Neeru Kumar

Antibody-targeted photodynamic therapy

PURPOSE To assess the feasibility of conjugation of verteporfin (Visudyne, Parkedale Pharmaceuticals, Rochester, Minnesota, USA) to antibody against vascular endothelial growth factor. DESIGN Experimental study. METHODS Rabbit antimouse vascular endothelial growth factor polyclonal antibody was conjugated to verteporfin. Fluorescence excitation-emission spectra of verteporfin and conjugate were examined. Vascular endothelial growth factor-expressing murine endothelial cells were incubated with saline, verteporfin, or conjugate, followed by laser exposure or no laser exposure. Cell viability at 1 and 24 hours was assessed via trypan blue exclusion. Results were analyzed by two-way analysis of variance with replication and the Bonferroni multiple comparison test. RESULTS The fluorescence excitation-emission spectrum of the conjugate was similar to that of verteporfin. After laser exposure, cell viability in conjugate-treated cells was reduced to 6% at 1 hour (P <.0001) and to 4% at 24 hours (P <.0001), compared with approximately 40% in nonlaser-exposed, conjugate-treated cells. The cytotoxicity in the conjugate-treated cells was higher than in verteporfin-treated cells exposed to laser, although the difference did not reach statistical significance. CONCLUSIONS The conjugation of verteporfin to polyclonal antibody is possible without the loss of its photosensitizing properties. Conjugated verteporfin destroys cellular targets at least as effectively as verteporfin alone.

An adaptable HPLC method for the analysis of frequently used antibiotics in ocular samples

Four different antibiotics, delivered individually to rabbit eyes via hydrophilic intraocular lenses soaked in the drug solution prior to implantation, were measured in aqueous and vitreous humor samples from the eyes. To meet this analytical need, we developed a sensitive, high performance liquid chromatographic (HPLC) method for measuring the concentrations of moxifloxacin, gatifloxacin, linezolid, and cefuroxime in the ocular tissue. Separations were carried out on a LichroSpher RP-18 column, maintained at room temperature. The fluoroquinolones were eluted with a mobile phase consisting of 20% acetonitrile, in 0.1% trifluoroacetic acid (pH 3.0) with 30 mM tetrabutylammonium chloride. Linezolid and cefuroxime were eluted with 25% acetonitrile in 25 mM Na acetate buffer, pH 5.0. All elutions were isocratic. With ultraviolet detection, the lower limit of quantitation (LLOQ) for these compounds approached 1 ng (on-column injection). By using fluorescence detection, the LLOQ for the fluoroquinolones improved to 200 pg. The overall accuracy of the method was >or=90%. With minor modifications, the method was optimized for each of the agents, and the resulting analytical sensitivity made the method suitable for clinical investigations of the ocular penetration of these drugs.

Ursolic acid differentially modulates apoptosis in skin melanoma and retinal pigment epithelial cells exposed to UV–VIS broadband radiation

The signaling pathways via mTOR (mammalian target of rapamycin) and AMPK (AMP-activated protein kinase) play key roles in transcription, translation and carcinogenesis, and may be activated by light exposure. These pathways can be modulated by naturally occurring compounds, such as the triterpenoid, ursolic acid (UA). Previously, the transcription factors p53 and NF-κB, which transactivate mitochondrial apoptosis-related genes, were shown to be differentially modulated by UA. UA-modulated apoptosis, following exposure to UV–VIS radiation (ultraviolet to visible light broadband radiation, hereafter abbreviated to UVR), is observed to correspond to differential levels of oxidative stress in retinal pigment epithelial (RPE) and skin melanoma (SM) cells. The cellular response to this phytochemical was characterized using western blot, flow cytometry, microscopy with reactive oxidative species probes MitoTracker and dihydroethidium, and membrane permeability assay. UA pretreatment potentiated cell cycle arrest and UVR-induced apoptosis selectively in SM cells while reducing photo-oxidative stress in the DNA of RPE cells presumably by antioxidant activity of UA. Mechanistically, the nuclear transportation of p65 and p53 was reduced by UA administration prior to UVR exposure while the levels of p65 and p53 nuclear transportation in SM cells were sustained at a substantially higher level. Finally, the mitochondrial functional assay showed that UVR induced the collapse of the mitochondrial membrane potential, and this effect was exacerbated by rapamycin or UA pretreatment in SM preferentially. These results were consistent with reduced proliferation observed in the clonogenic assay, indicating that UA treatment enhanced the phototoxicity of UVR, by modulating the activation of p53 and NF-κB and initiating a mitogenic response to optical radiation that triggered mitochondria-dependent apoptosis, particularly in skin melanoma cells. The study indicates that this compound has multiple actions with the potential for protecting normal cells while sensitizing skin melanoma cells to UV irradiation.

