Photodesorption of mitochondria

A photodesorption of mitochondria absorbed on a quartz plate was found. The rate of desorption depends on the wavelength, and the intensity and time of irradiation. The maximal rate of photodesorption was detected upon ultraviolet irradiation at the absorption band of mitochondrial proteins. Probably, the photodesorption is caused by a local photothermal effect: a heating of photoexcited surface-membrane proteins, which attach mitochondria to the quartz plate. Preliminary fixation of a smear by isopropanol preserves the spontaneous desorption. No photodesorption of either mitohondria or formazan was observed upon irradiation of the smear with formazan by visible light (wavelength 540 nm; formazan was formed in the NADH-pNTV:reductase reaction). The data obtained are important for the elaboration of technology of mitochondrial immobilization in measurements of the enzyme activity and for biocensors.     

Mitotic viability and metabolic competence in UV-irradiated yeast cells

Colony formation is the classic method for measuring survival of yeast cells. This method measures mitotic viability and can underestimate the fraction of cells capable of carrying out other DNA processing events. Here, we report an alternative method, based on cell metabolism, to determine the fraction of surviving cells after ultraviolet (UV) irradiation. The reduction of 2,3,5-triphenyl tetrazolium chloride (or TTC) to formazan in mitochondria was compared with cell colony formation and DNA repair capacity in wt cells and two repair-deficient strains (rad1Delta and rad7Delta). Both TTC reduction and cell colony formation gave a linear response with different ratios of mitotically viable cells and heat-inactivated cells. However, monitoring the formation of formazan in non-dividing yeast cells that are partially (rad7Delta) or totally (wt) proficient at DNA repair is a more accurate measure of cell survival after UV irradiation. Before repair of UV photoproducts (cis-syn cyclobutane pyrimidine dimers or CPDs) is complete, these two assays give very different results, implying that many damaged cells are metabolically competent but cannot replicate. For example, only 25% of the rad7Delta cells are mitotically viable after a UV dose of 12 J/m(2)75% of these cells are metabolically competent and remove over 55% of the CPDs from their genomic DNA. Moreover, repair of CPDs in wt cells dramatically decreases after the first few hours of liquid holding (L.H.; incubation in water) and correlates with a substantial decrease in cell metabolism over the same time period. In contrast, cell colony formation may be the more accurate indicator of cell survival after UV irradiation of rad1Delta cells (i.e., cells with little DNA repair activity). These results indicate that the metabolic competence of UV-irradiated, non-dividing yeast cells is a much better indicator of cell survival than mitotic viability in partially (or totally) repair proficient yeast cultures.     

Disruption of functional activity of mitochondria during MTT assay of viability of cultured neurons

The MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in the cell cytoplasm to a strongly light absorbing formazan is among the most commonly used methods for determination of cell viability and activity of NAD-dependent oxidoreductases. In the present study, the effects of MTT (0.1 mg/ml) on mitochondrial potential (ΔΨ_m), intracellular NADH, and respiration of cultured rat cerebellum neurons and isolated rat liver mitochondria were investigated. MTT caused rapid quenching of NADH autofluorescence, fluorescence of MitoTracker Green (MTG) and ΔΨ_m-sensitive probes Rh123 (rhodamine 123) and TMRM (tetramethylrhodamine methyl ester). The Rh123 signal, unlike that of NADH, MTG, and TMRM, increased in the nucleoplasm after 5-10 min, and this was accompanied by the formation of opaque aggregates of formazan in the cytoplasm and neurites. Increase in the Rh123 signal indicated diffusion of the probe from mitochondria to cytosol and nucleus due to ΔΨ_m decrease. Inhibition of complex I of the respiratory chain decreased the rate of formazan formation, while inhibition of complex IV increased it. Inhibition of complex III and ATP-synthase affected only insignificantly the rate of formazan formation. Inhibition of glycolysis by 2-deoxy-D-glucose blocked the MTT reduction, whereas pyruvate increased the rate of formazan formation in a concentration-dependent manner. MTT reduced the rate of oxygen consumption by cultured neurons to the value observed when respiratory chain complexes I and III were simultaneously blocked, and it suppressed respiration of isolated mitochondria if substrates oxidized by NAD-dependent dehydrogenases were used. These results demonstrate that formazan formation in cultured rat cerebellum neurons occurs primarily in mitochondria. The initial rate of formazan formation may serve as an indicator of complex I activity and pyruvate transport rate.     

