Improved Formazan Dissolution for Bacterial MTT Assay.

The MTT assay, based on the enzymatic reduction of the water-soluble, yellowish tetrazolium salt 3-(4,5-dimethylthiazol)-2,5-diphenyl-tetrazolium bromide (MTT) to purple formazan, is commonly used for assessment of cell viability and proliferation. Accurate performance by the MTT assay depends on complete solubilization of cells and formazan and stability of the colored solution. Comparison of different solubilization solutions revealed that dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), buffered with ammonia buffer, pH 10, and containing 5% SDS, produced the best results. These two solvents provided rapid and complete solubilization of formazan and cells, with minimal background absorbance at 700 nm, good reproducibility (low interassay coefficient of variation), high sensitivity, and color stability for at least 24 h. A linear relationship between viable-cell number and formazan absorbance was preserved for cell densities up to ∼1 × 109 cells/mL for Gram-negative and Gram-positive microorganisms. Since MTT can be reduced by medium components in the absence of cells, blanks containing all medium components but no cells should be run simultaneously. Measurements at two wavelengths, one corresponding to absorption peak of formazan (570 nm) and a background absorbance far from the peak (700 nm), are necessary to avoid artifacts due to incomplete solubilization and turbidity. IMPORTANCE Reduction of the water-soluble tetrazolium salt 3-(4,5-dimethylthiazol)-2,5 diphenyl-tetrazolium bromide (MTT) to purple, water-insoluble formazan is commonly used for assessment of cell viability and proliferation. Spectrophotometric detection of formazan requires its solubilization. The solubilization solvent has a strong influence on data acquisition and often introduces artifacts, leading to misreading of results. This study offers a choice of solvents that minimize solubilization artifacts when the MTT test is applied to microbiological cultures.

Nano-sized formazan analogues: Synthesis, structure elucidation, antimicrobial activity and docking study for COVID-19.

Three series of nanosized-formazan analogues were synthesized from the reaction of dithiazone with various types of α-haloketones (ester and acetyl substituted hydrazonoyl chlorides and phenacyl bromides) in sodium ethoxide solution. The structure and the crystal size of the new synthesized derivatives were assured based on the spectral analyses, XRD and SEM data. The antibacterial and antifungal activities were evaluated by agar diffusion technique. The results showed mild to moderate antibacterial activities and moderate to potent antifungal activities. Significant antifungal activities were observed for four derivatives 3a, 3d, 5a and 5g on the pathogenic fungal strains; Aspergillus flavus and Candida albicans with inhibition zone ranging from 16 to 20 mm. Molecular docking simulations of the synthesized compounds into leucyl-tRNA synthetase editing domain of Candida albicans suggested that most formazan analogues can fit deeply forming stable complexes in the active site. Furthermore, we utilized the docking approach to examine the potential of these compounds to inhibit SARS-CoV-2 3CLpro. The results were very promising verifying these formazan analogues as a hopeful antiviral agents.

Tetrazolium reduction assays under-report cell death provoked by clinically relevant concentrations of proteasome inhibitors.

High throughput cell viability screening assays often capitalize on the ability of active enzymes or molecules within viable cells to catalyze a quantifiable chemical reaction. The tetrazolium reduction (MTT) assay relies on oxidoreductases to reduce tetrazolium into purple formazan crystals that are solubilized so absorbance reflects viability, while other assays use cellular ATP to catalyze a luminescence-emitting reaction. It is therefore important to know how accurately these assays report cellular responses, as cytotoxic anti-cancer agents promote cell death via a variety of signaling pathways, some of which may alter how these assays work. In this study, we compared the magnitude of cytotoxicity to different cell types provoked by currently used anti-cancer agents, using three different cell viability assays. We found the three assays were consistent in reporting the viability of cells treated with chemotherapy drugs or the BH3 mimetic navitoclax, but the MTT assay underreported the killing capacity of proteasome inhibitors. Additionally, the MTT assay failed to confirm the induction of caspase-mediated cell death by bortezomib at physiologically relevant concentrations, thereby mischaracterizing the mode of cell death. While the cell viability assays used allow for the rapid identification of novel cytotoxic compounds, our study emphasizes the importance for these screening assays to be complemented with a direct measure of cell death or another independent measure of cell viability. We caution researchers against using MTT assays for monitoring cytotoxicity induced by proteasome inhibitors.

