Effect of growth phase on the Escherichia coli response to ultraviolet-A radiation: influence of conditioned media, hydrogen peroxide and acetate

The results reported herein indicate that the ultraviolet-A (UVA) radiation-induced effects in Escherichia coli depend on its growth phase. Stationary-phase cells recover faster from a sub-lethal UVA exposure and have a higher resistance to lethal effect of the radiation than exponential growing cells. Although pre-incubation in spent medium supernatant increased the resistance of log-phase cells to lethal UVA effects, this pre-treatment considerably prolonged the duration of the radioinduced sub-lethal growth delay. The aim of the present study was to investigate the effect exerted by the E. coli conditioned media and evaluate the influence of nutritional stress, hydrogen peroxide and acetate. Pre-incubated in conditioned medium, cells in exponential growth phase were irradiated and the induced effects were compared with those found when catalase, high culture densities and acetate were employed. Unexpectedly, the duration of the growth delay in cells submitted to these treatments was shortened in comparison with control cells incubated in conditioned medium with no modifications. Lengthening of the growth delay was mimicked when exponentially growing cells were incubated in fresh medium supplied with 5 microM H(2)O(2). The effects of spent medium on wild type and rpoS mutant strains were similar, indicating that this response is independent of RpoS controlled functions. We assumed that an oxidative component of the spent medium, probably H(2)O(2), could be involved in the observed phenomenon. This effect is specific of E. coli and independent of rpoS.     

Cell Cycle Effects and Concomitant p53 Expression in Hairless Murine Skin after Longwave UVA (365 nm) Irradiation: A Comparison with UVB Irradiation

Abstract— Ultraviolet A (UVA,315–400 nm) radiation is known to be a complete carcinogen, but in contrast to UVB (280-315 nm) radiation, much of the cell damage is oxygen dependent (mediated through reactive oxygen species), and the dominant premutational DNA lesion(s) remains to be identified. To investigate further the basic differences in UVA and UVB carcinogenesis, we compared in vivo cellular responses, viz. cell cycle progression and transient p53 expression in the epidermis, after UVA1 (340-400 nm) exposure with those after broadband UVB exposure of hairless mice. Using flow cytometry we found a temporary suppression of bromodeoxyuridine (BrdU) uptake in S-phase cells both after UVB and UVA1 irradiation, which only in the case of UVB is followed by an increase to well over control levels. With equally erythemogenic doses (1-2 MED), the modulation of BrdU uptake was more profound after UVB than after UVA1 irradiation. Also, a marked transient increase in the percentage of S-phase cells occurred both after UVB and after UVA1 irradiation, but this increase evolved more rapidly after UVA1 irradiation. Further, p53 expression increased both after UVB and UVA1 irradiations, with peak expression already occurring from 12 to 24 h after UVA1 exposure and around 24 h after UVB exposure. Overall, UVA1 radiation appears to have less of an impact on the cell cycle than UVB radiation, as measured by the magnitude and duration of changes in DNA synthesis and cells in S phase. These differences are likely to reflect basic differences between UVB and UVA1 in genotoxicity and carcinogenic action.     

Sublethal effects of ultraviolet A radiation on Enterobacter cloacae

We report the sublethal effects of ultraviolet A (UVA) on Enterobacter cloacae in comparison with those produced in Escherichia coli. UVA-induced sublethal effects were investigated in either bacterial membrane and at tRNA level. Limited dependence on oxygen concentration for photoinduced inhibition of biochemical membrane functions and low levels of oxidative damage during the irradiation period were found in En. cloacae. On the other hand, ultraviolet spectroscopy and reversed-phase HPLC analysis of hydrolysed tRNA showed that radio induced damage to tRNA is similar in En. cloacae and E. coli. Nevertheless, growth delay induced by UVA in En. cloacae was shorter than that found in E. coli submitted to the same experimental conditions. A limited post-irradiation ppGpp accumulation and the absence of any influence of the membrane damage on the growth delay extent seem to be responsible for the shortness of this effect in En. cloacae. Most of the differences between En. cloacae and E. coli could be attributed to an increased ability of En. cloacae to overcome oxidative stress during UVA exposure.     

