LETHAL EFFECTS OF NATURAL SOLAR ULTRAVIOLET RADIATION IN REPAIR PROFICIENT AND REPAIR DEFICIENT STRAINS OF ESCHERICHIA COLI: ACTIONS AND INTERACTIONS

Abstract— The inactivation of repair proficient ( Escherichia coli K12 AB 1157, E. coli B/r) and repair deficient ( E. coli K12 AB 1886 uvrA , AB 2463 recA and AB 2480 uvrA recA ) strains of bacteria by noon sunlight has been measured. The use of biological dosimetry based on an ultraviolet (UV) sensitive strain of Bacillus subtilis spores has allowed a quantitative comparison of bacterial inactivation by solar, 254 and 302 nm radiations. Our analysis indicates that: (1) uvrA and recA gene products are involved in repair of a substantial portion of the solar DNA damage, (2) 302 nm is a more appropriate wavelength than 254 nm to represent the DNA-damaging action of sunlight and that (3) repair proficient strains are inactivated by sunlight more rapidly than expected from the levels of DNA damage induced. When populations of repair proficient bacteria are exposed to noon sunlight for 20 min, they become sensitive to the lethal action of far-UV (254 nm), MMS (0.1 M ) and to a lesser extent, mild heat (52°C).     

Damage to UV-Sensitive Cells by Short UV in Photographic Flashes

Abstract— Light emitted by electronic photographic flash units is shown to damage bacteria and human skin fibroblasts deficient in repair systems, with survival curves very similar to those produced by 254 nm short UV. The lesions induced by these flashes are as photorepairable by the photolyase enzyme as those induced by 254 nm UV and result in equivalent survival rates. Biological dosimetry performed with microorganisms highly sensitive to UV ( Escherichia coli K12 AB2480, deficient in excision and recombinational-dependent repair systems and Bacillus subtilis UVSSP spores, deficient in excision and in a specific spore repair process) revealed that each 1 ms flash of light from the photographic unit used in this work contained the equivalent of 0.25 J m⁻² of 254 nm UV, when measured at a distance of 7.0 cm. This dose of UV was found to be lethal to both repair-deficient E. coli bacteria and repair-deficient human skin fibroblasts obtained from xeroderma pigmentosum donors, as well as mutagenic in B/r wild-type and HCR-mutant bacteria.     

INACTIVATION ACTION SPECTRA OF BACILLUS SUBTILIS SPORES IN EXTENDED ULTRAVIOLET WAVELENGTHS (50–300nm) OBTAINED WITH SYNCHROTRON RADIATION

Five types of Bacillus subtilis spores (UVR, UVS, UVP, RCE, and RCF) differing in repair and/or recombinational capabilities were exposed to monochromatic radiations at 13 wavelengths from 50 to 300 nm in vacuum. An improved biological irradiation system connected to a synchrotron radiation source was used to produce monochromatic UV radiation in this extended wavelength range with sufficient fluence to inactivate bacterial spores. From the survival curves obtained, the action spectra for the inactivation of the spores were depicted. Recombination-deficient RCE (recE) and RCF (recF) spores were more sensitive than the wild-type UVR spores in the entire range of wavelengths. This was considered to mean that DNA was the major target for the inactivation of the spores. Vacuum-UV radiations of 125-175 nm were effective in killing the spores, and distinct peaks of the sensitivity were seen with all types of the spores. Insensitivities at 190 and 100 nm were common to all five types of spores, indicating that these wavelengths were particularly impenetrant and absorbed by the outer layer materials. The vacuum-UV peaks centering at 150 nm were prominent in the spores defective in recombinational repair, while the far-UV peaks at around 235 and 270 nm were prominent in the UVS (uvrA ssp) and UVP (uvrA ssp polA) spores deficient in removal mechanisms of spore photoproducts. Thus, the profiles of the action spectra were explained by three factors; the penetration depth of each radiation in a spore, the efficiency of producing DNA damage that could cause inactivation, and the repair capacity of each type of spore.     

Action spectra for survival and spore photoproduct formation of Bacillus subtilis irradiated with short-wavelength (200-300 nm) UV at atmospheric pressure and in vacuo.

