Cytotoxic responses to 405 nm light exposure in mammalian and bacterial cells : involvement of reactive oxygen species

Light at wavelength 405 nm is an effective bactericide. Previous studies showed that exposing mammalian cells to 405 nm light at 36 J/cm2 (a bactericidal dose) had no significant effect on normal cell function, although at higher doses (54 J/cm2), mammalian cell death became evident. This research demonstrates that mammalian and bacterial cell toxicity induced by 405 nm light exposure is accompanied by reactive oxygen species production, as detected by generation of fluorescence from 6-carboxy-2’,7’- dichlorodihydrofluorescein diacetate. As indicators of the resulting oxidative stress in mammalian cells, a decrease in intracellular reduced glutathione content and a corresponding increase in the efflux of oxidised glutathione was observed from 405 nm light treated cells. The mammalian cells were significantly protected from dying at 54 J/cm2 in the presence of catalase, which detoxifies H2O2. Bacterial cells were significantly protected by sodium pyruvate (H2O2 scavenger) and by a combination of free radical scavengers (sodium pyruvate, dimethyl thiourea (OH scavenger) and catalase) at 162 and 324 J/cm2. Results herefore suggested that the cytotoxic mechanism of 405 nm light in mammalian cells and bacteria could be oxidative stress involving predominantly H2O2 generation, with other ROS contributing to the damage

The effects of 405 nm light on bacterial membrane integrity determined by salt and bile tolerance assays, leakage of UV absorbing material and SYTOX green labelling

Bacterial inactivation by 405nm light is accredited to the photo-excitation of intracellular porphyrin molecules which results in energy transfer and the generation of reactive oxygen species (ROS) which impart cellular oxidative damage. The specific mechanism of cellular damage, however, is not fully understood. Previous work has suggested that destruction of nucleic acids may be responsible for inactivation; however, microscopic imaging has suggested membrane damage as a major constituent of cellular inactivation. This study investigates the membrane integrity of Escherichia coli and Staphylococcus aureus exposed to 405nm light. Results indicated membrane damage to both species, with loss of salt and bile tolerance by S. aureus and E. coli, respectively, consistent with reduced membrane integrity. Increased nucleic acid release was also demonstrated in 405nm light-exposed cells, with up to 50% increase in DNA concentration into the extracellular media in the case of both organisms. SYTOX green fluorometric analysis however demonstrated contradictory results between the two test species. With E. coli, increasing permeation of SYTOX green was observed following increased exposure, with >500% increase in fluorescence, whereas no increase was observed with S. aureus. Overall, this study has provided good evidence that 405nm light exposure causes loss of bacterial membrane integrity in E. coli, but the results with S. aureus are more difficult to explain. Further work is required to gain greater understanding of the inactivation mechanism in different bacterial species, as there are likely to be other targets within the cell which are also impaired by the oxidative damage from photo-generated ROS

Differential sensitivity of osteoblasts and bacterial pathogens to 405-nm light highlighting potential for decontamination applications in orthopedic surgery

Healthcare associated infections pose a major threat to patients admitted to hospitals and infection rates following orthopedic arthroplasty surgery are as high as 4%. A 405-nm high-intensity narrow spectrum light has been proven to reduce environmental contamination in hospital isolation rooms, and there is potential to develop this technology for application in arthroplasty surgery. Cultured rat osteoblasts were exposed to varying light intensities and it was found that exposures of up to a dose of 36  J/cm2 had no significant effect on cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay], function (alkaline phosphatase activity), and proliferation rate (BrdU cell proliferation assay). High irradiance exposures (54  J/cm2) significantly affected the cell viability indicating that the effects of 405-nm light on osteoblasts are dose dependent. Additionally, exposure of a variety of clinically related bacteria to a dose of 36  J/cm2 resulted in up to 100% kill. These results demonstrating the differential sensitivity of osteoblasts and bacteria to 405-nm light are an essential step toward developing the technique for decontamination in orthopedic surgery

