Biological UV dosimeters in the assessment of the biological hazard from environmental radiation

To determine the impact of environmental UV radiation, biological dosimeters that weight directly the incident UV components of sunlight have been developed, improved and evaluated in the frame of the BIODOS project. Four DNA-based biological dosimeters ((i) phage T7, (ii) uracil thin layer, (iii) spore dosimeter and (iv) DLR-biofilm) have been assessed from the viewpoint of their biological relevance, spectral response and quantification of their biological effectiveness. The biological dosimeters have been validated by comparing their readings with weighted spectroradiometer data, by comparison with other biological doses, as well as with the determined amounts of DNA UV photoproducts. The data presented here demonstrate that the biological dosimeters are potentially reliable field dosimeters for measuring the integrated biologically effective irradiance for DNA damage.     

Influence of Phage Proteins on Formation of Specific UV DMA Photoproducts in Phage T7

Phage T7 can be used as a biological UV dosimeter. Its reading is proportional to the inactivation rate expressed in HT7 units. To understand the influence of phage proteins on the formation of DNA UV photoproducts, cyclobutane pyrimidine dimers (CPD) and (6-4)photoproducts ((6-4)PD) were determined in T7 DNA exposed to UV radiation under different conditions: intraphage T7 DNA, isolated T7 DNA and heated phage. To investigate the effects of various wavelengths, seven different UV sources have been used. The CPD and (6-4)PD were determined by lesion-specific antibodies in an immunodot-blot assay. Both photoproducts were HT7 dose-dependently produced in all three objects by every irradiation source in the biologically relevant UV dose range (1-10 HT7). The CPD to (6-4)PD ratios increased with the increasing effective wavelength of the irradiation source and were similar in intraphage T7 DNA, isolated DNA and heated phage with all irradiation sources. However, a significant decrease in the yield of both photoproducts was detected in isolated T7 DNA and in heated phage compared to intraphage DNA, the decrease was dependent on the irradiation source. Both photoproducts were affected the same way in isolated T7 DNA and heated phage, respectively. The yield of CPD and (6-4)PD was similar in B, C-like and A conformational states of isolated T7 DNA, indicating that the conformational switch in the DNA is not the decisive factor in photoproduct formation. The most likely explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in an increased rate of dimerization and (6-4)PD production of adjacent based in intraphage T7 DNA.     

Assessment of the Effects of Various UV Sources on Inactivation and Photoproduct Induction in Phage T7 Dosimeter

The correlation between the biologically effective dose (BED) of a phage T7 biological dosimeter and the induction of cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts ((6-4)PD) in the phage DNA was determined using seven various UV sources. The BED is the inactivation rate of phage T7 expressed in H^T7 units. The CPD and (6-4)PD were determined by lesion-specific monoclonal antibodies in an immunodot-blot assay. The various lamps induced these lesions at different rates; the relative induction ratios of CPD to (6-4)PD increased with increasing effective wavelength of irradiation source. The amount of total adducts per phage was compared to the BED of phage T7 dosimeter, representing the average number of UV lesions in phage. For UVC (200–280nm radiation) and unfiltered TL01 the number of total adducts approximates the reading; however, UV sources having longer effective wavelengths produced fewer CPD and (6-4)PD. A possible explanation is that although the most relevant lesions by UVC are the CPD and (6-4)PD, at longer wavelengths other photoproducts can contribute to the lethal damage of phages. The results emphasize the need to study the biological effects of solar radiation because the lesions responsible for the lethal effect may be different from those produced by various UV sources.     

