Investigation of comet assays under conditions mimicking ocular instillation administration in a three-dimensional reconstructed human corneal epithelial model.

Purpose: A comet assay is one of the genotoxicity methods for evaluating the potential of chemicals to induce DNA strand breaks. To investigate the usefulness of comet assays for evaluating the genotoxic potential of ophthalmic solutions, a three-dimensional (3D) reconstructed human corneal epithelial model (3D corneal model) was exposed to conditions mimicking topical ocular instillation administration. Methods: The 3D corneal model was exposed to acridine orange, ethidium bromide, hydrogen peroxide, 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat), 4-nitroquinoline 1-oxide (4-NQO), acrylamide and methyl methanesulfonate (MMS). To mimic the ocular surface condition to which ophthalmic solutions are administered, the exposure time was set to 1 minute. Likewise, human corneal epithelial (HCE-T) cells, as monolayer cultured cells, were exposed to the same chemicals, for comparison. Results: In the 3D corneal model, the amount of DNA fragments was statistically significantly increased in cells treated with each of the test chemicals except acrylamide. In HCE-T cells, the amount of DNA fragments was statistically significantly increased in acridine orange-, ethidium bromide-, hydrogen peroxide-, 4-NQO- and MMS-treated cells but not in paraquat- or acrylamide-treated cells. In the 3D corneal model, the lowest concentrations at which we observed DNA damage were about 100 times higher than the concentrations in HCE-T cells. Since the 3D corneal model is morphologically similar to human corneal tissue, form a multilayer and having tight junctions, it may be that the test chemicals only permeated about 1% into the 3D corneal model. Conclusion: These results suggest that the comet assay using 3D cell culture models may reflect in vivo conditions better than do monolayer cultured cells, and that the comet assay may be useful for the evaluation of genotoxic potential of topical ophthalmic solution.

Clinical Trial of Radiotherapy After Intravenous Injection of Acridine Orange for Patients with Cancer.

AIM: We previously found that low-dose X-ray treatment after systemic administration of acridine orange (AO), which is known to have a low toxicity in animals, inhibited tumor growth in experimental studies using mouse osteosarcoma. In this pilot study, we planned to verify the toxicity of intravenous injection of low-dose AO in humans and investigate the anticancer effect of radiation after systemic AO administration (iAOR) for human cancer. PATIENTS AND METHODS: Eight patients with terminal cancer were treated with iAOR. RESULTS: None of the patients exhibited an adverse effect from AO injection. Three out of the five patients who received a full course of iAOR exhibited clinical or image-based responses, whereas two patients did not. CONCLUSION: The systemic administration of AO was confirmed not to be toxic in humans, and iAOR was suggested to be potentially effective against radioresistant cancer.

Acridine Orange based platinum(II) complexes inducing cytotoxicity and cell cycle perturbation in spite of GSTP1 up-regulation.

A series of new ionic Pt(II) complexes of general formula [Pt(II)(A)n(Cl)(AO)]X (A=en, NH3; n=1, 2; X-=BF4-, NO3-, PF6-, CF3SO3-), 1-5, containing Acridine Orange (AO) bound to the metal atom through the endocyclic N atom, have been tested in human melanoma cells (M14, JR8 and PLF2), human neuroblastoma cell line SH-SY5Y and its cis-platin resistant subline SH-SY5Yres. The Pt(II) compounds, and in particular complexes 1 and 4, exhibit higher cytotoxic activity at lower concentration compared to cis-DDP in melanoma cells, affecting cell growth behavior and causing cell cycle perturbation. Moreover, M14 and JR8 cell lines were not able to rescue the impairment due to the new Pt(II) complexes since perturbation of cell cycle phases and cell proliferation inhibition were found after 72 h of recovery time. In order to evaluate whether GSTP1 may play a role in chemo-resistance of our melanoma model, we investigated the effect of the treatment with these Pt(II) compounds on GSTP1 gene expression. Up-regulation of GSTP1, evaluated by Qreal-time PCR was observed after treatment with complexes 1 and 4, showing that the effect of these Pt(II) compounds is GSTP1 indipendent. The lack of resistance of the new Pt(II)-AO complexes and their cytotoxicity, cell growth and cell cycle recovery in melanoma cells provide the basis for the development of new platinum anticancer compounds, directed to those tumors that over express GSTs enzymes.

Extracorporeal photodynamic image detection of mouse osteosarcoma in soft tissues utilizing fluorovisualization effect of acridine orange.