Spores of Bacillus subtilis are approximately ten times less likely to survive UV light irradiation in a vacuum than under atmospheric conditions. Photoproduct formation was studied in spores irradiated under ultrahigh vacuum (UHV) conditions and in spores irradiated at atmospheric pressure. In addition to the "spore photoproduct" 5-thyminyl-5,6-dihydrothymine (TDHT), which is produced in response to irradiation at atmospheric pressure, two additional photoproducts, known as the cis-syn and trans-syn isomers of thymine dimer, are produced on irradiation in vacuo. The spectral efficiencies for photoproduct formation in spores are reduced under vacuum conditions compared with atmospheric conditions by a factor of 2-6, depending on the wavelength. Because formation of TDHT does not increase after irradiation in vacuo, TDHT cannot be responsible for the observed vacuum effect. Vacuum specific photoproducts may cause a synergistic response of spores to the simultaneous action of UV light and UHV. An increased quantum efficiency, destruction of repair systems and formation of irreparable lesions are postulated for the enhanced sensitivity of B. subtilis spores to UV radiation in vacuo.     

ACTION SPECTRA IN ULTRAVIOLET WAVELENGTHS (150-250 nm) FOR INACTIVATION AND MUTAGENESIS OF Bacillus subtilis SPORES OBTAINED WITH SYNCHROTRON RADIATION

Bacillus subtilis spores were exposed in vacuo to monochromatic UV radiation from synchrotron radiation in the wavelength range of 150 nm to 250 nm. Survival and frequency of mutation to histidine-independent reversion were analysed for three types of spores differing in DNA-repair capabilities. UVR spores (wild-type DNA repair capability) exhibited nearly equal sensitivity to the lethal effects of far-UV (220 nm and 250 nm) and of vacuum-UV radiation (150 and 165 nm), but showed marked resistance to 190 nm radiation. UVS spores (excision-repair and spore-repair deficient) and UVP spores (a DNA polymerase I-defective derivative of UVS) exhibited similar action spectra; pronounced sensitivity at 250 and 220 nm, insensitivity at 190 nm and a gradual increase of the sensitivity as the wavelength decreased to 165 nm. In all strains, the action spectra for mutation induction paralleled those for the inactivation, indicating that vacuum-UV radiation induced lethal and mutagenic damages in the spore DNA. The insensitivity of the spores to wavelengths around 190 nm may be explicable by assuming that radiation is absorbed by materials surrounding the core in which DNA is situated.     

Overview of the inactivation by 254 nm ultraviolet radiation of bacteria with particular relevance to biodefense

Abstract Our goal was to ultimately predict the sensitivity of untested bacteria (including those of biodefense interest) to ultraviolet (UV) radiation. In this study, we present an overview and analysis of the relevant 254 nm data previously reported and available in the literature. The amount of variability in this data prevented us from determining an "average" response for any bacterium. Therefore, we developed particular selection criteria to include the data in our analysis and suggested future guidelines for reporting UV sensitivity results. We then compiled a table of the sensitivity to 254 nm UV for 38 bacteria and three bacterial spores. The UV sensitivity was quite similar (within 10%) among the spores of Bacillus anthracis (strains Vollum 1B and Sterne), Bacillus subtilis, and Bacillus megaterium. These data indicate that spores of B. subtilis and B. megaterium could be adequate simulants of B. anthracis spores in UVC experiments. Spores of B. anthracis, B. subtilis and B. megaterium were 5-10 times more resistant to UV than were their corresponding vegetative cells. The vegetative cells of B. anthracis showed similar UV sensitivity to those of Burkholderia pseudomallei, Shigella sonnei, and a wild-type strain of Escherichia coli. Yersinia enterocolitica and Vibrio cholerae appeared more sensitive to UV and Salmonella typhi slightly more resistant to UV than E. coli. The sensitivity (at 254 nm) of all vegetative bacteria ranged from 11 to 80 Jm(2) for a 1 Log(10) kill and from 25-200 Jm(2) for 4 Log(10) kill.     