Potential repair of Escherichia coli DNA following exposure to UV radiation from both medium- and low-pressure UV sources used in drinking water treatment

The increased use of UV radiation as a drinking water treatment technology has instigated studies of the repair potential of microorganisms following treatment. This study challenged the repair potential of an optimally grown nonpathogenic laboratory strain of Escherichia coli after UV radiation from low- and medium-pressure lamps. Samples were irradiated with doses of 5, 8, and 10 mJ/cm(2) from a low-pressure lamp and 3, 5, 8, and 10 mJ/cm(2) from a medium-pressure UV lamp housed in a bench-scale collimated beam apparatus. Following irradiation, samples were incubated at 37 degrees C under photoreactivating light or in the dark. Sample aliquots were analyzed for up to 4 h following incubation using a standard plate count. Results of this study showed that E. coli underwent photorepair following exposure to the low-pressure UV source, but no repair was detectable following exposure to the medium-pressure UV source at the initial doses examined. Minimal repair was eventually observed upon medium-pressure UV lamp exposure when doses were lowered to 3 mJ/cm(2). This study clearly indicates differences in repair potential under laboratory conditions between irradiation from low-pressure and medium-pressure UV sources of the type used in water treatment.     

Acidification induces Bax translocation to the mitochondria and promotes ultraviolet light-induced apoptosis

It has been suggested that Bax translocation to the mitochondria is related to apoptosis, and that cytosol acidification contributes to apoptosis events. However, the mechanisms remain obscure. We investigated the effect of acidification on Bax translocation and on ultraviolet (UV) light-induced apoptosis. The Bax translocation assay in vitro showed that Bax translocated to the mitochondria at pH 6.5, whereas no Bax translocation was observed at pH 7.4. VHDBB cells expressing the GFP-Bax fusion protein were treated for 12 h with a pH 6.5 DMEM medium, nigericin (5 μg/ml) and UV light (50 J/cm²), separately or in combination, and Bax translocation to the mitochondria was determined by SDS-PAGE and Western blot, and apoptotic cell death was detected by flow cytometry. The results showed that some of the Bax translocated to the mitochondria in the cells treated with the normal medium, nigericin and UV in combination, whereas all of the Bax translocated to the mitochondria in the cells treated with the pH 6.5 medium, nigericin and UV in combination. In VHDBB cells treated for 12 h with nigericin, UV alone, and UV and nigericin in combination, the respective rates of apoptotic cell death were 25.08%, 33.25% and 52.88%. In cells treated with pH 6.5 medium and nigericin, pH 6.5 medium and UV, and pH 6.5 medium, nigericin and UV in combination, the respective rates of apoptotic cell death increased to 37.19%, 41.42% and 89.44%. Our results indicated that acidification induces Bax translocation from the cytosol to the mitochondria, and promotes UV lightmediated apoptosis. This suggests that there is a possibility of improving cancer treatment by combining acidification with irradiation or chemotherapeutic drugs.     

Some acute effects of monochromatic ultraviolet B irradiation on mouse epidermis measured by the tetrazolium-reduction test and determination of DT-diaphorase activity, with reference to carcinogenesis

Some acute epidermal effects of monochromatic ultraviolet B (UVB) irradiation on hairless mouse skin were measured by the tetrazolium test (TZT), and by determining the DT-diaphorase activity in epidermal cells. Dose response and time course studies were carried out after UVB irradiation at 280, 290, 297 and 302 nm. Appropriate UV doses at all the wavelengths increased the cellular deposition of formazan (TZT). At higher doses the epidermal cells became too injured to react. Wavelengths at 280 and 290 nm seemed more injurious than those at 297 and 302 nm. There was, however, no increase in DT-diaphorase activity after UVB irradiation. This indicates that the increased formazan deposition (TZT) after UVB is more likely to be caused mainly by membrane effects. Detoxification mechanisms which activate DT-diaphorase, as often seen after cellular contact with chemical carcinogens, are not involved.     

Potential Repair of Escherichia coli DNA following Exposure to UV Radiation from Both Medium- and Low-Pressure UV Sources Used in Drinking Water Treatment

The increased use of UV radiation as a drinking water treatment technology has instigated studies of the repair potential of microorganisms following treatment. This study challenged the repair potential of an optimally grown nonpathogenic laboratory strain of Escherichia coli after UV radiation from low- and medium-pressure lamps. Samples were irradiated with doses of 5, 8, and 10 mJ/cm² from a low-pressure lamp and 3, 5, 8, and 10 mJ/cm² from a medium-pressure UV lamp housed in a bench-scale collimated beam apparatus. Following irradiation, samples were incubated at 37°C under photoreactivating light or in the dark. Sample aliquots were analyzed for up to 4 h following incubation using a standard plate count. Results of this study showed that E. coli underwent photorepair following exposure to the low-pressure UV source, but no repair was detectable following exposure to the medium-pressure UV source at the initial doses examined. Minimal repair was eventually observed upon medium-pressure UV lamp exposure when doses were lowered to 3 mJ/cm². This study clearly indicates differences in repair potential under laboratory conditions between irradiation from low-pressure and medium-pressure UV sources of the type used in water treatment.     