Development of gallic acid formazans as novel enoyl acyl carrier protein reductase inhibitors for the treatment of tuberculosis.

The enoyl acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis (MTB) is an attractive target for developing novel antitubercular agents. A series of gallic acid formazans, were computationally designed and docked into the active site of InhA to understand their binding mode and potential to inhibit InhA. Nine compounds from the designed series were identified as potential InhA inhibitors, on the basis of good Glide score. These compounds were synthesized in the laboratory and evaluated for in vitro antitubercular activity against drug-sensitive and multi-drug resistant strains of MTB. Out of nine compounds, three compounds exhibited the most promising MIC of <2µM against the sensitive strain of MTB, H37Rv. The compounds were evaluated against five resistant strains of MTB. Most of the compounds exhibited activity superior to the standard, linezolid, against all these resistant strains. The mechanism of action of these compounds was concluded to be InhA inhibition, through InhA enzyme inhibition study. Insignificant cytotoxicity of these compounds was observed on RAW 264.7 cell line. Inactivity of all these compounds against gram positive and gram negative bacteria indicated their specificity against MTB. The compounds were further analyzed for ADME properties and showed potential as good oral drug candidates. The results clearly identified some novel, selective and specific InhA inhibitors against sensitive and resistant strains of MTB.

Anti-inflammatory, Analgesic and Anti-ulcerogenic Activities of Novel bis-thiadiazoles, bis-thiazoles and bis-formazanes.

BACKGROUND: Indane-1,3-dione, thiazole, bis-thiazole and thiadiazoles rings are very interested moieties in anti-inflammatory and analgesic drugs. OBJECTIVE: The goal of this work is to synthesize new derivatives of bis-thiazoles and bis-1,3,4- thiadiazoles for the investigation of their anti-inflammatory, anti-ulcerogenic and analgesic activities. METHODS: 1,1'-(1,2-phenylene)bis(3-phenylthiourea) (1) reacts with a number of N-aryl arenecarbohydrazonoyl chlorides 2 to give a series of new bis-1,3,4-thiadiazoles 4. Also, reaction of bisthiosemicarbazone of 1,3-indanedione 6 with another type of hydrazonoyl halides namely, N-aryl-2- oxapropanehydrazonoyl chlorides 7 and ethyl-(N-arylhydrazono)chloroacetate 8 in dioxane under reflux in the presence of triethylamine give the respective bis-thiazole derivatives 9 and 10, respectively. The products 9 and 10 can exist in five and seven tautomeric forms for each one. Their actual tautomeric forms were deduced based on electronic absorption data (UV / Vis spectra). Moreover, a series of novel bis-formazans 12 and 13 have been synthesized by reaction of 1,3-dihydrazono-2,3- dihydro-1H-indene (11) with both hydrazonoyl chlorides 7 and 8. RESULTS: The structure of all the novel products was deduced by elemental analysis and spectral data. In addition, the biological activity of the newly synthesized compounds was evaluated and the results obtained indicate their potency as anti-inflammatory, anti-ulcerogenic and analgesic agents. CONCLUSION: In this context, we synthesize new derivatives of bis-thiazoles and bis-1,3,4-thiadiazoles as anti-inflammatory, anti-ulcerogenic and analgesic agents.

Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability.

The MTT assay (to a less degree MTS, XTT or WST) is a widely exploited approach for measuring cell viability/drug cytotoxicity. MTT reduction occurs throughout a cell and can be significantly affected by a number of factors, including metabolic and energy perturbations, changes in the activity of oxidoreductases, endo-/exocytosis and intracellular trafficking. Over/underestimation of cell viability by the MTT assay may be due to both adaptive metabolic and mitochondrial reprogramming of cells subjected to drug treatment-mediated stress and inhibitor off-target effects. Previously, imatinib, rottlerin, ursolic acid, verapamil, resveratrol, genistein nanoparticles and some polypeptides were shown to interfere with MTT reduction rate resulting in inconsistent results between the MTT assay and alternative assays. Here, to test the under/overestimation of viability by the MTT assay, we compared results derived from the MTT assay with the trypan blue exclusion assay after treatment of glioblastoma U251, T98G and C6 cells with three widely used inhibitors with the known direct and side effects on energy and metabolic homeostasis - temozolomide (TMZ), a DNA-methylating agent, temsirolimus (TEM), an inhibitor of mTOR kinase, and U0126, an inhibitor of MEK1/2 kinases. Inhibitors were applied shortly as in IC50 evaluating studies or long as in studies focusing on drug resistance acquisition. We showed that over/underestimation of cell viability by the MTT assay and its significance depends on a cell line, a time point of viability measurement and other experimental parameters. Furthermore, we provided a comprehensive survey of factors that should be accounted in the MTT assay. To avoid result misinterpretation, supplementation of the tetrazolium salt-based assays with other non-metabolic assays is recommended.