Revisiting the internal absorption coefficient of the retinal pigment epithelium melanosome

Retinal pigment epithelial (RPE) cells of the vertebrate eye contain melanin packaged in structures called melanosomes. Previously, we reported that the bovine melanosome has a laser photodisruption threshold of 153.6 mJ/cm2 at 25°C and 532 nm, and an internal absorption coefficient of 2237 cm-1. Those values used an estimate of melanosome density inferred from studies of skin laser effects. We now revisit that calculation using a density value obtained from density centrifugation analysis of bovine and baboon RPE melanosomes. Stepped-density gradients (60% to 80%) of the nonionic medium, Nycodenz, were formed, and samples of melanosomes were spun on the gradients in a swinging-bucket rotor at 10,000 rpm for 60 m. Bovine melanosomes formed two populations, one at the interface between 70% and 75%, and the other between 75% and 80%, corresponding to the densities of ~1.38 and ~1.41 gm/cm3, respectively. Baboon melanosomes migrated to within the same density region. Using a density value of 1.41 gm/cm3, and assuming a water content of 52% for hydrated melanosomes, the internal absorption coefficient was calculated as 2339 cm-1. Although this calculation uses an objective density measurement, the water content remains an estimate, and the actual value in situ may differ.

Modulation of photochemical damage in normal and malignant cells by naturally occurring compounds.

Certain phytochemicals, such as the stilbene, resveratrol (RES, found in red grapes and berries), and the triterpenoid, ursolic acid (UA, found in waxy berries and herbs such as rosemary and oregano), have antioxidant, anti‐inflammatory and antiproliferative effects. Two human‐derived cell lines, hTERT‐RPE with a nonmalignant phenotype derived from retinal pigment epithelium, and ATCC CRL‐11147 derived from a malignant skin melanoma, were used as in vitro models of photooxidative stress produced by exposure to the broadband output of a 150 W Hg vapor arc lamp at an irradiance of 19–26 mW cm−2. In untreated cells, UV–VIS broadband light exposure produced a loss of proliferative ability, an activation of NF‐κB and an increase in protein carbonyl adducts at 24 h postexposure. Pretreatment of the cells with RES or UA at 1–2 μmsignificantly reduced the amount of phosphorylated NF‐κB at 24 h postexposure. RES pretreatment reduced the burden of light‐induced protein carbonyl adducts by up to 25% in exposed cells. UA treatment markedly increased the sensitivity of melanoma cells to UV radiation, while conferring some photoprotection to RPE cells. These observations indicate that phytochemicals modulate the cellular response to photochemical stress by interacting with specific cell‐signaling pathways.

EFFECTS OF THREE REPUTED CARBOXYLESTERASE INHIBITORS UPON RAT SERUM ESTERASE ACTIVITY

Rats have very high endogenous levels of serum carboxylesterase (CAE) compared to primates. This difference accounts for the lower sensitivity of rats to toxic organophosphates, which interact with CAE instead of the more critical acetylcholinesterase. Pretreatment of rats with CAE inhibitors potentiates the effects of organophosphates. In this study, the effects of three putative CAE inhibitors, 2-(o-Cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide (CBDP), bis-p-nitrophenyl-phosphate (BNPP), and tetraisopropyl pyrophosphoramide (Iso-OMPA), on the hydrolysis of several commercially available substrates were determined. Respective kinetic constants Km and Vmax were derived and effects of inhibitors compared using saturating amounts of substrate. Data presented here indicate significant differences in substrate affinity (Km), reactivity (Vmax), as well as effects of inhibitors. CBDP inhibits hydrolysis of specific naphthyl and paranitrophenyl esters at relatively low concentrations (1-10 microM). In contrast, significantly higher concentrations (mM) of BNPP and Iso-OMPA were required for inhibition of serum esterase activity. Of the inhibitors tested, Iso-OMPA in general exhibited the smallest inhibitory effect on ester hydrolysis. Although inhibition of hydrolysis of specific paranitrophenyl and naphthyl esters occurred in the presence of similar amounts of CBDP, the degree of inhibition differed significantly (50-75% vs. greater than 90%, respectively). These data suggest that there exists in rat serum, a pool of naphthyl ester esterase activity that is very sensitive ex vivo (greater than 90% inhibition) to CBDP and may be very useful in validating a rodent model for soman toxicity.

Laser bioeffects associated with ultrafast lasers: Role of multiphoton absorption

Although laser-induced breakdown has the lowest energy threshold in the femtosecond domain, and appears to be responsible for production of threshold retinal lesions by ultrashort pulse lasers, previous findings suggested a role for multiphoton absorption in retinal damage. In this study, we investigated if near infrared ultrashort laser pulses produced DNA damage by exciting short wavelength-absorbing chromophores via multiphoton absorption. The experiments were carried out in nonpigmented and artificially pigmented hTERT-RPE cells, a line of human-derived retinal pigment epithelial (RPE) cells. Cells were exposed to 0.25 s pulses of CW and mode-locked 810 nm laser emissions, at irradiances equivalent to 0.5 and 1.0 times the ED50 for threshold damage in the primate eye, and DNA strand breaks were measured using the comet assay (single cell gel electrophoresis). At 0.5 X ED50, increased DNA strand breakage occurred after the ultrashort (mode-locked) exposures, but not the CW exposures, compared to contro...

