptors. First, we performed descriptor screening for intermolecular force and then developed a new in silico PLD prediction using descriptors related to molecular interactions. Based on the screening, we identified molecular refraction (MR) as a descriptor of intermolecular force. It is known that ClogP and most-basic pKa can be used for PLD prediction. Thereby, we developed an in silico prediction method using ClogP, most-basic pKa, and MR, which were related to hydrophobic interactions, electrostatic interactions, and intermolecular forces. In addition, a resampling method was used to determine the cut-off values for each descriptor. We obtained good results for 77 compounds as follows: sensitivity = 95.8%, specificity = 75.9%, and concordance = 88.3%. Although there is a concern regarding false-negative compounds for pKa calculations, this predictive ability will be adequate for PLD screening. In conclusion, the mechanism-based in silico PLD prediction method provided good prediction ability, and this method will be useful for evaluating the potential of drugs to cause PLD, particularly in the early stage of drug development, because this method only requires knowledge of the chemical structure.

Small-Molecule Stabilization of the 14-3-3/Gab2 Protein-Protein Interaction (PPI) Interface.

Small-molecule modulation of protein-protein interactions (PPIs) is one of the most promising new areas in drug discovery. In the vast majority of cases only inhibition or disruption of PPIs is realized, whereas the complementary strategy of targeted stabilization of PPIs is clearly under-represented. Here, we report the example of a semi-synthetic natural product derivative--ISIR-005--that stabilizes the cancer-relevant interaction of the adaptor protein 14-3-3 and Gab2. The crystal structure of ISIR-005 in complex with 14-3-3 and the binding motif of Gab2 comprising two phosphorylation sites (Gab2pS210pT391) showed how the stabilizing molecule binds to the rim-of-the-interface of the protein complex. Only in the direct vicinity of 14-3-3/Gab2pT391 site is a pre-formed pocket occupied by ISIR-005; binding of the Gab2pS210 motif to 14-3-3 does not create an interface pocket suitable for the molecule. Accordingly, ISIR-005 only stabilizes the binding of the Gab2pT391 but not the Gab2pS210 site. This study represents structural and biochemical proof of the druggability of the 14-3-3/Gab2 PPI interface with important implications for the development of PPI stabilizers.

Establishment of an in silico phototoxicity prediction method by combining descriptors related to photo-absorption and photo-reaction.

One of the mechanisms of phototoxicity is photo-reaction, such as reactive oxygen species (ROS) generation following photo-absorption. We focused on ROS generation and photo-absorption as key-steps, because these key-steps are able to be described by photochemical properties, and these properties are dependent on chemical structure. Photo-reactivity of a compound is described by HOMO-LUMO Gap (HLG), generally. Herein, we showed that HLG can be used as a descriptor of the generation of reactive oxygen species. Moreover, the maximum-conjugated π electron number (PENMC), which we found as a descriptor of photo-absorption, could also predict in vitro phototoxicity. Each descriptor could predict in vitro phototoxicity with 70.0% concordance, but there was un-predicted area found (gray zone). Interestingly, some compounds in each gray zone were not common, indicating that the combination of two descriptors could improve prediction potential. We reset the cut-off lines to define positive zone, negative zone and gray zone for each descriptor. Thereby we overlapped HLG and PENMC in a graph, and divided the total area to nine zones with cut-off lines of each descriptor. The rules to prediction were decided to achieve the best concordance, and the concordances were improved up to 82.8% for self-validation, 81.6% for cross-validation. We found common properties among false positive or negative compounds, photo-reactive structure and photo-allergenic, respectively. In addition, our method could be adapted to compounds rich in structural diversity using only chemical structure without any statistical analysis and complicated calculation.

Inhibition of rapamycin-induced Akt phosphorylation by cotylenin A correlates with their synergistic growth inhibition of cancer cells.

Cotylenin A, a plant growth regulator, and rapa-mycin, an inhibitor of the mammalian target of rapamycin, are potent inducers of differentiation in myeloid leukemia cells and also synergistically inhibit the proliferation of several human breast cancer cell lines including MCF-7 in vitro and in vivo. However, the mechanisms of the combined effects of cotylenin A and rapamycin are still unknown. Activated Akt induced by rapamycin has been suggested to attenuate the growth-inhibitory effects of rapamycin, serving as a negative feedback mechanism. In this study, we found that cotylenin A could suppress rapamycin-induced phosphorylation of Akt (Ser473) in MCF-7 cells and lung carcinoma A549 cells and that cotylenin A also enhanced the rapamycin-induced growth inhibition of MCF-7 and A549 cells. ISIR-005 (a synthetic cotylenin A-derivative) was able to enhance rapamycin­induced growth inhibition and could also markedly inhibit rapamycin-induced phosphorylation of Akt. We also found that the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) or arsenic trioxide (ATO) in combination with rapamycin markedly inhibited the growth of MCF-7 cells and 17-AAG or ATO suppressed rapamycin-induced phosphorylation of Akt. The PI3K inhibitor LY294002 also suppressed rapamycin-induced phosphorylation of Akt and combined treatment showed synergistic growth inhibition of MCF-7 cells. Rapamycin inhibited growth more significantly in Akt siRNA-transfected MCF-7 cells than in control siRNA-transfected MCF-7 cells. These results suggest that the inhibition of rapamycin-induced Akt phosphorylation by cotylenin A correlates with their effective growth inhibition of cancer cells.