Biosensor for the specific detection of a single viable B. anthracis spore

A simple membrane strip-based biosensor for the detection of viable B. anthracis spores was developed and combined with a spore germination procedure as well as a nucleic acid amplification reaction to identify as little as one viable B. anthracis spore in less than 12 h. The biosensor is based on identification of a unique mRNA sequence from the anthrax toxin activator (atxA) gene encoded on the toxin plasmid, pXO1. Preliminary work relied on plasmid vectors in both E. coli and B. thuringiensis expressing the atxA gene. Once the principle was firmly established, the vaccine strain of B. anthracis was used. After inducing germination and outgrowth of spores of B. anthracis (Sterne strain), RNA was extracted from lysed cells, amplified using nucleic acid sequence-based amplification (NASBA), and rapidly identified by the biosensor. While the biosensor assay requires only 15-min assay time, the overall process takes 12 h for the detection of as little as one viable B. anthracis spore, and is shortened significantly, if larger amounts of spores are present. The biosensor is based on an oligonucleotide sandwich-hybridization assay format. It uses a membrane flow-through system with an immobilized oligonucleotide probe that hybridizes with the target sequence. Signal amplification is provided when the target sequence hybridizes to a second oligonucleotide probe that has been coupled to dye-encapsulating liposomes. The dye in the liposomes then provides a signal that can be read visually or quantified with a hand-held reflectometer. The biosensor can detect as little as 1.5 fmol of target mRNA. Specificity analysis revealed no crossreactivity with closely related species such as B. cereus, B. megaterium, B. subtilis, B. thuringiensis etc.     

Sporicidal testing of commercial germicides using a chemical standard and a calibrated bioindicator

The AOAC sporicidal method uses as a standard the resistance of spores on carriers to 2.5N HCl. This resistance is variable at exposure times ranging from 2 to 20 min. The method described in this paper uses a glutaraldehyde standard and distinguishes various levels of sporicidal activity in the presence of 1-5% glutaraldehyde by using appropriate spore strains, spore preparations, and spore levels. The resistances of 2 Bacillus subtilis 19659 spore preparations cultured in 10% Columbia broth plus manganese and nutrient agar plus minerals, as well as that of B. subtilis var. niger cultured on Lab-Lemco agar, were tested. T-soy broth was a better recovery medium than fluid thioglycollate or modified fluid thioglycollate for B. subtilis 19659 spores exposed to HCl. Sporicidal tests were done on B. subtilis 19659 spores with 2 types of spore preparations. A commercial glutaraldehyde germicide was used for comparison of the sporicidal activity of the glutaraldehyde standard. Two strains of B. subtilis spores and 4 levels of spores (20,000-80,000, 100,000-400,000, 500,000-800,000, and 1,000,000 and up) were removed from check penicylinders from the same batches used for sporicidal tests. B. subtilis var. niger spores were the most resistant to HCl, while B. subtilis 19659 spores were more resistant to glutaraldehyde. Sporicidal activities of a commercial germicide containing 2.5% glutaraldehyde with additives and another containing 5% glutaraldehyde in phosphate buffer were similar. Both totally destroyed high levels of B. subtilis 19659 spores cultured in 10% Columbia broth plus manganese. Results indicate that use of a glutaraldehyde standard, calibrated numbers of spores on penicylinders (bioindicators), and appropriate spore strains and preparations can reduce the variability of sporicidal testing of commercial germicides.     

Experimental Correspondence between Spore Dosimetry and Spectral Photometry of Solar Ultraviolet Radiation

Abstract— The biologically effective dose of solar UV radiation was estimated from the inactivation of UV-sensitive Bacillus subtilis spores. Two types of independent measurements were carried out concurrently at the Aerological Observatory in Tsukuba: one was the direct measurement of colony-forming survival that provided the inactivation dose per minute (ID/min) and the other was the measurement of the spectral irradiance by a Brewer spectrophotometer. To obtain the effective spectrum, the irradiance for each 1 nm wavelength interval from 290 to 400 nm was multiplied with the efficiency for inactivation derived from the inactivation action spectrum of identically prepared spore samples. Integration of the effective spectrum provided the estimate for ID/min. The observed values of ID/min were closely concordant with the calculated values for the data obtained in four afternoons in 1993. The average ratio (±SD) between them was 1.24 (±0.16) for 14 data points showing high inactivation rates (<0.05 ID/min). Considering difficulties in the absolute dosimetry of UV radiation, the concordance was satisfactory and improved credibility of the two types of monitoring systems of biologically effective dose of solar UV radiation.     