Intracellular signaling mechanisms responsive to laser-induced photochemical and thermal stress

The cells of the retinal pigment epithelium (RPE) are subject to photo-oxidative stress arising from the interaction of incident light with lipofuscin, melanin, and other pigment granules in the RPE cytoplasm. Specific genotypic responses to these stressors are controlled by transcription factors, such as NF-κB (RelA/p50 dimer). The effects of CW laser exposures on NF-κB nuclear translocation have been studied in a line of human-derived RPE cells (hTERT-RPE) that develop melanin pigmentation in culture. The cells were exposed to the CW emission of an Argon-ion laser for 10 m at 0.5 W/cm2, a range previously shown to produce oxidation of cellular proteins, DNA, and antioxidants. NF-κB dimer was measured in nuclear extracts by an electrophoretic mobility shift assay. NF-κB nuclear translocation exhibited a modest, early peak at 1 h, and a larger, late peak at 24 h. NF-κB activation could be reduced only by some antioxidants; for example, 20 mM N-acetyl-L-cysteine or 100 μM pyrrolidine dithiocarbamate were ineffective, while 500 μM ascorbic acid was highly effective. These results indicate that interaction of the laser with the RPE melanin granules is a likely source of oxidative reactions, and that the induction of photoxidative stress activates NF-κB, but it remains to be determined if NF-κB is pro- or anti-apoptotic in the RPE cell.

Pulsewidth-dependent nature of laser-induced DNA damage in RPE cells

Ultrashort pulse laser radiation may produce cellular damage through unique mechanisms. Primary cultures of bovine retinal pigment epithelial (RPE) cells were exposed to the out put of a Ti:Sapphire laser producing 30 fs (mode-locked) pulses, 44 amplified fs pulses, or continuous wave exposures at 800 nm. Laser exposures at and below the damage threshold were studied. DNA damage was detected using single cell gel electrophoresis (comet assay). Unexposed (control) cells produced short tails with low tail moments. In contrast, all laser-exposed cells showed some degree of DNA fragmentation, but the size and shape of the resulting comets differed among the various modalities. CW-exposed cells produced generally light and relatively compact tails, suggesting fewer and larger DNA fragments, while mode-locked laser exposures (30 fs pulses) resulted in large and diffuse comets, indicating the DNA was fragmented into many very small pieces. Work is continuing to define the relationship of laser pulsewidth and intensity with the degree of DNA fragmentation. These results suggest that DNA damage may result from multiple mechanisms of laser-cell interaction, including multiphoton absorption.

Ursolic acid mediates photosensitization by initiating mitochondrial-dependent apoptosis

The signaling pathways PI3K/Akt and MAPK play key roles in transcription, translation and carcinogenesis, and may be activated by light exposure. These pathways may be modulated or inhibited by naturally-occurring compounds, such as the triterpenoid, ursolic acid (UA). Previously, the transcription factors p53 and NF-kB, which transactivate mitochondrial apoptosis-related genes, were shown to be differentially modulated by UA. Our current work indicates that UA causes these effects via the mTOR and insulin-mediated pathways. UA-modulated apoptosis, following exposure to UV radiation, is observed to correspond to differential levels of oxidative stress in retinal pigment epithelial (RPE) and skin melanoma (SM) cells. Flow cytometry analysis, DHE (dihydroethidium) staining and membrane permeability assay showed that UA pretreatment potentiated cell cycle arrest and radiation-induced apoptosis selectively on SM cells while DNA photo-oxidative damage (i.e. strand breakage) was reduced, presumably by some antioxidant activity of UA in RPE cells. The UA-mediated NF-κB activation in SM cells was reduced by rapamycin pretreatment, which indicates that these agents exert inter-antagonistic effects in the PI3K/Akt/mTOR pathway. In contrast, the antagonistic effect of UA on the PI3K/Akt pathway was reversed by insulin leading to greater NF-κB and p53 activation in RPE cells. MitoTracker, a mitochondrial functional assay, indicated that mitochondria in RPE cells experienced reduced oxidative stress while those in SM cells exhibited increased oxidative stress upon UA pretreatment. When rapamycin administration was followed by UA, mitochondrial oxidative stress was increased in RPE cells but decreased in SM cells. These results indicate that UA modulates p53 and NF-κB, initiating a mitogenic response to radiation that triggers mitochondria-dependent apoptosis.