Mitochondria are a primary target of hypericin phototoxicity: synergy of intracellular calcium mobilisation in cell killing

Hypericin, a naturally occurring anthraquinone synthesised by hypericum, upon light activation exhibits photodynamic cytotoxicity attributed mainly to the production of reactive oxygen species. This study aimed to elucidate the primary subcellular targets and mechanistic aspects of hypericin photosensitization in human prostate carcinoma cells. Depletion of intracellular glutathione (>85%) via inhibition of gamma-glutamyl-cysteine synthase had no effect on hypericin (5 microM) phototoxicity, thus precluding any direct oxidative involvement of H2O2. There was no change in intracellular SOD activity immediately after hypericin irradiation (1.5-5 J cm(-2)). Evaluation of the lysosomal enzyme hexosaminidase activity showed: (a) 60% cell loss 22 h following irradiation (1.5 J cm(-2)) and (b) a steady rate of lysosomal leakage to the cytosol (25%), at the same time and irradiation. However, lysosomal damage appears to be a slower process compared to the rapid loss of mitochondrial function, as reflected from parallel tetrazolium to formazan assays. The activity of cytosolic and mitochondrial aconitase, an enzyme exquisitely sensitive to oxidation, revealed a dose correlated loss of activity in the mitochondria immediately following hypericin photoactivation. The use of ionomycin, which modulates both internal Ca2+ stores and external Ca2+ transport during hypericin photosensitization, profoundly enhanced photocytotoxicity. Our data supports a direct mitochondrial hypericin phototoxicity that does not involve glutathione/H2O2 homeostasis. Further a potential synergistic treatment combining mitochondrial targeting of photosensitisers and Ca2+ mobilisation was identified.     

Degradation of mitochondria to lipofuscin upon heating and illumination

In the present work, it has been shown that isolated mitochondria can undergo transformation to lipofuscin granules without any additional factors (oxygen saturation or prooxidants). The process occurs spontaneously and slowly at low temperature and rapidly upon heating (thermolipofuscin) or UV irradiation (photolipofuscin). The main contribution to the formation of mitochondrial lipofuscin is made by denatured proteins. The formation of thermolipofuscin depends on lipid peroxidation, while the presence of lipids is not required for photolipofuscin formation. It has been shown that the use of a detergent that is able to degrade mitochondria is necessary to measure the lipofuscin content properly.     

A permeability test for the study of mitochondrial injury in in vitro cultured heart muscle and endothelioid cells.

A cytochemical permeability test for the detection of injury to in situ mitochondria of cultured heart cells is presented. The test is based on the increased rate at which injured mitochondria stain for succinate dehydrogenase activity. Whereas an intact inner mitochondrial membrane limits the rate at which Nitro Blue tetrazolium and phenazine methosulphate reach succinate dehydrogenase, injured mitochondria allow these reactants to reach the enzyme more rapidly to form microscopically-observable formazan granules. The extent of staining at fixed durations of incubation with the reactants was assessed on a blind basis with pseudo dark-field microscopy, using a standardized rating scale. Differences in the staining of control and treated cells were analysed statistically by a semi-quantitative method. Treatment of the cultures with either vitamin A or chlorpromazine, resulted in more rapid mitochondrial staining. Brief pre-fixation of the cells with cold acetone also labilized the mitochondria as did a delay in the change of culture medium.     

UV-induced cross-linking of Tet repressor to DNA containing tet operator sequences and 8-azidoadenines.

The synthesis of 8-azido-2'-deoxyadenosine-5'-triphosphate is described. The photoreactive dATP analog was characterized by thin layer chromatography, proton resonance spectroscopy, infrared spectroscopy and UV spectroscopy. Its photolysis upon UV irradiation was studied. After incorporation of this dATP analog into DNA containing the tet operator sequence the investigation of the interactions between tet operator DNA and Tet repressor protein by UV photocross-linking becomes possible. Photocross-linking of protein to DNA was demonstrated by the reduced migration of the DNA in SDS polyacrylamide gel electrophoresis. Addition of the inducer tetracycline prior to UV irradiation significantly reduces the DNA-protein cross-linking rate. The long wave UV light applied here does not significantly alter the DNA or the protein under the photocross-linking conditions.     