Cytotoxicity assessment of modified bioactive glasses with MLO-A5 osteogenic cells in vitro.

The primary objective of this study was to evaluate in vitro responses of MLO-A5 osteogenic cells to two modifications of the bioactive glass 13-93. The modified glasses, which were designed for use as cell support scaffolds and contained added boron to form the glasses 13-93 B1 and 13-93 B3, were made to accelerate formation of a bioactive hydroxyapatite surface layer and possibly enhance tissue growth. Quantitative MTT cytotoxicity tests revealed no inhibition of growth of MLO-A5 cells incubated with 13-93 glass extracts up to 10 mg/ml, moderate inhibition of growth with 13-93 B1 glass extracts, and noticeable inhibition of growth with 13-93 B3 glass extracts. A morphology-based biocompatibility test was also performed and yielded qualitative assessments of the relative biocompatibilities of glass extracts that agree with those obtained by the quantitative MTT test. However, as a proof of concept experiment, when MLO-A5 cells were seeded onto 13-93 B3 scaffolds in a dynamic in vitro environment, cell proliferation occurred as evidenced by qualitative and quantitative MTT labeling of scaffolds. Together these results demonstrate the in vitro toxicity of released borate ion in static experiments; however borate ion release can be mitigated in a dynamic environment similar to the human body where microvasculature is present. Here we argue that despite toxicity in static environments, boron-containing 13-93 compositions may warrant further study for use in tissue engineering applications.

Citrus auraptene suppresses cyclin D1 and significantly delays N-methyl nitrosourea induced mammary carcinogenesis in female Sprague-Dawley rats.

BACKGROUND: Breast cancer is a major problem in the United States leading to tens of thousands of deaths each year. Although citrus auraptene suppresses cancer in numerous rodent models, its role in breast cancer prevention previously has not been reported. Thus, our goal was to determine the anticarcinogenic effects of auraptene against breast cancer. METHODS: The effects of auraptene on cell proliferation of MCF-7 and MDA-MB-231 human breast carcinoma cells in culture was assessed by measuring metabolism of a substrate to a formazan dye. Dietary effects of auraptene on tumor incidence, multiplicity and latency were studied in the N-methyl nitrosourea (MNU) induced mammary carcinogenesis model in female Sprague Dawley rats. The concentration of auraptene in rat tissues was analyzed by reverse phase HPLC. Cyclin D1 expression in MCF-7 cells and rat tumors was measured by western blot. RESULTS: Auraptene (500 ppm) significantly delayed median time to tumor by 39 days compared to the MNU only group (p < 0.05, n = 24-26). Auraptene (10 microM) reduced Insulin like Growth Factor-1 (IGF-1, 10 ng/mL)-induced cyclin D1 expression by 40% in MCF-7 cells. In comparison, western blot analysis of rat mammary tumors (n = 10 per group) confirmed that auraptene (500 ppm) significantly reduced (p < 0.05) cyclin D1 expression by 49% compared to the MNU only group. Analysis of rat mammary tissue extract by HPLC with fluorescence detection indicated an average concentration (means +/- S.E.) of 1.4 +/- 0.5 microM and 1.8 +/- 0.3 microM in the normal mammary glands of the auraptene 200 ppm and 500 ppm groups, respectively. The concentration (means +/- S.E.) of auraptene in the mammary tumors of the auraptene 200 ppm group was 0.31 +/- 0.98 microM. CONCLUSION: Overall, these observations suggest that the predominant effect of auraptene was to delay the development of tumors possibly through the suppression of cyclin D1 expression. These results point to the potential chemopreventive effects of auraptene in mammary carcinogenesis.

Screen anticancer drug in vitro using resonance light scattering technique.