Cell Cycle Kinetics Following UVA Irradiation in Comparison to UVB and UVC Irradiation

Abstract— There is limited information about the carcinogenic effect of longwave ultraviolet radiation (UVA: 315-400 nm). In particular very little is known about the relevant genotoxic damage caused by physiological doses of UVA radiation. A general response of cells to DNA damage is a delay or arrest of the cell cycle. Conversely, such cellular responses after UVA irradiation would indicate significant genotoxic damage. The aim of this study is to compare cell cycle kinetics of human fibroblasts after UVC (190-280 nm radiation), UVB (280-315 nm radiation) and UVA irradiation. Changes in the cell cycle kinetics were assessed by bivariate flow cytometric analysis of DNA synthesis and of DNA content. After UVC, UVB or UVA irradiation of human fibroblasts a suppression was seen of bromodeoxyuridine (BrdU) incorporation at all stages of S phase. The magnitude of this suppression appeared dose dependent. Maximum suppression was reached at 5-7 h after UVB exposure and directly after UVA exposure, and normal levels were reached 25 h after UVB and 7 h after UVA exposure. The lowered BrdU uptake corresponded with a lengthening of the S phase. No dramatic changes in percentages of cells in G₁, S and G₂/M were seen after the various UV irradiations. Apparently, UVA irradiation, like UVB and UVC irradiation, can temporarily inhibit DNA synthesis, which is indicative of genotoxic damage.     

COMPARATIVE EFFECT OF UVA AND UVB ON CULTURED RABBIT LENS

Abstract Effects on lens physiology of UVB and UVA used separately and sequentially were investigated using 4 week old rabbit lenses in organ culture. Narrowband UVB at 0.3 J/cm²= joules/lens (1 h exposure) has little effect on sodium and calcium concentrations in the lens interior or transparency of lenses subsequently cultured for 20 h after a 1 h exposure. With an incident energy of 3 J/cm² of broadband UVB (295–330 nm), lenses become opaque and slightly swollen with significant ion imbalances during culture over a 1 day period. In contrast, lenses exposed to approximately 6–24 J/cm² of UVA (330–400 nm) remain transparent after 1 day of culture. Extended culture up to 4 days reveals no signs of opacification. Ion homeostasis and normal lens hydration are also maintained in UVA-irradiated lenses. The presence of 95% oxygen during UVA irradiation is also without effect. Broadband UVA irradiation is damaging, however, if lenses are first exposed to subthreshold doses of narrowband UVB (307 ± 5 nm) irradiation, viz . 0.3 J/cm². Thus, sequential UVB/UVA irradiation at subthreshold doses causes impaired active cation transport and accumulation of sodium and calcium accompanying lens opacification.     

UVA irradiation induces energy-independent phospholipid-flip in mammalian plasma membrane

Translocation from the outer to the inner membrane leaflet (flip) of phospholipids after ultraviolet A (UVA) irradiation was investigated in Chinese hamster ovary cells. Fluorescent 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzox- adiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoserine (NBD-labeled phosphatidylserine [NBD-PS]) was used to assay transbilayer lipid movement. A marked increase in flip of NBD-PS was observed immediately after low-dose UVA irradiation which was not lethal and returned to the basal level after 6 h. UVA-induced flip was not attributed to the increase of permeability by UVA irradiation because cells that were negative for staining with propidium iodide also showed increased flip of NBD-PS. Furthermore, the enhancement was independent of adenosine 5'-triphosphate, demonstrating the lack of involvement of phospholipid translocase. Marked increases were also observed in flip of both NBD-phosphatidylethanolamine and NBD-phosphatidylcholine immediately after UVA irradiation, showing that the increase was independent on the head groups of phospholipids. These findings indicated that UVA changes the flip-flop of phospholipids and that the cell membrane is a molecular and cellular target of UVA.     