Various imaging methods have been employed for the extracorporeal detection of malignant tumors in the human body, such as scintigraphy and PET; however, none is sufficiently accurate and all are also very expensive. To resolve these issues, we attempted to develop a new imaging technique of photodynamic diagnosis (PDD) with acridine orange (AO). AO has the ability to rapidly and specifically accumulate in malignant tumors and emit brilliant green fluorescence after blue light excitation. In this study, we investigated the feasibility of PDD utilizing the fluorovisualization effect of AO, for the extracorporeal detection of mouse osteosarcoma inoculated into the soft tissues. At 2 h after intravenous administration of 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 mg/kg AO, the tumor and the surrounding normal tissues were illuminated by blue light. The visual fluorescence contrast and ratio (X) of the difference in fluorescence intensity between the tumor and the surrounding normal tissues were evaluated using a high-resolution digital camera equipped with an absorption filter. In addition, the fluorescence contrast was also detected sequentially at 0.5, 1, 2, 3, 6 and 12 h after intravenous administration of AO at 1.0 mg/kg. The results revealed that the optimal condition for clear detection of the tumor was evaluation 2 h after intravenous injection of AO at 0.1 mg/kg, because it provided the best visual contrast on the digital images, and the fluorescence intensity as well as the value of X were higher as compared to the values under other conditions of dose and timing. Based on the results of an acute toxicity study of AO, the estimated LD50 of this substance following intravenous administration was 27.30 mg/kg. In conclusion, we believe that PDD using AO administered intravenously may be feasible for the detection of human musculoskeletal sarcomas in the soft tissues at extremities, and this technique might be a less invasive, less expensive, quicker and more accurate imaging modality than other previously reported imaging methods for this purpose.

An evaluation of novel vital dyes for intraocular surgery.

PURPOSE: To evaluate systematically the staining characteristics and safety of potential new dyes for intraocular surgery. METHODS: Six dyes were included in the investigation: light green SF (LGSF) yellowish, E68, bromophenol blue (BPB), Chicago blue (CB), rhodamine 6G, rhodulinblau-basic 3 (RDB-B3). All dyes were dissolved and diluted in a balanced saline saline solution. The light-absorbing properties of each dye were measured at a concentration of 0.05% between 200 and 1000 nm. Staining characteristics were examined by staining lens capsule tissue and epiretinal membranes (ERMs), removed intraoperatively, with dye concentrations of 1.0%, 0.5%, 0.2%, and 0.05%. Enucleated porcine eyes (postmortem time, 9 hours) were also stained. Dye-related toxicity was evaluated by a colorimetric test (MTT) measuring the inhibition of retinal pigment epithelium (RPE) cell proliferation (ARPE-19 and primary human RPE cells, passages 3-6). Cell viability was also quantified based on a two-color fluorescence cell-viability assay. Dyes were investigated in concentrations of 0.2% and 0.02%. RESULTS: All dyes investigated in this study stained human lens capsules, removed intraoperatively; ERMs, peeled during macular pucker surgery; and enucleated porcine eyes, depending on the concentration applied. The long-wavelength absorption maximum of the dyes was within the range of 527 to 655 nm at concentrations of 0.05%. Rhodamine G6 and RDB-B3 showed adverse effects on ARPE-19 cell proliferation at a concentration of 0.2% and were excluded from further investigation in primary RPE cells. The remaining four dyes showed no toxic effect on ARPE-19 and primary RPE cell proliferation at concentrations of 0.2% and 0.02%. Cell viability was affected by LGSF yellowish (0.2%) and CB (0.2% and 0.02%). Two dyes (E68 and BPB) showed no relevant toxicity in vitro. CONCLUSIONS: The systematic evaluation of dyes for intraocular use seems mandatory. In this study four dyes were identified with effective staining characteristics, with two of these dyes having no detectable toxic effect on RPE cells in vitro.

De-intercalation of ethidium bromide and acridine orange by xanthine derivatives and their modulatory effect on anticancer agents: a study of DNA-directed toxicity enlightened by time correlated single photon counting.