Recovery and sporicidal resistance of various B. subtilis spore preparations on porcelain penicylinders compared with results from AOAC test methods

Sporicidal test results obtained from carriers inoculated with 4 types of defined Bacillus subtilis spore preparations were compared with the standard AOAC sporicidal test using soil extract nutrient broth (SENB) B. subtilis 19659 spores. Recoveries of spores inoculated on penicylinders from B. subtilis clean spores (washed and suspended in water) and B. subtilis 19659 spores inoculated from culture filtrates according to the AOAC method were compared. Spores were exposed to 6 concentrations (0.5-3.0% w/v) of glutaraldehyde in phosphate buffer (pH 7.5) for 10 h. Concentrations were established by titrimetry and liquid chromatography. Recoveries of surviving spores were determined for 3 types of clean B. subtilis var. niger preparations, one clean B. subtilis 19659 preparation, and the SENB B. subtilis 19659 filtrates. Spore carriers, inoculated by the standard AOAC protocol, resulted in as much as a 2-log number difference in runs 1-12, but not more than 0.5 log number for each clean spore preparation. The SENB spores varied most in resistance to glutaraldehyde, with no growth in recovery media from 3 different batches of 1, 1.5, and 2% glutaraldehyde. Separate batches of SENB preparations of B. subtilis 19659 were resistant and destroyed by 1.0% glutaraldehyde, with 3.98 and 6.0 log numbers of spores on penicylinders, respectively. Clean spore preparations of B. subtilis 19659 on porcelain penicylinders were more resistant to glutaraldehyde than were SENB spores. Nutrient agar/Mg/Ca and nutrient agar/Mg spore preparations of B. subtilis var. niger showed the most uniform resistance to glutaraldehyde. Spores with calcium added showed increased resistance to glutaraldehyde. B. subtilis 19659 spores from the Columbia broth spore preparation were the most resistant and were recovered after exposure to 3.0% glutaraldehyde.     

Effects of the binding of alpha/beta-type small, acid-soluble spore proteins on the photochemistry of DNA in spores of Bacillus subtilis and in vitro

The main lesion produced in DNA by UV-C irradiation of spores of Bacillus subtilis is 5-thyminyl-5,6-dihydrothymine (spore photoproduct [SP]). In contrast, cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP) are the main photolesions in other cell types. The novel photochemistry of spore DNA is accounted for in part by its reduced hydration, but largely by the saturation of spore DNA with alpha/beta-type small, acid-soluble spore proteins (SASP). Using high-performance liquid chromatography-mass spectrometry analysis of the photoproducts, we showed that in wild-type B. subtilis spores (1) UV-C irradiation generates almost exclusively SP with little if any CPD and 6-4PP; (2) the SP generated is approximately 99% of the intrastrand derivative, but approximately 1% is in the interstrand form; and (3) there is no detectable formation of the SP analog between adjacent C and T residues. UV-C irradiation of spores lacking the majority of their alpha/beta-type SASP gave less SP than with wild-type spores and significant levels of CPD and 6-4PP. The binding of an alpha/beta-type SASP to isolated DNA either in dry films or in aqueous solution led to a large decrease in the yield of CPD and 6-4PP, and a concomitant increase in the yield of SP, although levels of interstrand photoproducts were extremely low.     

Biological monitoring of solar UV radiation at 17 sites in Asia, Europe and South America from 1999 to 2004

A small and robust dosimeter for determining the biologically effective dose of ambient UV radiation has been developed using UV-sensitive mutant spores of Bacillus subtilis strain TKJ6312. A membrane filter with four spots of the spores was snapped to a slide mount. The slide was wrapped and covered with two or more layers of polyethylene sheet to protect the sample from rain and snow and to reduce monthly-cumulative doses within the measurable range. From 1999, monthly data were collected at 17 sites for more than 1 year, and data for 4 to 6 consecutive years were obtained from 12 sites. Yearly total values of the spore inactivation dose (SID) ranged from 3200 at subarctic Oulu to 96000 at tropical Denpasar, and the mean yearly values of SID exhibited an exponential dependence on latitude in both hemispheres with a doubling for about every 14 degrees of change. During the observation period, increasing trends of UV doses have been observed at all sites with more than 5 years of data available. Year-to-year variations at high and middle latitude sites are considered due mostly to climatic variation. At three tropical sites, negative correlations between the yearly doses and the column ozone amounts were observed. The results verified the applicability of spore dosimetry for global and long-time monitoring of solar UV radiation, in particular at tropical sites where no monitoring is taking place.     