Induction of multinucleated cells caused by UVA exposure in different stages of the cell cycle

Fibroblasts of the line 3T3 from swiss albino mice were exposed to ultraviolet A (UVA) irradiation. The cells were synchronized by treatment with nocodazole and mitotic shake-off, and then exposed to UVA irradiation in different stages of the cell cycle. Their photosensitivity varied through the cell cycle, being greatest in the G2 phase. UVA irradiation was found to induce the formation of multinucleated cells. Cells in the G1 phase were found to be most prone to multinucleation 15 min after UVA irradiation, while cells exposed to UVA irradiation in S and G2 phases contained the largest fractions of multinucleated cells 24 h after treatment. The present results indicate that multinucleated cells are formed by fusion of two or more cells shortly after UVA irradiation of early G1 cells, while impairment of cytokinesis is a possible explanation for the delayed formation of multinucleated cells after irradiation in S and G2.     

Transient reduction in the tRNA 4-thiouridine content induced by ultraviolet A during post-irradiation growth in Enterobacter cloacae

The influence of previous exposure to ultraviolet-A radiation (UVA) was studied on the susceptibility of Enterobacter cloacae to undergo the growth delay effect. Comparison of growth curves corresponding to irradiated and control cells showed that a previous treatment with UVA almost abolished the growth delay effect. UV absorption spectra of tRNA, and reverse phase HPLC analysis of hydrolysed tRNA, demonstrated a low content of 4-thiouridine in E. cloacae cells grown after UVA exposure at low doses. Since 4-thiouridine is the UVA target responsible for initiation of growth delay, this observation explained the influence of previous exposure to UVA on the susceptibility of this organism to undergo growth delay. A similar but weaker alteration was found when Escherichia coli was assayed. The results suggest that, in addition to cross-linking with cytidine residues, the content of 4-thiouridine in tRNA may be modified by UVA by an unknown mechanism.     

Effect of cyclobutane pyrimidine dimers on apoptosis induced by different wavelengths of UV.

Ultraviolet radiation within three different wavelength ranges, UVA (340-400 nm), UVB (290-320 nm) or UVC (200-290 nm), was shown to induce apoptosis in OCP13 cells, derived from the medaka fish. Morphological changes such as cell shrinkage and a decrease in the number of nucleoli appeared 4 h after UVA, UVB or UVC irradiation, although with different relative efficiencies. Doses required to induce apoptosis with similar efficiencies were about 2500-fold higher for UVA and 10-fold higher for UVB than for UVC. The following phenomena occurred after UVA irradiation but not after UVB or UVC irradiation. (1) Ultraviolet-A-induced cell detachment occurred with or without cycloheximide pretreatment. (2) Cells attached to plastic showed morphological changes such as rounding up of nuclei without a change in the cell distribution. (3) Morphological changes after UVA irradiation could not be evaded by photorepair treatment. (4) Morphological changes did not occur in cells attached to glass coverslips but only those in plastic dishes. (5) Apoptosis occurred without detectable increase of caspase-3-like activity. (6) Morphological changes were inhibited by N-acetylcysteine, a scavenger of active oxygen species. These results suggest the existence of two different pathways leading to apoptosis, one for long- (UVA) and the other for short- (UVB or UVC) wavelength radiation.     

