The Effects of Low-Level Laser Light Exposure on Sperm Motion Characteristics and DNA Damage.
Objective: To determine the effects of low-level laser light exposure on the motility of spermatozoa and on DNA damage.
Methods: Thirty-three semen samples were collected for routine analysis and were classified as normospermic, oligospermic, or asthenospermic. After routine semen analysis was performed residual semen was divided into treated and control aliquots. Treated samples were exposed to a 30 second infrared laser pulse of 50 mW/cm(2) at 905 nm, a wavelength thought to increase light sensitive cytochrome c oxidase in the mitochondrial electron transport chain. Samples were then incubated at 37°C and aliquots analyzed at 30 minutes and 2 hours using Computer Assisted Semen Analysis (CASA). After incubation, 250 µL of each sample was frozen at -80°C until DNA fragmentation analysis by flow cytometry.
Results: A significant increase in motility, most prominent in oligospermic and asthenospermic samples (85% increase), was observed 30 minutes after the treatment (p<0.0001). No significant increase in DNA damage compared to control samples was observed. Significant changes in sperm motion kinetics were observed.
Conclusions: Low-level laser light exposure appears to have a positive short-term effect on the motility of the treated spermatozoa and did not cause any increase in DNA damage measured at 2 hours. We conclude that some cases of asthenospermia may be related to mitochondrial dysfunction. The implications of this study in terms of future clinical applications needs further investigation.
Low-level infrared laser effect on plasmid DNA.
Departamento de Ciências Fisiológicas, Instituto Biomédico, Universidade Federal do Estado do Rio de Janeiro, Rua Frei Caneca, 94, Rio de Janeiro, 20211040, Brazil, email@example.com
Low-level laser therapy is used in the treatment of many diseases based on its biostimulative effect. However, the photobiological basis for its mechanism of action and adverse effects are not well understood. The aim of this study, using experimental models, was to evaluate the effects of laser on bacterial plasmids in alkaline agarose gel electrophoresis and Escherichia coli cultures. The electrophoretic profile of bacterial plasmids in alkaline agarose gels were used for studying lesions in DNA exposed to infrared laser. Transformation efficiency and survival of Escherichia coli AB1157 (wild-type), BH20 (fpg/mutM ( – )), BW9091 (xth(-)), and DH5?F’Iq (recA ( – )) cells harboring pBSK plasmids were used as experimental models to assess the effect of laser on plasmid DNA outside and inside of cells. Data indicate low-level laser: (1) altered the electrophoretic profile of plasmids in alkaline gels at 2,500-Hz pulsed-emission mode but did not alter at continuous wave, 2.5- and 250-Hz pulsed-emission mode; (2) altered the transformation efficiency of plasmids in wild-type and fpg/mutM(-) E. coli cells; (3) altered the survival fpg/mutM(-), xthA(-) and recA(-) E. coli cultures harboring pBSK plasmids. Low-level infrared laser with therapeutic fluencies at high frequency in pulsed-emission modes have effects on bacterial plasmids. Infrared laser action can differently affect the survival of plasmids in E. coli cells proficient and deficient in DNA repair mechanisms, therefore, laser therapy protocol should take into account fluencies, frequencies and wavelength of laser, as well as tissue conditions and genetic characteristics of cells before beginning treatment.
Low-level laser therapy: a useful technique for enhancing the proliferation of various cultured cells.
Department of Dermatology, Vitiligo Research Chair, College of Medicine, King Saud University, PO Box 240997, Riyadh, 11322, Saudi Arabia, firstname.lastname@example.org
The aim of this work is to review the available literature on the details of low-level laser therapy (LLLT) use for the enhancement of the proliferation of various cultured cell lines including stem cells. A cell culture is one of the most useful techniques in science, particularly in the production of viral vaccines and hybrid cell lines. However, the growth rate of some of the much-needed mammalian cells is slow. LLLT can enhance the proliferation rate of various cell lines. Literature review from 1923 to 2010. By investigating the outcome of LLLT on cell cultures, many articles report that it produces higher rates of ATP, RNA, and DNA synthesis in stem cells and other cell lines. Thus, LLLT improves the proliferation of the cells without causing any cytotoxic effects. Mainly, helium neon and gallium-aluminum-arsenide (Ga-Al-As) lasers are used for LLLT on cultured cells. The results of LLLT also vary according to the applied energy density and wavelengths to which the target cells are subjected. This review suggests that an energy density value of 0.5 to 4.0 J/cm(2) and a visible spectrum ranging from 600 to 700 nm of LLLT are very helpful in enhancing the proliferation rate of various cell lines. With the appropriate use of LLLT, the proliferation rate of cultured cells, including stem cells, can be increased, which would be very useful in tissue engineering and regenerative medicine.