Biological responses to the simulated Martian UV radiation of bacteriophages and isolated DNA

Mars is considered as a main target for astrobiologically relevant exploration programmes. In this work the effect of simulated Martian solar UV radiation was examined on bacteriophage T7 and on isolated T7 DNA. A decrease of the biological activity of phages, characteristic changes in the absorption spectrum and in the electrophoretic pattern of isolated DNA/phage and the decrease of the amount of PCR products were detected indicating damage of isolated and intraphage T7 DNA by UV radiation. Further mechanistic insights into the UV-induced formation of intraphage/isolated T7 DNA photoproducts were gained from the application of appropriate enzymatic digestion and neutral/alkaline agarose gel electrophoresis. Our results showed that intraphage DNA was about ten times more sensitive to simulated Martian UV radiation than isolated T7 DNA indicating the role of phage proteins in the DNA damage. Compared to solar UV radiation the total amount of DNA damage determined by QPCR was about ten times larger in isolated DNA and phage T7 as well, and the types of the DNA photoproducts were different, besides cyclobutane pyrimidine dimers (CPD), double-strand breaks (dsb), and single-strand breaks (ssb), DNA-protein cross-links were produced as well. Surprisingly, energy deposition as low as 4-6eV corresponding to 200-400nm range could induce significant amount of ssb and dsb in phage/isolated DNA (in phage the ratio of ssb/dsb was approximately 23%/12% and approximately 32%/19% in isolated DNA). 5-8% of the CPD, 3-5% of the AP (apurinic/apyrimidinic) sites were located in clusters in DNA/phage, suggesting that clustering of damage occur in the form of multiple damaged sites and these can have a high probability to produce strand breaks. The amount of total DNA damage in samples which were irradiated in Tris buffer was reduced by a factor approximately 2, compared to samples in phosphate buffer, suggesting that some of the photoproducts were produced via radicals.     

Construction of spectral sensitivity function using polychromatic UV sources.

A procedure is presented for constructing the spectral sensitivity functions of biological dosimeters, using five polychromatic UV sources possessing different emission spectra. Phage T7 and uracil biological dosimeters have been used for measuring the dose rates of the lamps. Their spectral sensitivity functions consisting of two exponential terms have been constructed. The parameters of the spectral sensitivity functions have been determined by comparing the directly measured and calculated dose-rate values. The parameters of the sensitivity function are accepted as correct values when the deviation of the measured and calculated values is a minimum. Based on the deviations between the constructed and the experimentally determined spectral sensitivities with monochromatic sources, the differences between the measured and calculated results are interpreted. The importance of the correct spectral sensitivity data is demonstrated through the effectiveness spectra of a TL 01 lamp for phage T7 killing, uracil dimerization and erythema induction.     

DNA as a solar dosimeter in the ocean.

Stratospheric ozone depletion may result in increased solar UV-B radiation to the ocean's upper layers and may cause deleterious effects on marine organisms. The primary UV-B damage induced in biological systems is to DNA. While physical measurements of solar UV-B penetration into the sea have been made, the effective depth and magnitude of actual DNA damage have not been determined. In the experiments reported here, UV-B-induced photoproducts (cyclobutane pyrimidine dimers) have been quantified in DNA molecules exposed to solar UV at the surface and at various depths in clear, tropical marine waters off Lee Stocking Island (23 degrees 45' N, 76 degrees 0.7' W), Exuma Cays, Bahamas. (14C)thymidine-labeled DNA or unlabeled bacteriophage phi X174 DNA was placed in specially designed quartz tubes at various depths for up to five days. Following exposure, DNA samples were removed to the laboratory where UV-B-induced pyrimidine dimers were quantified using a radiochromatographic assay, and bacteriophage DNA inactivation by solar UV-B was assayed by plaque formation in spheroplasts of Escherichia coli. Pyrimidine dimer induction was linear with time but the accumulation of dimers in DNA with time varied greatly with depth. Attenuation of dimer formation with depth of water was exponential. DNA at 3 m depth had only 17% of the pyrimidine dimers found at the surface. Bacteriophage phi X174 DNA, while reduced 96% in plaque-forming ability by a one day exposure to solar UV at the surface of the water, showed no effect on plaque formation after a similar exposure at 3 m. The data collected at the water's surface showed a "surface-enhanced dose" in that DNA damages at the real surface were greater than at the imaginary surface, which was obtained by extrapolating the data at depth to the surface. These results show the sensitivity of both the biochemical (dimers) and biological (phage plaques) DNA dosimeters. DNA dosimeters offer a sensitive, convenient and relatively inexpensive monitoring system, having both biochemical and biological endpoints for monitoring the biologically effective UV-B flux in the marine environment. Unlike physical dosimeters, DNA dosimeters do not have to be adjusted for biological effectiveness since they are sensitive only to DNA-mediated biologically effective UV-B radiation. Results of pyrimidine dimer induction in DNA by solar UV accurately predicted UV doses to the phage DNA.     