UVA and UVB Radiation Differentially Regulate Vascular Endothelial Growth Factor Expression in Keratinocyte-derived Cell Lines and in Human Keratinocytes

Vascular endothelial growth factor (VEGF) is a central regulator of neoangiogenesis in inflammatory and neoplastic conditions. Ultraviolet irradiation is one of the mainstays of dermatological therapy for various inflammatory skin diseases. In the present study we have compared the effects of UV irradiation on the production of VEGF by keratinocytes (KC) and by the KC-derived cell lines A431 and HaCaT. Irradiation of A431 and HaCaT cells with both UVA (10 J/cm2 and 20 J/cm2) and UVB (8 mJ/cm2 and 16 mJ/cm2) led to strong upregulation of VEGF mRNA and protein. Induction of VEGF by UVA and UVB in these cells was mediated by different pathways, i.e. the generation of free radicals and the secretion of (a) soluble factor(s), respectively. Unlike KC-derived cell lines, no increase in VEGF production was observed in KC in primary culture after irradiation with the same UV doses. Increasing the irradiation dose in these cells of UVA to 40 J/cm2 led to a marked decrease in soluble VEGF, whereas doses as high as 32 mJ/cm2 UVB only minimally affected VEGF levels. Reduction of VEGF production by KC might contribute to the effect of UVA irradiation in inflammatory skin diseases. The differential response of primary KC and autonomously growing KC-derived cell lines to the induction of VEGF by UV light could favor neoangiogenesis in the vicinity of epidermal tumor cells in vivo, thereby endowing them with a growth advantage over normal cells.     

Differential effects of UVA1 and UVB radiation on Langerhans cell migration in mice

The UVB (280-315 nm)- and UVA1 (340-400 nm)-induced migration of Langerhans cells (LC) from the epidermis and accumulation of dendritic cells (DC) in the lymph nodes draining the exposed skin site of C3H/HeN mice have been investigated. One minimum erythemal dose (MED) of UVB (1.5 kJ/m2) and of UVA1 (500 kJ/m2) were chosen, which have been shown previously to suppress delayed hypersensitivity (DTH). UVB irradiation resulted in a reduction in epidermal LC numbers, local to the site of the exposure, which was most apparent 12 h after exposure, but, in contrast, UVA1 had no significant effect even at 72 h after exposure. UVA1 did not exert any protection against the UVB-mediated depletion in LC numbers. The reduction in local LC following UVB exposure was prevented by systemic (intraperitoneal) treatment of mice with neutralising antibodies to either tumor necrosis factor (TNF)-alpha or interleukin (IL)-beta 2 h prior to the irradiation. It has been reported previously that UVB exposure caused an increase in the number of dendritic cells (DC) in the lymph nodes draining the irradiated skin site. In the present study we have shown that UVA1 had a similar effect. Pretreatment of the mice with neutralising antibodies to IL-1beta (by intraperitoneal injection) substantially inhibited DC accumulation induced by both UV regimens. However, anti-TNF-alpha antibodies affected only the UVB-induced increase, and did not alter the elevation in DC numbers observed following UVA1 exposure. These results indicate that UVB causes the migration of LC from the epidermis and an accumulation of DC in the draining lymph nodes by a mechanism that requires both TNF-alpha and IL-1beta. In contrast, UVAI does not cause LC migration from the epidermis and the accumulation of DC in the draining lymph nodes observed following UVA1 exposure requires IL-1beta, but not TNF-alpha. It is likely therefore that UVA1 acts through a different mechanism from UVB and may target a cutaneous antigen presenting cell other than LC, such as the dermal DC.     

Microchemostat-microbial continuous culture in a polymer-based, instrumented microbioreactor

In a chemostat, microbial cells reach a steady state condition at which cell biomass production, substrates and the product concentrations remain constant. These features make continuous culture a unique and powerful tool for biological and physiological research. We present a polymer-based microbioreactor system integrated with optical density (OD), pH, and dissolved oxygen (DO) real-time measurements for continuous cultivation of microbial cells. Escherichia coli (E. coli) cells are continuously cultured in a 150 microL, membrane-aerated, well-mixed microbioreactor fed by a pressure-driven flow of fresh medium through a microchannel. Chemotaxisial back growth of bacterial cells into the medium feed channel is prevented by local heating. Using poly(ethylene glycol) (PEG)-grafted poly(acrylic acid) (PAA) copolymer films, the inner surfaces of poly(methyl methacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS) of the microbioreactor are modified to generate bio-inert surfaces resistant to non-specific protein adsorption and cell adhesion. The modified surfaces of microbioreactor effectively reduce wall growth of E. coli for a prolonged period of cultivation. Steady state conditions at different dilution rates are demonstrated and characterized by steady OD, pH, and DO levels.     