Aviakosm Ekolog Med. 2009 May-Jun;43(3):60-4.
Infrared cold laser radiation as an antimutagen
[Article in Russian]
Shurygina IP, Belichenko NI, Mashkina EV, Pavlov NB, Bogacheva MA, Shkurat TP.
Effects of infrared cold laser radiation (IRCLR) on mutagenesis and proliferation of the corneal epithelium were studied with laboratory white mice subjected to instigated circulatory hypoxia of the brain. The experiment was to reveal whether IRCLR influences the frequency of chromosomal rearrangements and to allow calculation of the corneal cells mitotic index for circulatory brain hypoxia. Laser radiation was shown to reconstitute the normal frequency of chromosomal aberrations as well as the mitotic cycle in epithelial cells of the mice cornea. Data of the experiment are promising from the standpoint of antihypoxic use of IRCLR in ophthalmology.
Vestn Oftalmol. 2009 Mar-Apr;125(2):24-6.
Effect infrared low-intensity laser radiation on a mutation process and proliferative corneal activity in experimental hypoxia
[Article in Russian]
Shurygina IP, Galenkina NM, Shkurat TP.
The paper deals with the impact of infrared low-intensity laser radiation (IRLILR) on a mutation process and the proliferative activity of the animal cornea during stimulation of circulatory brain hypoxia. During an experiment on laboratory albino rats, IRLILR was studied for its impact on the level of chromosomal rearrangements and the mitotic index in the corneal cells was calculated in circulatory brain hypoxia. Laser exposure during stimulation of circulatory brain hypoxia favors normalization of the level of chromosomal aberrations and a mitotic cycle in the rat corneal epithelial cells. The experimental findings suggest that IRLILR may be used in ophthalmological care for antihypoxic purposes.
J Photochem Photobiol B. 2009 Feb 9;94(2):131-7. Epub 2008 Nov 21.
DNA damage after phototherapy in wounded fibroblast cells irradiated with 16 J/cm(2).
Mbene AB, Houreld NN, Abrahamse H.
Laser Research Group, Faculty of Health Sciences, University of Johannesburg, P.O. Box 17011, Doornfontein, Johannesburg 2028, South Africa.
BACKGROUND AND OBJECTIVE: Phototherapy or biomodulation is a remarkable therapy that has become more popular and widely used in the treatment of a variety of medical conditions, such as slow to heal wounds, pain, soft tissue injuries and skin trauma. It has been shown to induce DNA damage; however this damage appears to be repairable. This study aimed to determine the effects of phototherapy induced DNA damage and activation of the DNA repair gene methylpurine DNA glycosylase (MPG).
MATERIALS AND METHODS: DNA integrity was assessed using the comet assay, with and without formamidopyrimidine glycosylase (Fpg). For the comet assay, wounded human skin fibroblast cells (WS1) were irradiated twice, once at 30 min and again at 72 h with 5 or 16 J/cm(2) using a diode laser at 636 nm and cellular responses were assessed 1 or 24h post-irradiation. Real time reverse transcriptase polymerase chain reaction (RT-PCR) assessed MPG expression and three reference genes namely; beta Actin (ACTB), Glyceraldehyde three phosphate dehydrogenase (GAPDH) and Ubiquitin c (UBC). Wounded cells were irradiated once (30 min) with 16 J/cm(2), and MPG expression was assessed at 0, 3 and 8h post-laser irradiation.
RESULTS: At both 1 and 24h, wounded cells irradiated with 5 J/cm(2) showed insignificant DNA damage compared to control cells, while irradiation with 16 J/cm(2) showed significant damage. However, 24h post-irradiation these cells showed a significant decrease in damage compared to cells left to incubate for 1h. This observation was attributed to activation of DNA repair mechanisms. Real time RT-PCR showed that ACTB was not influenced by cell culture conditions or laser irradiation, and MPG expression was not detected.
CONCLUSION: In conclusion, irradiation with 5 J/cm(2) did not produce additional DNA damage, while damage to cells irradiated with 16 J/cm(2) was repairable by mechanisms other than MPG. This study also showed that ACTB can be used as a reference gene in laser experiments, using parameters set out in this study.