Sunlight-induced DNA Damage in Marine Micro-organisms Collected Along a Latitudinal Gradient from 70°N to 68°S

We examined ultraviolet radiation (UVR)-induced DNA damage in marine micro-organisms collected from surface seawater along a latitudinal transect in the Central Pacific Ocean from 70°N to 68°S. Samples were collected predawn and incubated under ambient UVR in transparent incubators at in situ temperatures until late afternoon at which time they were filtered into primarily bacterioplankton and eukaryotic fractions. Cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts [(6-4)PDs] were quantified in DNA extracts using radioimmunoassays. UVB was lowest in the polar regions and highest near the equator and correlations between UVB and DNA damage were observed. The eukaryotic fraction showed significant CPDs across the entire transect; (6-4)PDs were detected only in the tropics. The bacterial fraction showed no accumulation of (6-4)PDs at any latitude, although residual (6-4)PDs were observed. Bacterial cell volumes were greatest in the sub-Arctic and northern temperate latitudes and lower in the tropics and southern hemisphere, a unique observation that parallels Bergmann's rule. A strong negative correlation was observed between cell volume and CPDs. The environmental impact of solar UVR on marine micro-organisms in the open ocean is complex and our results suggest that several factors such as DNA repair, cell size, temperature, salinity, nutrients and species composition are important in determining relative sensitivity.     

Validation of phage T7 biological dosimeter by quantitative polymerase chain reaction using short and long segments of phage T7 DNA

Phage T7 can be used as a biological dosimeter; its reading, the biologically effective dose (BED), is proportional to the inactivation rate |ln (n/n0)|. For the measurement of DNA damage in phage T7 dosimeter, a quantitative polymerase chain reaction (QPCR) methodology has been developed using 555 and 3826 bp fragments of phage T7 DNA. Both optimized reactions are so robust that an equally good amplification was obtained when intact phage T7 was used in the reaction mixture. In the biologically relevant dose range a good correlation was obtained between the BED of the phage T7 dosimeter and the amount of ultraviolet (UV) photoproducts determined by QPCR with both fragments under the effect of five various UV sources. A significant decrease in the yield of photoproducts was detected by QPCR in isolated T7 DNA and in heated phage compared with intraphage DNA with all irradiation sources. Because the yield of photoproducts was the same in B, C and A conformational states of T7 DNA, a possible explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in increased induction of photoproducts.     

Use of Uracil Thin Layer for Measuring Biologically Effective UV Dose

Abstract— Dimerization of uracil monomers in a polycrystalline state by UV radiation changes the absorption characteristics of a thin layer of the material. The change in optical density, measured by spectrophotometry in the250–400 nm range, as a function of the exposure time is evaluated in terms of the biologically effective UV dose. A statistical evaluation of a great number of uracil dosimeters irradiated with a TL01 lamp from Philips establishes the possibility of evaluating the biologically effective UV dose using a uracil dosimeter. Nonlinear regression procedures were introduced to correct the absorption spectra for contributions due to light scattering and to determine the optical density values required to calculate the UV dose expressed in H^Uunits. Comparison of cumulative daily doses and long-term monitoring measured by the uracil thin-layer dosimeter and a phage T7 dosimeter are given, which allow the determination of conversion factors between various biological dosimeters under different irradiation conditions.     