Quantitation of mitochondrial injury in cultured rat heart endothelioid cells with nitroblue tetrazolium

Synopsis A sensitive method is presented for measurement of changes in the permeability of mitchondria in cultured cells. Rat heart endothelioid cells were used to determine the penetration rate of nitroblue tetrazolium (NitroBT) or other reactants into mitochondriain situ. Nitroblue formazan, produced as a consequence of succinate dehydrogenase activity in the mitochondria, was eluted and measured with a spectrophotometer. Prior injury of cells with hypo-osmolar solutions increased the rate of formazan production. Several methods are described or suggested for the statistical analysis of the data.     

UV-Induced destruction of light-harvesting complexes from purple bacterium Chromatium minutissimum

We studied UV-induced photodestruction of the native forms of bacteriochlorophyll a (Bchl a) from chromatophores and light harvesting complexes (LHC) of the sulphur photosynthetic bacterium Chromatium minutissimum. Irradiation of chromato- phores with 365-nm light (Soret band) or 280-nm light (absorption region of aromatic amino acids) led to the destruction of all long-wavelength forms of Bchl a. The quantum yields of photodestruction produced by the 280-nm light was higher than that produced by the 365-nm light. For the spectral forms of Bchl a absorbing at 850 nm and 890 nm, the difference was about one order of magnitude, and for the form absorbing at 800 nm the difference was almost two orders of magnitude. Similar UV sensitivity was observed for the Bchl a forms from isolated LHC. As a rule, the quantum yields of photodestruction induced by UV irradiation at 280 nm were about 100-1000 times higher (approximately 10(-3)-10(-4)) than that upon red light irradiation (approximately 10(-6)-10(-7)). We found that irradiation of chromatophores at 280 nm resulted in a crosslink between the core and peripheral LHC.     

Mitotic recombination and inactivation in Saccharomyces cerevisiae induced by UV-radiation (254 nm) and hyperthermia depend on UV fluence rate

In experiments with wild-type diploid yeast cells of Saccharomyces cerevisiae, the synergistic interaction of ultraviolet (UV) light (wavelength, 254 nm) and heat (45--60 degrees C) was studied both for mutagenic and inactivation effects. Simultaneous hyperthermia and UV light treatments increase the frequency of UV-induced mitotic intergenic recombination (crossing-over) and cell inactivation. The enhancing effect was a function of UV light fluence rate. It is concluded that the effect of hyperthermia on low fluence UV or high fluence UV irradiation results in comparable effects on survival and mitotic recombination suggesting similar modulation by hyperthermia of the effects induced by UV at different fluence rates. The interpretation of the data obtained was carried out within the widely accepted point of view considering the synergistic effects as a result of repair ability damage.     

Beta-carboline-carbohydrate hybrids: molecular design, chemical synthesis and evaluation of novel DNA photocleavers

Beta-carboline present in beta-carboline alkaloids from marine organisms was found, for the first time, to cleave DNA at the guanine site upon irradiation with UV light with a long wavelength without any additive, and beta-carboline-carbohydrate hybrid system was effective for DNA cleavage.     

THE EFFECT OF IONIZING RADIATION UPON MITOCHONDRIA OF THE CENTRAL NERVOUS SYSTEM

After irradiation of rats with a linear electron accelerator, the respiratory rate in rat brain mitochondria was studied in the presence of substrate + ADP and after the conversion of ADP → ATP. After 20,000 rads of irradiation to the head there was a transient diminution of mitochondrial respiratory control when glutamate was used as the substrate, but no changes were observed when succinate was the substrate. Irradiation with 10,000 rads had no effect upon respiratory control. The addition of NADH₂ to irradiated mitochondria had no effect upon mitochondrial respiration. Irradiation of the brain with 20,000 rads failed to produce mitochondrial peroxidation or swelling, even in the presence of FeNH₄(SO₄)₂ or ascorbate. The slight changes in respiratory control of brain mitochondria following irradiation is in marked contrast to the susceptibility of mitochondria from other organs. The comparative radioresistance of brain mitochondria may be the result of greatly diminished radiation-induced peroxidation of cerebral mitochondrial membranes.     