Explaining Program Execution in Deductive Systems

. Programs in deductive database and programming systems have a natural meaning that is based upon their mathematical reading as logical rules. High-level `explanations' of a program evaluation/execution can be constructed to provide added functionality: (1) To debug a program by following a chain of deductions leading to an unexpected (and possibly incorrect) conclusion; (2) To follow the derivation of certain correct conclusions to determine why and how they are reached; (3) To identify consequences of a (typically, incorrect or unexpected) fact. This functionality can be utilized either to perform post-mortem analysis of a session, or to interactively develop programs by running queries and viewing their deductions simultaneously. `Explanations' of programs are especially important in the context of deductive databases for three reasons: (1) These programs could involve recursion, and hence, the chain of inferences is often not evident. (2) When the input data set is l..

Genetic analysis of OCT1 gene polymorphisms in an Indian population

Background: Genetic variants of the organic cation transporter (OCT1) gene could influence interindividual variation in clinical response to metformin therapy. The genetic basis for the single-nucleotide polymorphism (SNP) of OCT1 gene has been established in other populations, but it remains to be elucidated in the Indian population. This study is focused on OCT1 gene variants rs2282143 (P341L, 1022C>T), rs628031 (M408V, 1222A>G) and rs622342 (1386C>A) frequency distributions in the South Indian Tamilian population. Materials and Methods: A total of 112 unrelated healthy subjects of South Indian Tamilian origin, aged 18-60 years, of either sex were recruited for the study. Genotyping was determined using the quantitative real time-polymerase chain reaction and polymerase chain reaction followed by restriction fragment length polymorphism methods. Results: Allele frequencies of rs2282143, rs628031and rs622342 polymorphisms were 8.9%, 80.3% and 24.5%, respectively. Interethnic differences in the genotype and allele frequencies of OCT1 gene polymorphism were observed when compared with other major populations. The SNPs rs2282143, T allele and rs628031, G allele were more common in Asians (5.5-16.8% and 76.2-81%) and African Americans (8.2% and 73.5%) than in Caucasians (0-2% and 57.4-60%). Conclusion: This is the first time the frequency of OCT1 gene polymorphism was determined in the Indian population, and is similar to the frequencies observed in African-Americans and other Asian populations but different from those in Caucasians. The data observed in this study would justify further pharmacogenetic studies to potentially evaluate the role of OCT1 gene polymorphism in the therapeutic efficacy of metformin

Genetic analysis of OCT1 gene polymorphisms in an Indian population

Background: Genetic variants of the organic cation transporter (OCT1) gene could influence interindividual variation in clinical response to metformin therapy. The genetic basis for the single-nucleotide polymorphism (SNP) of OCT1 gene has been established in other populations, but it remains to be elucidated in the Indian population. This study is focused on OCT1 gene variants rs2282143 (P341L, 1022C>T), rs628031 (M408V, 1222A>G) and rs622342 (1386C>A) frequency distributions in the South Indian Tamilian population. Materials and Methods: A total of 112 unrelated healthy subjects of South Indian Tamilian origin, aged 18-60 years, of either sex were recruited for the study. Genotyping was determined using the quantitative real time-polymerase chain reaction and polymerase chain reaction followed by restriction fragment length polymorphism methods. Results: Allele frequencies of rs2282143, rs628031and rs622342 polymorphisms were 8.9%, 80.3% and 24.5%, respectively. Interethnic differences in the genotype and allele frequencies of OCT1 gene polymorphism were observed when compared with other major populations. The SNPs rs2282143, T allele and rs628031, G allele were more common in Asians (5.5-16.8% and 76.2-81%) and African Americans (8.2% and 73.5%) than in Caucasians (0-2% and 57.4-60%). Conclusion: This is the first time the frequency of OCT1 gene polymorphism was determined in the Indian population, and is similar to the frequencies observed in African-Americans and other Asian populations but different from those in Caucasians. The data observed in this study would justify further pharmacogenetic studies to potentially evaluate the role of OCT1 gene polymorphism in the therapeutic efficacy of metformin