Used by permission of the Czech Society for the Use of Laser in Medicine, www.laserpartner.org
Low level laser therapy (LLLT) – Does it damage DNA?
K.O.Greulich, Inst. Mol. Biotech, Jena, D
Published jointly in Laser Partner and Laser World (www.laser.nu)
Low level laser therapy (LLLT) has been found beneficial in a wide variety of therapeutic applications (see for example 1). However, some concern has arisen on possible DNA damage. May it be possible that it benefits the patient only at a first glance but damages DNA and therefore increases the risk of therapy induced disease up to an increased cancer risk ?
What are the facts ? LLLT is usually performed with red (630 nm) or near infrared (830nm ) laser light. Typical accumulated doses per area are of the order of a few Joules per square centimeter. What an effect may such irradiation may have on DNA ? Unfortunately, most studies on the effects of radiation on DNA are performed with ionizing radiation (alpha, beta , gamma rays) or with UV light. There, DNA damage may be dramatic, although such studies have revealed a surprisingly strong DNA repair capacity of otherwise healthy human cells. Even when the overall integrity of a cell’s genome is seriously degraded, the damaged DNA can be repaired without directly detectable consequences (although long term mutational damage cannot be completely excluded).
One efficient and comparably simple technique, requiring basically only a fluorescence microscope and a gel electrophoresis device, to study DNA damage and repair is “Single Cell Gel Electrophoresis” (SCGE). Cells are embedded in an electrophoresis gel, their cell nuclei are perforated chemically and subsequently an electric field is applied (2-5). Since under suitable physicochemical conditions DNA is negatively charged, it migrates towards the electrically positive side of the gel. In a given time, small DNA fragments migrate a long distance (10-20 micrometers), large molecules migrate a correspondingly shorter distance. Very large DNA molecules cannot leave the cell nucleus. When the DNA of a cell is undamaged, it remains in the nucleus, which appears in a microscope, after staining with a fluorescence dye, as a sphere, or two dimensionally as a circle. When part of the DNA is damaged, the latter migrates out of the nucleus.After staining, such a cell has the appearance of a comet with bright head and a tail whose length (or a more quantitative parameter called tail moment) is a measure for the degree of damage. Therefore, SCGE is also called the COMET assay.
Using this COMET assay, light induced DNA damage has been studied in the wavelength range from 308 nm (UV) to approx. 450 nm (blue) (6) . While at 308 nm (UV) 0.0001 Joules per square centimeter were sufficient to induce detectable DNA damage, 1 Joule per square centimeter was required at 450 nm. The damage declined exponentially with wavelength. When one extrapolates this to the wavelengths which are used for LLLT, one can estimate that at least a thousandfold dose for 630 nm and a millionfold dose for 830 nm would be required to induce DNA damage detectable by the COMET assay. Probably the effects are even smaller, since in ref.6 a pulsed laser source was used, which often generates more damage than a corresponding continuous laser.
There is still the possibility that the COMET assay is just not sensitive enough to detect minor, but harmful DNA damages. However, one can compare the amount of radiation with that of sunlight. Bright sunlight has a power per area (=intensity) of 0.1345 Watts per square centimeter, which. after irradiation for only ten seconds, results in a dose per area of 1.345 Joule per square centimeter, comparable to what is used in LLLT, integrated over the whole spectral range. When one filters out a wavelength band of +/- 10 nm, Sun radiation for a few minutes is required to accumulate a few Joules per square centimeter. Such an irradiation is not generally supposed to cause disease. Since we are on the red side of the optical spectrum which, as mentioned above, is less DNA damaging than the average sunlight, we are on the safe side when we assume that the doses per area as they are used in LLLT correspond to the DNA damaging effects of a few minutes sunbath. If any DNA breaks are induced by such irradiation, they will be repaired immediately, otherwise even a short sunbath would cause mutations and finally cancer.
May that mean that LLLT does produce no effect at all, that everything is placebo effect ? Again, COMET assay experiments give a hint: when one first irradiates cells of the bacterium Escherichia coli (7) or human lymphocytes (8) with red (He-Ne) laser light (0.054 – 0.27 Joules per square centimeter) and then tries to damage DNA by UV irradiation, DNA fragmentation is much lower than without pre- irradiation with red light. An interpretation of this effect is that the pre-irradiation activates enzymes of the DNA repair machinery which immediately repairs possible UV damages. Since the effect is similar for cells as different as bacterial and mammalian cells one may conclude that it is evolutionarily conserved. In addition, these experiments indicate that low level laser illumination indeed can cause beneficial effects.