Temperature-dependent formation and photorepair of DNA damage induced by UV-B radiation in suspension-cultured tobacco cells

Two photoproducts of DNA damage, i.e. cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs), induced by UV-B radiation in suspension-cultured tobacco cells were quantified by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. CPDs and 6-4PPs were induced in tobacco cells by UV-B radiation. Photorepair of CPDs was faster than that of 6-4PPs. UV-B radiation induces formation of CPDs and 6-4PPs even at 0 degrees C, but low temperature significantly decreases the UV-B-induced (in contrast to UV-C-induced) formation of CPDs and 6-4PPs. Low temperature also retarded the removal of CPDs and 6-4PPs under white light, and almost no photorepair of CPDs and 6-4PPs was detected at 0 degrees C. When purified DNA from tobacco cells grown in darkness was irradiated with UV-B, formation of CPDs and 6-4PPs took place at the same speed at different temperatures. It indicated that formation of CPDs and 6-4PPs induced by UV-B was temperature-independent in a non-cellular system. Based on our results for suspension-cultured tobacco cells, not only the photorepair but also UV-B-induced formation of CPDs and 6-4PPs are temperature-dependent.     

Influence of spectral and angular sensitivity on the readout of biological dosimeters

Biological systems used as biological dosimeters can possess different angular sensitivities from the detectors usually used in physical devices. A simple experimental setup has been developed and used to measure the angular sensitivity of uracil thin-layer biological dosimeters. Results of angular sensitivity measurements for uracil thin-layer dosimeters are presented using a Xe arc lamp as the UV source. According to the experiments described here, uracil thin-layer dosimeters show a cosine-type angular dependence. In several indoor experiments broadband UV meters are used to control the applied dose rate from a given artificial UV source. The experimental setup has been designed and used to verify experimentally the importance of spectral and angular sensitivity differences of biological and physical UV meters applied in biological experiments. Model calculations for two different irradiation systems, using different geometrical arrangements of artificial UV sources, are also presented. For these arrangements relative dose rates that could be measured with dosimeters of arbitrary spectral, but different angular sensitivity have been calculated.     

Further insight in the photochemistry of DNA: structure of a 2-imidazolone (5-4) pyrimidone adduct derived from the mutagenic pyrimidine (6-4) pyrimidone photolesion by UV irradiation

Pyrimidine (6-4) pyrimidone photoproducts represent one of the major mutagenic and carcinogenic class of DNA damage produced by UV exposure. At present, besides their conversion to their Dewar valence isomer, (6-4) photoproducts are generally believed to be photostable, and the observed biological properties of Paterno-Büchi-derived photoproducts are, thus far, exclusively attributed to these two types of compounds. Using a model system (2) relevant to DNA photochemistry, we have observed that the 5'-base moiety of the (6-4) thymine dimer 3, under far-UV radiation, is able to undergo a ring contraction leading to a 2-oxoimidazoline, 1. This unprecedented secondary photochemical reaction constitutes the first report of a major photomodification affecting (6-4) photoproducts and strongly questions the biological stability of the (6-4) adducts under UV light with 2-imidazolone (5-4) pyrimidone adducts being possibly another source of endogenous DNA damage.     

SOLAR DOSIMETRY WITH REPAIR DEFICIENT BACTERIAL SPORES: ACTION SPECTRA, PHOTOPRODUCT MEASUREMENTS AND A COMPARISON WITH OTHER BIOLOGICAL SYSTEMS