An in vitro screening model using resonance light scattering (RLS) technique with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent as the reactive probe to target cancer cell was firstly developed. In this model, MTT was reduced by viable cancer cells to produce a purple formazan. Cell viability was proportional to the number of formazan induced strong light scattering signal. The inhibition rate of anticancer drug was found to vary inversely with the H(22)-MTT system RLS intensity. So it was intuitive to see the sequence of the tumor suppressive activity of six anticancer drugs without data processing by RLS/MTT screening spectra. Compared with the traditional MTT method, this method has high sensitivity, low detection limit and quite intuitive screening results which were identical to those obtained from the MTT colorimetric assay.

Blockade of the aryl hydrocarbon receptor pathway triggered by dioxin, polycyclic aromatic hydrocarbons and cigarette smoke by Phellinus linteus.

Environmental pollutants including halogenated and polycyclic aromatic hydrocarbons activate the aryl hydrocarbon receptor (AhR) and thereby cause a wide range of pathological changes. Development of AhR antagonists will be useful for prevention and treatment of diseases related to AhR activation. Towards this end, we aimed in the present study at seeking for potential inhibitors of the AhR pathway in mycelial extracts using the dioxin responsive element-based sensing via secreted alkaline phosphatase (DRESSA). Through the screening of 13 mycelia, extracts prepared from Phellinus linteus, Cordyceps militaris and Hericium erinaceum inhibited activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin, benzo[a]pyrene or 3-methylcholanthrene. Subsequent studies revealed that only Phellinus linteus suppressed activation of AhR and AhR-dependent gene expression triggered by all of these agonists. Cigarette smoke is known to contain a number of halogenated and polycyclic aromatic hydrocarbons. We found that Phellinus linteus has the potential to block activation of AhR and AhR-dependent gene expression triggered by cigarette smoke. Furthermore, the inhibitory effect of Phellinus linteus on the AhR pathway was independent of; 1) depression of AhR or AhR nuclear translocator, and 2) induction of AhR repressor. We conclude that Phellinus linteus contains potent inhibitor(s) of AhR activation and may be useful for prevention of pathologies associated with aberrant activation of AhR.

The use of High Performance Thin-Layer Chromatography to determine the role of membrane lipid composition in bile salt-induced kidney cell damage.

INTRODUCTION: In obstructive liver disease bile salts are known to accumulate in and damage specific kidney cells. High Performance Thin-Layer Chromatography (HPTLC) was used to determine the membrane lipid composition of a range of kidney cells. METHODS: Kidney cells were exposed to three hydrophobic bile salts (lithocholic, deoxycholic and chenodeoxycholic acids) and cytotoxicity was determined. In addition membrane lipids from the cells were extracted in a chloroform:methanol (2:1, v/v) solution and quantified by HPTLC. RESULTS: The results reveal a differential toxicity to the bile acids with IC(50) values ranging from 79+/-5 microM to 394+/-13 microM. When the lipid composition of the most and least susceptible cells were assayed, the least susceptible cells had a much higher lipid composition (46.6+/-3.7 microg/mg protein compared to 28.1+/-5.2 microg/mg protein for the least susceptible cells). DISCUSSION: These results suggest that HPTLC may be a useful technique when determining the mechanisms of toxicity of compounds which cause the disruption of the cell membrane.

Microplate Alamar blue assay for susceptibility testing of Candida albicans biofilms.

Although biofilm-based fungal infections are an important cause of morbidity and mortality in patients, there is no standardized method for the in vitro evaluation of the drug susceptibility of biofilms. We investigated a high-throughput method for determining the susceptibility of Candida albicans biofilms that uses the oxidation reduction indicator Alamar blue (AB). Biofilms from the tested Candida albicans strains were markedly resistant to amphotericin B (AMB), nystatin (NYT), fluconazole (FLC) and 5-fluorouracil (5FC), but susceptible to Conflikt disinfectant. The latter was used in comparative studies of AB reduction with two other methods for assessing in vitro drug susceptibility i.e., 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction and enumeration of viable colony counts (CFU/ml). AB results correlated well with XTT (r=0.88-0.93) and CFU/ml (r=0.93-0.99) for all four C. albicans test strains. This simple, reproducible method for determining in vitro drug susceptibility should facilitate discovery of antifungals active against Candida biofilms.

