S. Gáspár

Phages T7 in biological UV dose measurements

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.

ULTRAVIOLET DOSIMETRY IN OUTDOOR MEASUREMENTS BASED ON BACTERIOPHAGE T7 AS A BIOSENSOR

A simple method has been worked out for measuring the biologically effective dose (BED) of solar radiation The method uses phage T7 as a biosensor and it includes field measurements of global and direct UV radiation from the sun in the air; it has been applied to underwater measurements as well. Results of field measurements are presented with discussion of the angle‐dependent sensitivity of the biosensor. A model of spectral irradiance based on the measured values is presented. Relevance of the HT7 unit—derived earlier by us from T7 phage inactivation upon UV radiation—as a measure of the BED is also discussed.

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 HT7 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.

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 HUunits. 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.

Biological UV dosimetry-a comprehensive problem

Abstract The biologically effective dose from environmental radiation was measured using phage T7 and uracil thin layer sensors in a dosimeter developed previously (Gy. Ronto, S. Gaspar and A. Berces, Phages T7 in biological UV dose measurement, J. Photochem. Photobiol. B: Biol., 12 (1992) 285–294; P. Grof, S. Gaspar and A. Berces, Uracil thin layers in dosimetry of UV radiation, Proc. Int. Symp. Budapest Biomedical Optics, Europe, 1993 , to be published). Examples of measured daily and annual profiles are presented to demonstrate the possibilities and limits of biological dosimetry. In addition, phage T7, uracil sensor and Robertson-Berger meter results are compared. A comparison of model calculation data with the measured values is presented. On the basis of the results obtained, the application of biological dosimeters in both long-term field measurements and laboratory experiments is suggested.

New trends in photobiology. Phage nucleoprotein-psoralen interaction: quantitative characterization of dark and photoreactions

The irradiation of the phage T7 system containing psoralen as photosensitizer causes many processes, each of them leading to phage inactivation. These processes include the UV-induced photoreactions in the phage nucleic acid, and photoreactions in the nucleic acid sensitized by either psoralen or psoralen photobreakdown products. In addition the intercalation of the psoralen molecule itself in the phage nucleic acid as well as the psoralen photobreakdown products cause phage inactivation. Under appropriate experimental conditions these reactions can be studied and characterized separately. The quantitative characteristics (e.g. inactivation cross-section, action spectra and index for dark genotoxicity) are demonstrated for different linear and angular psoralens. Some theoretical and practical consequences of the results obtained are discussed.

Influence of phage proteins on formation of specific UV DNA photoproducts in phage T7

Abstract— 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 immunodotblot 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 bases in intraphage T7 DNA.

Action spectra for photoinduced inactivation of bacteriophage T7 sensitized by 8-methoxypsoralen and angelicin

The action spectrum (240-300 nm) for photoinactivation of unsensitized phage T7 and the action spectra (310-380 nm) for photoinactivation of phage T7 sensitized with 8-methoxypsoralen (8-MOP) and angelicin were measured by an automated method. For unsensitized phage T7 the action spectrum is in good agreement with the absorption spectrum. For sensitization with angelicin the action spectrum is similar to the absorption spectrum, but for sensitization with 8-MOP the spectra are different. The agreement between the T7 absorption and action spectra in the far-UV region is due to photodamage of DNA, leading to phage inactivation. The similarity in the action and absorption spectra in the near-UV region for sensitization with angelicin seems to be in accordance with the monofunctional photobinding of angelicin to DNA. The action spectrum for sensitization with 8-MOP has a maximum at about 320 nm and this suggests that, in addition to the monoadducts, the biadducts play a role in the inactivation of phage T7. Taking the number of bound furocoumarin molecules into consideration, the quantum efficiencies were estimated. Furocoumarin increases the quantum efficiency in the near-UV region and the values are similar to those obtained in far-UV light without psoralens.

Monitoring of environmental UV radiation by biological dosimeters

As a consequence of the stratospheric ozone layer depletion biological systems can be damaged due to increased UV-B radiation. The aim of biological dosimetry is to establish a quantitative basis for the risk assessment of the biosphere. DNA is the most important target molecule of biological systems having special sensitivity against short wavelength components of the environmental radiation. Biological dosimeters are usually simple organisms, or components of them, modeling the cellular DNA. Phage T7 and polycrystalline uracil biological dosimeters have been developed and used in our laboratory for monitoring the environmental radiation in different radiation conditions (from the polar to equatorial regions). Comparisons with Robertson-Berger (RB) meter data, as well as with model calculation data weighted by the corresponding spectral sensitivities of the dosimeters are presented. Suggestion is given how to determine the trend of the increase in the biological risk due to ozone depletion.

Stability of nucleic acid under the effect of UV radiation

Nucleic acids (combined with protein molecules) are essential constituents of the living systems playing an important role in the early evolution of life as well. A specific feature of these molecules has been found and directly confirmed recently: under the influence of short-wavelength UV radiation bipyrimidine photoproducts (cyclobutane dimers and 6-4 bipyrimidines) are induced and the reversion of them can be provoked by the same photons. However, reversion is preferred by the shorter wavelengths. With increasing ratio of the longer wavelength components of the radiation (using artificial UV sources and solar light on the Earths surface) the impact of the reversible photoproducts in the harmful biological effect decreases and other photoproducts are dominant. Assuming the photoinduced reactions (dimerisation and reversion) are statistical events, during the irradiation the chance for a number of nucleoprotein molecules to survive the radiation damage can be reality. The theoretical and experimental basis of these assumptions will be discussed in the case of bacteriophage T7 nucleoprotein.