Exacerbating effect of vitamin E supplementation on DNA damage induced in cultured human normal fibroblasts by UVA radiation

The effects of vitamin E supplementation were evaluated in cultured human normal fibroblasts exposed to ultraviolet A radiation (320-380 nm) (UVA). Cells were incubated in medium containing alpha-tocopherol, alpha-tocopherol acetate or the synthetic analog Trolox for 24 h prior to UVA exposure. DNA damage in the form of frank breaks and alkali-labile sites, collectively termed single-strand breaks (SSB), was assayed by the technique of single cell gel electrophoresis (comet assay), immediately following irradiation or after different repair periods. The generation of hydrogen peroxide (H2O2) and superoxide ion (O2.-) was measured by flow cytometry through the oxidation of indicators into fluorescent dyes. It was observed that pretreatment of cells with any form of vitamin E resulted in an increased susceptibility to the photoinduction of DNA SSB and in a longer persistence of damage, whereas no significant change was observed in the production of H2O2 and O2.- reactive oxygen species, compared to untreated controls. These findings indicate that in human normal fibroblasts, exogenously added vitamin E exerts a promoting activity on DNA damage upon UVA irradiation and might lead to increased cytotoxic and mutagenic risks.     

Induction of apoptosis by photoexcited tetracyclic compounds derivatives of benzo[b]thiophenes and pyridines

The antiproliferative activity, upon UVA irradiation, of two tetracyclic derivatives of benzo[b]thiophenes and pyridines, a benzo[b]thienopyridopyrimidone (1) and a thienocarboline (2), has been investigated in a panel of human tumor cell lines. The two compounds present a remarkable cytotoxicity after UVA irradiation (365 nm), reaching an IC50 of 0.1 microM in the leukaemia cell lines and 0.3-0.5 microM in the solid tumour cell lines. Their effect on the cell cycle was measured by flow cytometry in Jurkat cells. The compounds induce cell cycle perturbations and trigger a massive apoptosis as revealed by the externalisation of Annexin V-targeted residues at the outer plasmatic membrane. Furthermore the drugs induce, upon UVA irradiation significant variations of the mitochondrial potential (Deltapsi(mt)) measured by flow cytometry using the fluorochrome JC-1. In addition we characterized the mitochondrial production of reactive oxygen species (ROS) using the probe dihydroethidine (HE) and the oxidations of the mitochondrial phospholipid cardiolipin using the interacting probe nonyl acridine orange (NAO). Both compounds stimulate the production of ROS, and remarkably induce oxidation of cardiolipin. We have investigated the DNA-binding properties of these two compounds by means of UV-Vis spectroscopy and fluorescence. The two compounds exhibit a low affinity toward the macromolecule. The mode of binding was also investigated by means of flow linear dichroism (LD) which has revealed that the two compounds do not efficiently intercalate into DNA. Finally, the DNA-photocleavaging properties of the test compounds were studied on pBR322 plasmid DNA as a model. Only compound 1 is able to induce a significant production of single strand breaks only after digestion with the base excision repair enzyme Endo III. Altogether these data suggest that DNA is not a preferential target of these molecules and other subcellular structures may be responsible for their high phototoxic activity.     

DOES LOW-INTENSITY He-Ne LASER RADIATION PRODUCE A PHOTOBIOLOGICAL GROWTH RESPONSE IN Escherichia coli?