In conclusion, COMET assay experiments reveal possible therapeutic effects of LLLT but do not indicate a risk of DNA damage.
- Z.Simunovic editor Lasers in Medicine and Dentistry : LLLT 2000 Eur.Med.Laser Association. Access via www.lasermedico.ch
- O.Östling, K.J. Johanson Microelectrophoretic study of radiation induced DNA damages in individual mammalian cells 1984 Bioch.Bioph.Res.Comm.123,291-2982
- N.Singh, M.Mc Coy, R.Tice, E.Schneider A simple technique for quantification of low levels of DNA damage in individual cells 1988 Exp.Cell Res. 175,184-191
- P.L.Olive,D.Wlodek,J.P.Banath DNA double strand breaks measured in individual cells subjected to gel electrophoresis 1991 Cancer Res.51, 4671-4676
- A.Rapp, C.Bock, A.Rapp, H.Dittmar, K.O.Greulich 2000 J.Photochem. Photobiol. in press UV-A breakage sensitivity of human chromosomes measured by COMET-FISH depends on gene density and is not dependent on chromosome size,
- A.de With, K.O.Greulich Wavelength dependence of laser induced DNA damages in lymphocytes observed by single cell gel electrophoresis 1995 J.Photochem.Photobiol.B,30,71-76
- R.Kohli, P.K.Gupta, A.Dube He-Ne laser pre-irradiation induces protection against UV C radiation in wild type E coli strain K12B1157 2000 Rad.Res.153, 181-185
- A.Dube, C.Bock, E.Bauer, R.Kohli, P.K.Gupta, K.O.Greulich He-Ne laser protects B-lymphoblasts from UV induced DNA damage 2000 Rad.Env. Bioph.submitted
|Photomed Laser Surg. 2005 Aug;23(4):355-61.|
Exact action spectra for cellular responses relevant to phototherapy
Karu TI, Kolyakov SF.
Institute of Laser and Information Technologies, Russian Academy of Sciences, Troitsk, Moscow Region, Russian Federation. email@example.com
OBJECTIVE: The aim of the present work is to analyze available action spectra for various biological responses of HeLa cells irradiated with monochromatic light of 580-860 nm. BACKGROUND DATA: Phototherapy (low-level laser therapy or photobiomodulation) is characterized by its ability to induce photobiological processes in cells. Exact action spectra are needed for determination of photoacceptors as well as for further investigations into cellular mechanisms of phototherapy. METHODS: Seven action spectra for the stimulation of DNA and RNA synthesis rate and cell adhesion to glass matrix are analyzed by curve fitting, followed by deconvolusion with Lorentzian fitting. Exact parameters of peak positions and bandwidths are presented. RESULTS: The peak positions are between 613.5 and 623.5 nm (in one spectrum, at 606 nm), in the red maximum. The far-red maximum has exact peak positions between 667.5 and 683.7 nm in different spectra. Two near infrared maxima have peak positions in the range 750.7-772.3 nm and 812.5-846.0 nm, respectively. CONCLUSIONS: In the wavelength range important for phototherapy (600-860 nm), there are four “active” regions, but peak positions are not exactly the same for all spectra.
Photomed Laser Surg. 2004 Dec;22(6):504-8.
Cell survival, DNA, and protein damage in B14 cells under low-intensity near-infrared (810 nm) laser irradiation.
Kujawa J, Zavodnik IB, Lapshina A, Labieniec M, Bryszewska M.
Department of Rehabilitation, Medical University of Lodz, Lodz, Poland.firstname.lastname@example.org
OBJECTIVE: The aim of this study was to reveal the possible cytotoxic and genotoxic effects of low-intensity (200 mW) near-infrared (810 nm) laser irradiation, using B14 cell line.
BACKGROUND DATA: Laser therapy is widely used in biomedical treatment of many diseases, but the possible molecular mechanisms of laser actions remain unclear and the damaging effects of laser irradiation are still controversial. The side effects of laser therapy involve the generation of reactive oxygen and nitrogen species which in turn initiate lipid peroxidation, protein damage or DNA modification.