A transmissible cytotoxic activity isolated from a patient with brain ischemia causes microglial cell activation and dysfunction.

1. Microglial cell activation occurs during brain injury, ischemia, and in several neurologic disorders. Recently, we isolated a transmissible cytotoxic activity (TCA) from the cerebrospinal fluid of a patient with brain ischemia. Such a TCA, associated with one or more protein(s) that supposedly had undergone in vivo misfolding, causes apoptosis in vitro in different cell lines, including microglial cells. The TCA producing cells and the potential in vivo role of such cytotoxic activity remains to be elucidated. Here, we investigated the in vitro effects of TCA on microglial cell immune functions.2. The murine microglial cell line RR4 was exposed to TCA, and then its response was evaluated as: (a) phagocytosis and antifungal activity against Candida albicans; (b) secretory pattern; and (c) levels of p38 phosphorylation.3. Unlike mock-treated controls, microglial cells exposed to TCA showed an increase in phagocytic activity. Unexpectedly, their capability to kill the ingested fungi significantly diminished. Moreover, TCA-treated cells produced amounts of macrophage inflammatory protein 1-alpha, tumor necrosis factor-alpha, and nitric oxide significantly higher than mock-treated cells. Finally, phosphorylation of p38 mitogen-activated protein kinase (MAPK) was detected in TCA-treated but not in mock-treated controls as early as 30 min after treatment.4. Overall, these results indicate that TCA causes a rapid molecular response in microglial cells, by the time, leading to an intriguing effector and secretory dysfunction.

Connective tissue growth factor and fibronectin secretion in renal tubular epithelial cells induced by TGF-beta1: suppressive effects of troglitazone.

Although some studies have suggested that troglitazone could retard the progression of glomerulosclerosis, its effects on renal tubulointerstitial fibrosis have not been completely clarified. The aim of this study was to investigate the effects of troglitazone on the secretion of connective tissue growth factor (CTGF) and fibronectin (FN) in human renal proximal tubular epithelial (HK-2) cells induced by transforming growth factor-beta1 (TGF-beta1). The mRNA of CTGF and FN were measured by semi-quantitative RT-PCR. CTGF and FN protein were detected by Western blot and ELISA, respectively. Our results revealed that troglitazone could inhibit CTGF and FN expression in a dose-dependent manner in human renal proximal tubular epithelial cells induced by TGF-beta1, which may be one of the mechanisms of troglitazone contributing to retard the progression of renal tubulointerstitial fibrosis.

Immune modulatory effects of Prunella vulgaris L. on monocytes/macrophages.

Prunella vulgaris L. (Labiatae), a popular Western and Chinese herbal medicine, has long been associated with anti-viral and anti-bacterial effects. While its anti-viral effects are attributed mainly to the inhibition of virus replication, the biological mechanisms of its anti-bacterial effects remain unknown. As a biological response modifier (BRM), the polysaccharides isolated from P. vulgaris have been shown to up-regulate the immune responses of monocytes/macrophages. However, the immune stimulatory effects seem to contradict its well-known anti-inflammatory properties. We hypothesized that the anti-microbial effects exhibited by the polysaccharides isolated from P. vulgaris encompass both anti-inflammatory and immune stimulatory effects. One of the polysaccharide fractions PV2IV markedly stimulated the production of superoxide and nitrite representing nitric oxide from murine macrophage RAW264.7 and brain macrophage BV2 cells. The amount of nitrite and superoxide produced after PV2IV stimulation was as high as that stimulated by bacterial endotoxin lipopolysaccharide (LPS) in a dose-dependent manner. In addition, PV2IV also increased cellular protein levels of inducible nitric oxide synthase (iNOS) and mRNA for tumor necrosis factor-alpha (TNFalpha). Similar to the effects of a high dose of LPS, the fraction PV2 could trigger activation-induced cell death (AICD) by stimulating caspase-3 activity and reduction of MTT uptake in monocytes/macrophages. These results may help our understanding of the molecular mechanism of P. vulgaris, which exhibited both immune stimulatory and anti-inflammatory effects against microbial invasion.

Differentiation of human bone marrow stromal cells into neural-like cells induced by sodium ferulate in vitro.