Bacterial Inactivation by Solar Ultraviolet Radiation Compared with Sensitivity to 254 nm Radiation

Our goal was to derive a quantitative factor that would allow us to predict the solar sensitivity of vegetative bacterial cells to natural solar radiation from the wealth of data collected for cells exposed to UVC (254 nm) radiation. We constructed a solar effectiveness spectrum for inactivation of vegetative bacterial cells by combining the available action spectra for vegetative cell killing in the solar range with the natural sunlight spectrum that reaches the ground. We then analyzed previous studies reporting the effects of solar radiation on vegetative bacterial cells and on bacterial spores. Although UVC-sensitive cells were also more sensitive to solar radiation, we found no absolute numerical correlation between the relative solar sensitivity of vegetative cells and their sensitivity to 254 nm radiation. The sensitivity of bacterial spores to solar exposure during both summer and winter correlated closely to their UVC sensitivity. The estimates presented here should make it possible to reasonably predict the time it would take for natural solar UV to kill bacterial spores or with a lesser degree of accuracy, vegetative bacterial cells after dispersion from an infected host or after an accidental or intentional release.     

EFFECT OF DIPICOLINIC ACID ON THE ULTRAVIOLET RADIATION RESISTANCE OF BACILLUS CEREUS SPORES

Abstract— The ultraviolet radiation (UV) resistance of B. cereus spores was shown to depend on their content of dipicolinic acid (DPA). Wild-type spores with decreasing amounts of DPA exhibited increased UV resistance. Similarly, spores devoid of DPA (DPA-minus), produced by a mutant strain of B. cereus unable to synthesize DPA, were more resistant to UV than mutant spores (DPA-plus) produced in the presence of exogenously supplied DPA. Resistance of both the wild type and mutant strains to ionizing radiation, however, was unaffected by DPA content. Comparison of the resistance of DPA-minus and DPA-plus mutant spores to UV of various wavelengths showed that the greater sensitivity of the latter DPA-plus spores appeared at wavelengths corresponding to the region of the first molecular absorption band of the calcium chelate of DPA. In the wild type and mutant, thymine photoproducts were produced at a greater rate and to a greater extent in spores with high levels of DPA than in spores with low DPA.
The data indicate that DPA transfers energy to DN A in vivo , which leads to the conclusion that DPA occurs in the spore protoplast.     

Label-free flow-enhanced specific detection of Bacillus anthracis using a piezoelectric microcantilever sensor

Differentiation between species of similar biological structure is of critical importance in biosensing applications. Here, we report specific detection of Bacillus anthracis (BA) spores from that of close relatives, such as B. thuringiensis (BT), B. cereus (BC), and B. subtilis (BS) by varying the flow speed of the sampling liquid over the surface of a piezoelectric microcantilever sensor (PEMS). Spore binding to the anti-BA spore IgG coated PEMS surface is determined by monitoring the resonance frequency change in the sensor's impedance vs. frequency spectrum. Flow increases the resonance frequency shift at lower flow rates until the impingement force from the flow overcomes the binding strength of the antigen and decreases the resonance frequency shift at higher flow rates. We showed that the change from increasing to decreasing resonance frequency shift occurred at a lower fluid flow speed for BT, BC, and BS spores than for BA spores. This trend reduces the cross reactivity ratio of BC, BS, and BT to the anti-BA spore IgG immobilized PEMS from around 0.4 at low flow velocities to less than 0.05 at 3.8 mm s(-1). This cross reactivity ratio of 0.05 was essentially negligible considering the experimental uncertainty. The use of the same flow that is used for detection to further distinguish the specific binding (BA to anti-BA spore antibody) from nonspecific binding (BT, BC, and BS to anti-BA spore antibody) is unique and has great potential in the detection of general biological species.     

Detection of Bacillus subtilis spores using peptide-functionalized cantilever arrays

We move beyond antibody-antigen binding systems and demonstrate that short peptide ligands can be used to efficiently capture Bacillus subtilis (a simulant of Bacillus anthracis) spores in liquids. On an eight-cantilever array chip, four cantilevers were coated with binding peptide (NHFLPKV-GGGC) and the other four were coated with control peptide (LFNKHVP-GGGC) for reagentless detection of whole B. subtilis spores in liquids. The peptide-ligand-functionalized microcantilever chip was mounted onto a fluid cell filled with a B. subtilis spore suspension for approximately 40 min; a 40 nm net differential deflection was observed. Fifth-mode resonant frequency measurements were also performed before and after dipping microcantilever arrays into a static B. subtilis solution showing a substantial decrease in frequency for binding-peptide-coated microcantilevers as compared to that for control peptide cantilevers. Further confirmation was obtained by subsequent examination of the microcantilever arrays under a dark-field microscope. Applications of this technology will serve as a platform for the detection of pathogenic organisms including biowarfare agents.     