METHODS: B14 cells and suspension of human erythrocyte membranes were irradiated with near-infrared (810 nm) therapy laser at different radiant exposures (3.75-15.0 J/cm(2)) and light power (fluency rate) 200 mW at 22 degrees C. Laser induced cellular oxidative damage was measured in terms of cell survival, DNA damage, measured using the method of single cell gel electrophoresis (Comet assay), protein damage measured as protein carbonyls formation.
RESULTS: No substantial changes of cell survival under B14 cells irradiation at radiant exposures 3.75-11.25 J/cm(2) were observed. Similarly, neither considerable light-induced DNA damage or protein carbonyls accumulation was revealed. On the contrary, laser irradiation has led to decrease of cell protein carbonyl groups level in a dose-dependent manner. Additionally, using human red blood cell membranes as model membranes and biological oxidant HOCl we observed that laser irradiation resulted in a decrease of the level of membrane protein carbonyl groups accumulated under oxidative HOCl treatment.
CONCLUSIONS: We can conclude that laser irradiation used (810 nm, 200 mW, 3.75-11.25 J/cm(2)) did not produce any considerable cytotoxic or genotoxic effects in B14 cells. Moreover, laser irradiation reduced cellular protein damage (protein carbonyl groups) produced by biological oxidant HOCl.
Izv Akad Nauk Ser Biol. 2004 May-Jun;(3):280-93.
Regenerative capacity of muscle tissue and thymus state in irradiated rats exposed to long-term He-Ne laser radiation
[Article in Russian]
Buliakova NV, Azarova VS.
We studied the effect of He-Ne laser on regeneration of damaged gastrocnemius muscle in rats irradiated at 6 Gy in conditions of fractional laser energy spread (10 exposures, 3 min for each limb, within 30 days after the operation; 2-3 exposures weekly; 2.5-3.0 mW/cm2 power density; and 9.0-10.8 J/cm2 total dose per animal). Laser radiation stimulated regenerative activity of the skeletal muscle and favored a more even distribution of load on the thymus (a smooth decrease in its weight and slow aplasia). The level of chromosomal aberrations in the thymocytes demonstrated certain instability although remain.
Lasers Med Sci. 2001;16(3):213-7.
Stimulation of MCM3 gene expression in osteoblast by low level laser irradiation.
Yamamoto M, Tamura K, Hiratsuka K, Abiko Y.
Department of Biochemistry, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Biostimulatory effect of cell proliferation and bone formation by laser irradiation has been reported, however, very little is known about the molecular basis of mechanisms. We previously constructed the cDNA library of mouse osteoblastic cells (MC3T3-E1) which enhanced gene expression by laser irradiation using a subtracted gene cloning procedure. In the present study, we focused on a gene clone, designated as MCL-140, which exhibited the high homology of DNA sequence with mouse minichromosome maintenance (MCM) 3 gene. MCM3 is involved in the initiation of DNA replication as licensing factor in eukaryotic cells. Nucleotide sequence of MCL-140 insert was determined and assessed in the nucleic acid databases. The transcription level of MCL-140 was examined by Northern blot analysis. The DNA sequences of clone MCL-140 insert exhibited 96.2% homology with MCM 3 gene coding P1 protein. Higher MCM3 mRNA levels were observed in laser-irradiated cells compared to the levels in non-irradiated cells: furthermore, radiolabelled thymidine incorporation was increased by laser irradiation. These findings suggest that low-level laser irradiation may enhance DNA replication and play a role in stimulating proliferation of osteoblast through the enhancement of the MCM3 gene expression.
|Radiat Environ Biophys. 2001 Mar;40(1):77-82.|
He-Ne laser irradiation protects B-lymphoblasts from UVA-induced DNA damage.
Dube A, Bock C, Bauer E, Kohli R, Gupta PK, Greulich KO.
Biomedical Applications Section, Laser R & D, Block D, Center for Advanced Technology, Indore 452013, India. email@example.com
The effect of He-Ne laser (632.8 nm) pre-irradiation on UVA (343 nm)-induced DNA damage in the human B-lymphoblast cell line NC37 was investigated using the comet assay. He-Ne laser pre-irradiation was observed to result in a dose-dependent decrease in UVA-induced DNA damage. This effect was also found to be dependent on the incubation period between He-Ne laser pre-irradiation and the UVA exposure. Whereas the control cells with a higher DNA damage point to an initial ability of faster repair, both the control and the He-Ne laser pre-irradiated cells subsequently show the same rate of DNA repair. The results suggest that He-Ne laser irradiation protect the cells from UVA-induced DNA damage primarily through an influence on processes that prevent an initial DNA damage.