Abstract— Populations of radiation sensitive spores ( Bacillus subtilis UVSSP), vegetative bacteria ( E. coli K12-AB2480) and bacteriophage ( E. coli phage T4vx) have been considered as possible biological dosimeters to integrate DNA-absorbed solar energy incident on the Earth's surface.
Irradiation of spores of B. subtilis UVSSP with monochromatic far- and near-UV radiation and solar radiation have indicated that these radiations have a similar efficiency in inducing spore photoproducts per lethal event. Action spectra for lethality taken with the three radiation sensitive biological systems show a similar pattern in each case with a broad shoulder in the 334–365 nm wavelength region. This finding indicates a relatively high susceptibility of the DNA to chemical alteration in this wavelength range. Although less sensitive to sunlight than the other biological systems tested, the B. subtilis UVSSP spore mutant has the advantage of temperature independence of inactivation, stability between irradiation and assay and a simple, reproducible irradiation and assay procedure. Field measurements have supported the utility of this mutant as a sunlight dosimeter.     

DNA Damage Induced by Late Spring Sunlight in Antarctica

Sunlight ultraviolet (UV) radiation constitutes an important environmental genotoxic agent that organisms are exposed to, as it can damage DNA directly, generating pyrimidine dimers, and indirectly, generating oxidized bases and single-strand breaks (SSBs). These lesions can lead to mutations, triggering skin and eye disorders, including carcinogenesis and photoaging. Stratospheric ozone layer depletion, particularly in the Antarctic continent, predicts an uncertain scenario of UV incidence on the Earth in the next decades. This research evaluates the DNA damage caused by environmental exposure to late spring sunlight in the Antarctic Peninsula, where the ozone layer hole is more pronounced. These experiments were performed at the Brazilian Comandante Ferraz Antarctic Station, at King’s George Island, South Shetlands Islands. For comparison, tropical regions were also analyzed. Samples of plasmid DNA were exposed to sunlight. Cyclobutane pyrimidine dimers (CPDs), oxidized base damage and SSBs were detected using specific enzymes. In addition, an immunological approach was used to detect CPDs. The results reveal high levels of DNA damage induced by exposure under the Antarctic sunlight, inversely correlated with ozone layer thickness, confirming the high impact of ozone layer depletion on the DNA damaging action of sunlight in Antarctica.     

Biological consequences of cyclobutane pyrimidine dimers.

In the skin many molecules may absorb ultraviolet (UV) radiation upon exposure. In particular, cellular DNA strongly absorbs shorter wavelength solar UV radiation, resulting in various types of DNA damage. Among the DNA photoproducts produced the cyclobutane pyrimidine dimers (CPDs) are predominant. Although these lesions are efficiently repaired in the skin, this CPD formation results in various acute effects (erythema, inflammatory responses), transient effects (suppression of immune function), and chronic effects (mutation induction and skin cancer). The relationships between the presence of CPD in skin cells and the subsequent biological consequences are the subject of the present review.     

Field testing of biological spectral weighting functions for induction of UV-absorbing compounds in higher plants

Action spectra are typically used as biological spectral weighting functions (BSWF) in biological research on the stratospheric ozone depletion issue. Despite their critical role in determining the amount of UV supplied in experiments, there has been only limited testing of different functions under realistic field conditions. Here, we calculate effective radiation according to five published BSWF and evaluate the appropriateness of these BSWF in representing the induction of UV-absorbing compounds. Experiments were carried out in the field using both ultraviolet-B radiation (280-320 nm) supplementation and selective filtering of solar UV radiation. For the four species tested, BSWF that extend into the ultraviolet-A radiation (320-400 nm) (UV-A) with moderate effectiveness best represented the observed results. When compared with the commonly used generalized plant response, these BSWF suggest that simulations of ozone depletion will require more radiation than in the past experiments. However, they imply lower radiation supplements than a new plant growth BSWF that has a greater emphasis on UV-A wavelengths.     

Phage T7 in biological UV dose measurement.