Both solar UVA and UVB radiation impair conidial culturability and delay germination in the entomopathogenic fungus Metarhizium anisopliae.

The entomopathogenic hyphomycete Metarhizium anisopliae has been used in programs of agricultural pest and disease vector control in several countries. Exposure to simulated solar radiation for a few hours can completely inactivate the conidia of the fungus. In the present study we determined the effect of exposures to full-spectrum sunlight and to solar ultraviolet A radiation at 320-400 nm (UVA) on the conidial culturability and germination of three M. anisopliae strains. The exposures were performed in July and August 2000 in Logan, UT. The strains showed wide variation in tolerance when exposed to full-spectrum sunlight as well as to UVA sunlight. Four-hour exposures to full-spectrum sunlight reduced the relative culturability by approximately 30% for strain ARSEF 324 and by 100% for strains ARSEF 23 and 2575. The relative UV sensitivity of the two more sensitive strains was different under solar UV from that under ultraviolet B radiation at 280-320 nm (UVB) in the laboratory. Four-hour exposures to solar UVA reduced the relative culturability by 10% for strain ARSEF 324, 40% for strain ARSEF 23 and 60% for strain ARSEF 2575. Exposures to both full-spectrum sunlight and UVA sunlight delayed the germination of the surviving conidia of all three strains. These results, in addition to confirming the deleterious effects of UVB, clearly demonstrate the negative effects of UVA sunlight on the survival and germination of M. anisopliae conidia under natural conditions. The negative effects of UVA in sunlight also emphasize that the biological spectral weighting functions for this fungus must not neglect the UVA wavelengths.     

DAMAGE BY SOLAR RADIATION AT DEFINED WAVELENGTHS. INVOLVEMENT OF INDUCIBLE REPAIR SYSTEMS

Abstract The susceptibility of bacteriophage damaged by solar-ultraviolet (UV, 290-380 nm) radiations at denned wavelengths and by radiation at a visible wavelength (405 nm) to the Weigle reactivation system induced by far-UV (254 nm) irradiation of the host cell has been studied in a repair competent strain of Escherichia coli . The sector of inducible repair diminishes with wavelength, being very small after 313 nm irradiation and absent after irradiation at longer wavelengths. However, irradiation of bacteria at wavelengths as long as 313 nm induces a bacteriophage reactivation system as effectively as radiation at 254 nm in both the repair competent and an excision deficient host cell. At longer wavelengths pre-irradiation of the repair competent host cell enhances reactivation of 254 nm irradiated bacteriophage but the reactivation is smaller and the process quite distinct from that induced in the 254-313 nm region. We conclude that, with increasing wavelength, damage induced by solar UV radiations becomes increasingly less susceptible to repair systems induced by far-UV (pyrimidine dimers) and that this type of inducible repair system is no longer induced by wavelengths longer than 313 nm.     

Comparisons of spore dosimetry and spectral photometry of solar-UV radiation at four sites in Japan and Europe

In order to develop monitoring and assessment systems of biologically effective doses of solar-UV radiation, concurrent measurements of spectral photometry and spore dosimetry were conducted in summer months at four sites in Japan and Europe. Effectiveness spectra were derived by multiplying spectral irradiance in 0.5 nm steps between 290 and 400 nm with the inactivation efficiency of the spores determined using monochromatic radiation of fine wavelength resolution. Shapes of the effectiveness spectra were very similar at the four sites exhibiting major peaks at 303.5, 305.0, 307.5 and 311.0 nm. The dose rates for spore inactivation from direct survival measurements and from calculations by the integration of the effectiveness spectra were compared for 174 data points. The ratios (observed/calculated) of the two values were concordant with a mean of 1.26 (+/- 0.24 standard deviation [SD]). The possible causes for the variations and slightly larger observed values are discussed.     

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.