Time Correlated Single Photon Counting (TCSPC) was used for the first time to analyze the effect/changes in the mode of intercalation of ethidium bromide (EtBr) and acridine orange (AO) to calf thymus DNA brought about due to interaction of naturally occurring methylxanthines such as theophylline (X1), theobromine (X2) and caffeine (X3). UV absorption and fluorescence studies were also carried to observe the behaviour of these xanthines on the modulation of the binding mode of anticancer agents (cisplatin, novantrone, and actinomycin D) and certain intercalating dyes (EtBr and AO) to DNA. In TCSPC analysis we found that when the concentration of the drugs (X1, X2 and X3) increased from 0.025 mM to 2 mM i.e. P/D 2.4 to P/D 0.03 reduction in intercalation of EtBr and AO was observed, suggesting that xanthine derivatives could play very important role in reducing the DNA-directed toxicity in a dose dependent manner. In TCSPC, the amplitude of smaller lifetime component A(1) and higher lifetime component A(2) are attributed to free and intercalated dye concentration and their variation could indicate the process of intercalation or reduced intercalation of EtBr and AO by xanthine derivatives. We found that at the maximum drug concentration the smaller lifetime component A(1) was increased by 7-8% and 17-37% in EtBr and AO intercalated complex respectively. Also the changes in lifetime and fluorescence decay profile were observed for the DNA-intercalated dyes before and after treatment with xanthines. Especially, at maximum P/D 0.03 the lifetime of DNA-intercalated EtBr and AO reduced by 1-2 ns. The present analysis reveals that xanthines are able to interact with free dyes and also with intercalated dyes, suggesting that when they interact with free dyes they might inhibit the further intercalation of dye molecules to DNA and the interaction with intercalated dyes might lead to displacement of the dyes resulting in de-intercalation. The results obtained from UV and fluorescence spectroscopy also support the present investigation of probable interaction of xanthines with the DNA damaging agents in modulating/reducing the DNA-directed toxicity.

Polyploidization induced by acridine orange in mouse osteosarcoma cells.

This study was undertaken to clarify the in vitro effect of acridine orange (AO) on the cell kinetics of mouse osteosarcoma cells, as well as the mechanism of cell growth inhibition induced by AO. A mouse osteosarcoma cell line (MOS), established from a radiation-induced mouse osteosarcoma, was cultured under exposure to 0.05, 0.5, 5, and 50 micrograms/ml of AO, either continuously or for 10 minutes. The cell kinetic analysis was performed using the following parameters: tumor cell growth by trypan blue exclusion test, mitotic activity, DNA synthetic activity by BrdU labeling and DNA ploidy by cytofluorometry. The results showed that continuous exposure to 5 and 50 micrograms/ml of AO or 10 minute exposure to 50 micrograms/ml of AO quickly killed the tumor cells within 12 hours, whereas continuous exposure to 0.5 microgram/ml of AO or 10 minute exposure to 5 micrograms/ml of AO gradually inhibited tumor cell growth. Under the latter conditions, mitotic activity was rapidly and completely inhibited within 48 hours but DNA synthetic activity was not completely inhibited even after 96 hours. DNA ploidy analysis demonstrated that most of the tumor cells arrested at the S-G2 phase after 12 hours, followed by G2 phase arrest after 24 hours and progressive DNA synthesis to a higher DNA ploidy class after 48 to 96 hours. We therefore concluded that a high concentration of AO has a strong cytocidal effect due to cytotoxicity whilst a moderate concentration of AO induces progressive and synchronous polyploidization by mitotic inhibition without DNA damage in MOS cells. We presume that this in vitro effect on MOS cells may be caused by protein synthetic inhibition after transfer RNA inactivation caused by AO binding.

Antimutagenicity of lignin in vitro.

The inhibitory activity of lignin against nitrosoguanidine (MNNG)- and acridine orange (AO)- induced mutagenesis was examined using two microbial systems: green unicellular flagellate Euglena gracilis and Salmonella typhimurium TA100 and TA97. To verify the hypothesis that the above mentioned mutagens may generate some oxidant species and subsequently free radicals, or they may interact with lignin, two physico-chemical measurements were performed. Lignin at a tested concentration (100 micrograms/ml) decreases Euglena-bleaching activity of MNNG by 67.7% and AO by 99.7%. Percentage of MNNG-induced revertants of S. typhimurium was also decreased substantially by lignin. We conclude that our results indicate the possible mechanisms behind the antimutagenic/anticarcinogenic effects of lignin: namely, scavening of reactive oxygen species produced by MNNG and binding of AO itself.

Comparison of acridine orange and Giemsa stains in several mouse bone marrow micronucleus assays--including a triple dose study.

Data from four mouse bone marrow micronucleus assays are presented in which sister slides were stained with Giemsa or Acridine Orange (AO). These data are used to support our decision to change from Giemsa stain to AO when conducting rodent bone marrow micronucleus assays. The AO method leads to definitive assessments of the incidence of micronucleated polychromatic erythrocytes on a slide, and in so doing, it saves reading time and increases confidence in data. Preliminary experiments with DMBA using the triple-dose/single sample protocol of MacGregor are also described, and it is concluded that this protocol shows great promise.