An experimental method complete with theoretical considerations is presented for the measurement of different biological UV doses. The method is based on the high sensitivity of phage T7 activity to UV light. A precisely determined T7 inactivation action spectrum is presented over a wide optical range (240-514 nm). Using the T7 spectral sensitivity in relation to the minimal erythema dose (MED) and the effective spectral irradiance from solar radiation for the MED, an example is given to determine the MED value based on the measurement of T7 inactivation for a given case. The advantages and applicability of the method are discussed.     

Exposure of phage T7 to simulated space environment: the effect of vacuum and UV-C radiation

The experiment "Phage and Uracil Response" (PUR) will be accommodated in the EXPOSE facility of the International Space Station (ISS). Its objective is to examine and quantify the effect of specific space conditions on bacteriophage T7 and isolated T7 DNA thin films. In order to define the environmental and technical requirements of the EXPOSE, the samples were subjected to the Experiment Verification Test (EVT). During EVT the samples were exposed to selected space conditions: high vacuum (10(-4) to 10(-6) Pa) and UV-C radiation (254 nm) alone and in combination. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA/phage and the decrease of the amount of PCR products have been detected indicating the damage of isolated and intraphage T7 DNA. Intraphage DNA is more sensitive to simulated space parameters than isolated T7 DNA in thin layers as well. We obtained substantial evidence that DNA lesions accumulate throughout exposure, and the amount of damage depends on the thickness of the layers. According to our preliminary results, the damages by exposure to conditions of dehydration and UV irradiation are larger than the sum of vacuum alone, or radiation alone case, suggesting a synergistic action of space vacuum and UV radiation with DNA being the critical target.     

Seasonal fluctuation of DNA photodamage in marine plankton assemblages at Palmer Station, Antarctica

Ultraviolet radiation-induced DNA damage frequencies were measured in DNA dosimeters and natural plankton communities during the austral spring at Palmer Station, Antarctica, during the 1999-2000 field season. We found that the fluence of solar ultraviolet-B radiation (UV-B) at the earth's surface correlated with stratospheric ozone concentrations, with significant ozone depletion observed because of "ozone hole" conditions. To verify the interdependence of ozone depletion and DNA damage in natural microbial communities, seawater was collected daily or weekly from Arthur Harbor at Palmer Station, Antarctica, throughout "ozone season," exposed to ambient sunlight between 0600 and 1800 h and fractionated using membrane filtration to separate phytoplankton and bacterioplankton populations. DNA from these fractions was isolated and DNA damage measured using radioimmunoassay. Under low-ozone conditions cyclobutane dimer concentrations in bacterioplankton and phytoplankton communities were maximal. DNA damage measured in dosimeters correlated closely with ozone concentrations and UV-B fluence. Our studies offer further support to the theory that stratospheric deozonation is detrimental to marine planktonic organisms in the Southern Ocean.     

Assessment of UV Biological Spectral Weighting Functions for Phenolic Metabolites and Growth Responses in Silver Birch Seedlings

In research concerning stratospheric ozone depletion, action spectra are used as biological spectral weighting functions (BSWFs) for describing the effects of UV radiation on plant responses. Our aim was to evaluate the appropriateness of six frequently used BSWFs that differ in effectiveness with increasing wavelength. The evaluation of action spectra was based on calculating the effective UV radiation doses according to 1–2) two formulations of the generalized plant action spectrum, 3) a spectrum for ultraviolet induced erythema in human skin, 4) a spectrum for the accumulation of a flavonol in Mesembryanthemum crystallinum , 5) a spectrum for DNA damage in alfalfa seedlings and 6) the plant growth action spectrum. We monitored effects of UV radiation on the concentration of individual UV absorbing metabolites and chlorophyll concentrations in leaves and growth responses of silver birch ( Betula pendula ) seedlings. Experiments were conducted outdoors using plastic films attenuating different parts of the UV spectrum. Chlorophyll concentrations and growth were not affected by the UV treatments. The response to UV radiation varied between and within groups of phenolics. In general, the observed responses of phenolic groups and individual flavonoids were best predicted by action spectra extending into the UV-A region with moderate effectiveness.