Change in the reaction of the antioxidant system of wheat sprouts after UV-irradiation of seeds

The response of the antioxidant system of sprouts of wheat Triticum aestivum L. to preliminary irradiation of seeds with UV light was studied. The dependence of lipid peroxidation and the extent of antioxidant activity on the duration of irradiation was studied. It was shown that low doses of UV radiation (5-15 min) stimulate the antioxidant protection of green wheat sprouts grown for eight days. Increasing the irradiation time to 30-60 min leads to the inhibition of lipid peroxidation by the antioxidant system. A more prolonged irradiation of seeds with UV light (for 1-6 h) led to an increase in the level of lipid peroxidation in sprouts. However, 1-2-day-old sprouts from seeds irradiated for 5-6 h, adapted themselves to the influence due to the compensatory mechanisms. By the 8th day of germination of preliminarily irradiated seeds, the content of antioxidants and malone dialdehyde returned to the norm. The dynamics of activity of peroxidase in seeds irradiated with low doses of UV light for 30 min was studied. It was found that on the third day of seed germination, a decrease in peroxidase activity followed by its slight increase occurred. The maximum activity of the enzyme in the endosperm was observed on day 5-6, and in roots and green sprouts, on day 3-5 of germination. It was concluded that antioxidants and peroxidase are involved in the compensatory mechanisms of inhibition of free radicals formed upon UV irradiation of seeds.     

The effect of local UVB skin irradiation on the rate of formazan deposition in the epidermis of hairless mice studied by means of a tetrazolium-reduction method

One-hundred-and-twenty hairless mice were irradiated with UVB (310 nm, exposure 60 mJ/cm2) on a limited area of the dorsal skin. At different time intervals after irradiation, the rate of endogenous dehydrogenase activity per mg dry epidermis was measured by the tetrazolium reduction method. The amount of formazan deposited remained normal for 18 h, and then increased, reaching a peak significantly higher than normal at 24 h, and thereafter returned to normal. At day 8 there was a new, probably significant peak. The reaction was followed for 14 days. It was concluded that UVB irradiation provokes a period of increased formazan deposition in the epidermis, similar to what has been observed after ionizing radiation and chemical carcinogens. The validity of the tetrazolium test for skin carcinogenic irritaments was thus also confirmed.     

A cytochemical staining procedure for succinate dehydrogenase activity in pre-ovulatory mouse oocytes embedded in low gelling temperature agarose.

We describe a cytochemical staining procedure for succinate dehydrogenase (SDH) activity in pre-ovulatory mouse oocytes. The oocytes were embedded in low gelling temperature agarose and treated with caffeine before cytochemical staining in the presence of nitro blue tetrazolium (NBT), phenazinemethosulfate (PMS), and succinate. This resulted in intense staining of the oocytes by formazan precipitate. The level of aspecific formazan production in the absence of succinate was very low. We applied the procedure to oocytes matured in vitro and found that the location of the formazan precipitate as a result of SDH activity correlated well with the location of mitochondria. The chromatin of the cytochemically stained oocytes could subsequently be analyzed by means of the DNA-specific fluorochrome DAPI. In pre-ovulatory oocytes, we found a correlation between chromatin organization and the location of mitochondria: in oocytes with an intact germinal vesicle the mitochondria were uniformly distributed in the cytoplasm, as shown by fine grains of formazan precipitate. In oocytes with condensed chromatin the mitochondria apparently had clustered, because the formazan precipitate was more coarse in these cells.     

Isolation and Characterization of an Escherichia coli ruv Mutant Which Forms Nonseptate Filaments After Low Doses of Ultraviolet Light Irradiation

Two ultraviolet light (UV)-sensitive mutants have been isolated from Escherichia coli K-12. These mutants, designated RuvA(-) and RuvB(-), were controlled by a gene located close to the his gene on the chromosome map. They were sensitive to UV (10- to 20-fold increase) and slightly sensitive to gamma rays (3-fold increase). Host cell reactivation, UV reactivation and genetic recombination were normal in these mutants. Irradiation of the mutants with UV resulted in the production of single-strand breaks in deoxyribonucleic acid, which was repaired upon incubation in a growth medium. After UV irradiation, these mutants resumed deoxyribonucleic acid synthesis at a normal rate, as did the parent wild-type bacteria, and formed nonseptate, multinucleate filaments. From these results we concluded that the mutants have some defect in cell division after low doses of UV irradiation, similar to the lon(-) or fil(+) mutant of E. coli. The ruv locus was divided further into ruvA and ruvB with respect to nalidixic acid sensitivity and the effect of minimal agar or pantoyl lactone on survival of the UV-irradiated cell. The ruvB(-)mutant was more sensitive to nalidixic acid than were ruvA(-) and the parent strain. There was a great increase in the surviving fraction of the UV-irradiated ruvB(-) mutant when it was plated on minimal agar or L agar containing pantoyl lactone. No such increase in survival was observed in the ruvA(-) mutant.