Human marrow stromal cells (hMSCs) are multipotential stem cells, capable of differentiating into bone, cartilage, fat and muscle. Several recent reports demonstrated that hMSCs have been also differentiated into neural cells. However, only a few reported inducers are applicable for clinical use. This work is to explore the effects of sodium ferulate (SF) on differentiation of hMSCs into neural cells in vitro. We found that hMSCs could be induced to the cells with typical neural morphology when cultured with SF. The cells express neural proteins, such as nestin, neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP). About 30% of the hMSC-derived cells expressed nestin when cultured with SF for 3 h, but no expression was detected after 24 h. The percentages of positive cells for NSE or GFAP were about 67% and 39% separately at 6 h, and reached the plateau phage after treatment with SF for 3 days. The data suggest that SF can induce hMSCs to differentiate into neural-like cells in vitro.

Induction of manganese superoxide dismutase (MnSOD) mediates cardioprotective effect of tamoxifen (TAM).

Tamoxifen (TAM), a synthetic nonsteroidal antiestrogen effectively and widely used for breast cancer treatment, is known to have antioxidant and cardioprotective effects, but whether the beneficial cardiovascular effect of TAM is linked to its antioxidant effect is unknown. In this study, we investigated the effect of TAM on the levels of manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, in cardiac tissues and cardiomyocytes. TAM treatment induced MnSOD expression in vitro and in vivo. Cardiomyocytes isolated from TAM-pretreated mice also had higher MnSOD levels and fewer apoptotic cells compared to cardiomyocytes from control mice after adriamycin (ADR) treatment. To further confirm the role of MnSOD in the protection against ADR in cardiomyocytes, we used cardiomyocytes isolated from MnSOD knock-out (MnSOD(+/-)), wild-type (NTg) and human MnSOD transgenic (TgH) mice. TUNEL assay indicated that the percentage of cells undergoing apoptosis after ADR treatment was significantly greater in MnSOD(+/-) than in NTg or TgH cardiomyocytes. 3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay showed that basal level of mitochondrial function was lower in MnSOD(+/-) cardiomyocytes than in NTg or TgH, and that MnSOD(+/-) was more sensitive to ADR. ADR treatment increased caspase activity, which was significantly higher in MnSOD(+/-) than in NTg or TgH cardiomyocytes. These results suggested that TAM-induced MnSOD expression is at least, in part, contribute to the cardioprotective effects of TAM.

Loss of functional E-cadherin renders cells more resistant to the apoptotic agent taxol in vitro.

Experimental evidence supports a role for E-cadherin in suppressing invasion, metastasis, and proliferation. Germline mutations of the E-cadherin represent the genetic cause of hereditary diffuse gastric cancer (HDGC). In this type of tumor, isolated cancer cells permeate the basal membrane and paradoxically survive in the gastric wall in the absence of contact with neighbor epithelial cells or with the extracellular matrix. This suggests that upon E-cadherin deregulation, cells acquired resistance to apoptosis. To test this hypothesis, CHO cells stably expressing either wild-type E-cadherin or the HDGC-related germline mutations T340A and V832M were seeded either on a thin layer of collagen type I or on plastic and then subjected to the apoptotic agent taxol. We found that in vitro functional E-cadherin renders cells more sensitive to the effect of taxol. Our results also indicate that this effect is associated to decreased level of the anti-apoptotic bcl-2 protein.

UV induced bystander signaling leading to apoptosis.

Human keratinocytes (HaCaT) were exposed to UV (A+B) (UVA-350-400 mJ/cm2 and UVB-30 mJ/cm2) which induces apoptosis as evidenced by MTT assay, DNA laddering, Bax and Fas up-regulation. UV induced apoptotic conditioned media (6 h or earlier) did not cause apoptosis in unexposed cells. However, treatment with conditioned medium collected post UV exposure (1 h) induced Bax in unexposed cells as observed by RT-PCR. The induction of cell death was initiated by conditioned medium collected 12 h after UV exposure and the extent of death was increased progressively when conditioned medium collected 24 or 72 h post UV exposure was used. Medium collected 24 h after UV exposure also increased mitochondrial membrane permeability as determined by rhodamine uptake. Conditioned medium induced apoptosis did not involve reactive oxygen species (ROS) unlike UV induced apoptosis indicating that the apoptosis pathway could be different. Interestingly, at high dilution apototic conditioned medium did not induce apoptosis but actually protected cells from UV insult. The role of nerve growth factor (NGF) in UV induced bystander effects are also discussed.