Abstract A photobiological study was camed out on the bacterium Escherichia coli in order to determine whether stimulation of growth occurred after irradiation of an inoculum with coherent red light. No enhancement or inhibition of growth was observed for cultures of the bacterium following irradiation of inocula with a Helium-neon laser (continuous wave, λ= 632.8 nm) at irradiances of 7.7 × 10¹⁵ and 1.8 × 10¹⁶ photons cm⁻² s⁻¹ using fluences of 4.5 × 10^−-1 and 4.5 J cm⁻² at each irradiance. Bacterial growth in irradiated and control cultures was monitored during a growth period of ca 2 h using a viable count technique after inocula in the early exponential phase had been diluted with fresh growth medium. These results do not provide support for the work of Kam et al . (1983, Nuov. Cim . 2D, 1138–1144), and Tiphlova and Karu (1988, Photochem. Photobiol . 48 , 467–471), which appear to show substantial enhancement of E. coli growth under these conditions.     

Wavelength dependence of ultraviolet-induced DNA damage distribution: involvement of direct or indirect mechanisms and possible artefacts.

DNA damage profiles have been established in plasmid DNA using purified DNA repair enzymes and a plasmid relaxation assay, following exposure to UVC, UVB, UVA or simulated sunlight (SSL). Cyclobutane pyrimidine dimers (CPDs) are revealed as T4 endonuclease V-sensitive sites, oxidation products at purine and pyrimidine as Fpg- and Nth-sensitive sites, and abasic sites are detected by Nfo protein from Escherichia coli. CPDs are readily detected after UVA exposure, though produced 10(3) and 10(5) times less efficiently than by UVB or UVC, respectively. We demonstrate that CPDs are induced by UVA radiation and not by contaminating UVB wavelengths. Furthermore, they are produced at doses compatible with human exposure and are likely to contribute to the mutagenic specificity of UVA [E. Sage et al., Proc. Natl. Acad. Sci. USA 93 (1996) 176-180]. Oxidative damage is induced with a linear dose dependence, for each region of the solar spectrum, with the exception of oxidized pyrimidine and abasic sites, which are not detectable after UVB irradiation. The distribution of the different classes of photolesions varies markedly, depending on wavelengths. However, the unexpectedly high yield of oxidative lesions, as compared to CPDs, by UVA and SSL led us to investigate their production mechanism. An artificial formation of hydroxyl radicals is observed, which depends on the material of the sample holder used for UVA irradiation and is specific for long UV wavelengths. Our study sheds light on a possible artefact in the production of oxidative damage by UVA radiation. Meanwhile, after eliminating some potential sources of the artefact ratios of CPDs to oxidized purine of three and five upon irradiation with UVA and SSL, respectively, are still observed, whereas these ratios are about 140 and 200 after UVC and UVB irradiation.     

The bystander effect is a novel mechanism of UVA-induced melanogenesis

We successfully identified the bystander effect in B16 murine melanoma cells exposed to UVA irradiation. The effect was identified based on melanogenesis following the medium transfer of the B16 cells, which had been cultured for 24 h after being exposed to UVA irradiation, to nonirradiated cells (bystander cells). Our confirmation study of the functional mechanism of bystander cells confirmed the reduced levels of mitochondrial membrane potential 1-4 h after the medium transfer. In addition, we observed increased levels of intracellular oxidation after 9-12 h, and the generation of melanin radicals, including long-lived radicals, 24 h after medium transfer. Further analysis of bystander factors revealed that the administration of EGTA treatment at the time of medium transfer led to an inhibition of melanogenesis and to neutralization of the mitochondrial membrane potential level, as well as to the restoration of intracellular oxidation levels to those of controls. The results demonstrated that the UVA irradiation bystander effect in B16 cells, as indicated by melanogenesis, was induced by the increase in intracellular oxidation due to the mitochondrial activity of calcium ions, which were among the bystander factors involved in the increase.