Robert B. Webb

Lethal and Mutagenic Effects of Near-Ultraviolet Radiation

Ultraviolet (UV) radiation* of wavelengths longer than 295 nm from sunlight is a ubiquitous part of the natural environment of most organisms. Although beneficial and possibly beneficial aspects of sunlight have long been recognized and studied (Daniels, 1974; Wurtman, 1975), harmful effects, except for sunburn, have received relatively little attention until recently. Photochemical possibilities of natural components of the cell suggest that efficient mechanisms for the partial prevention or repair of resultant damage from exposure to solar radiation must exist for biological entities to survive regular exposure to natural sunlight. Recent work has identified DNA lesions induced by near-UV radiation (Section 2.4). Furthermore, mechanisms of protection and repair of near-UV-induced lesions have been reported (Sections 2.3 and 2.6).

Induction of single-strand breaks (alkali-labile bonds) in bacterial and phage DNA by near UV (365 nm) radiation.

Abstract—Irradiation at 365 nm results in the induction of approximately 2–4 times 10‐6 and 1‐2times 10‐6 single‐strand breaks (alkali‐labile bonds) per 108 daltons per J m‐2 in extracted phage T4 DNA and in Escherichia coli bacterial DNA, respectively. The rate of break induction in DNA of intact phage is approximately one‐fourth that for extracted phage DNA. 2‐aminoethylisothiouronium bromide‐HBr protects against break induction in both phage systems. No breaks are induced in the DNA of bacteria irradiated under anaerobic conditions over the dose range tested. Possible induction mechanisms are suggested. Consideration is given to the relative importance of pyrimidine dimers and single‐strand breaks in the bactericidal action of 365 nm radiation.

SENSITIVITY OF STRAINS OF ESCHERICHIA COLI DIFFERING IN REPAIR CAPABILITY TO FAR UV, NEAR UV AND VISIBLE RADIATIONS†

Abstract— In stationary phase, strains of Escherichia coli deficient in excision (B/r Her) or recombination repair (K.12 AB2463) were more sensitive than a repair proficient strain (B/r) to monochromatic near‐ultraviolet (365 nm) and visible (460 nm) radiations. The relative increase in sensitivity of mutants deficient in excision or recombination repair, in comparision to the wildtype, was less at 365 nm than at 254 nm. However, a strain deficient in both excision and recombination repair (K12 AB2480) showed a large, almost equal, increase in sensitivity over mutants deficient in either excision or recombination repair at 365 nm and 254 nm. All strains tested were highly resistant to 650 nm radiation. Action spectra for lethality of strains B/r and B/r Her in stationary phase reveal small peaks or shoulders in the 330–340, 400–410 and 490–510 nm wavelength ranges. The presence of 5μg/ml acriflavine (an inhibitor of repair) in the plating medium greatly increased the sensitivity of strain B/r to radiation at 254, 365 and 460 nm, while strains E. coli B/r Her and K12 AB2463 were sensitized by small amounts. At each of the wavelengths tested, acriflavine in the plating medium had at most a small effect on E. coli K.12 AB2480. Acriflavine failed to sensitize any strain tested at 650 nm. Evidence supports the interpretation that lesions induced in DNA by 365 nm and 460 nm radiations play the major role in the inactivation of E. coli by these wavelengths. Single‐strand breaks (or alkali‐labile bonds), but not pyrimidine dimers are candidates for the lethal DNA lesions in uvrA and repair proficient strains. At high fluences lethality may be enhanced by damage to the excision and recombination repair systems.

Mutagenesis in Escherichia coli by Visible Light

Mutation to resistance to bacteriophage T5 in continuous cultures of Escherichia coli was induced by visible light (wavelength longer than 408 nanometers) and by black light (300 to 400 nanometers). Mutation rates more than 18 times greater than the spontaneous rate (no light) were obtained with moderate, nonlethal intensities of visible light. Mutation rates for both visible and black light were proportional to irradiance.

OYGEN DEPENDENCE AND REPAIR OF LETHAL EFFECTS OF NEAR ULTRAVIOLET AND VISIBLE LIGHT

Abstract— –Lethality in a repairable strain (WP2) and an excision repair deficient strain (WP2hcr) of Escherichia coli was studied at wavelengths of 254, 313, 365, and 390–750 nm. Survival curves were empirically fitted to the expression S= 1 ‐ (1‐e‐kl)“, where S is the fraction surviving, D is the incident dose in ergs mm‐2, k is the inactivation constant in units of (erg mm‐2)‐1 and n is the ‘shoulder constant’. The repairable sector (k(hcr‐)–k(hcr‐)lk(hcr‐), a conservative estimate of the repair capability of E. coli WP2, was 0.91 at 254 nm, 0.92 at 313 nm, 0.60 at 365 nm, and 0.13 at 390–750 nm. Although there was no oxygen enhancement of inactivation at 254 nm and 313 nm, a strong enhancement was identified at 365 nm and 390–750 nm. These results suggest that oxygen‐dependent damage induced by near u.v. (365 nm) can be partially repaired by the excision‐repair system in E. coli.

Reduced dimer excision in bacteria following near ultraviolet (365 nm) radiation

The experiment consisted of uv-irradiating Escherichia coli at 365 mn and then inducing substrate for the excision system in DNA by 254 nm radiation. The samples were then assayed for excision. The ability of E. coli to excise dimers induced in DNA by 254 nm radiation was progressively reduced as a function of the dose of 365 nm radiation. The dose of radiation required for the elimination of detectable excising ability was approximately the same as the threshold dose for exponential inactivation. The chemical evidence supports the hypothesis that excision repair rapidly becomes ineffective at biologically significant doses of 365 nm radiation.

ACTION SPECTRA FOR OXYGEN‐DEPENDENT AND INDEPENDENT INACTIVATION OF ESCHERZCHZA COLZ WP2s FROM 254 TO 460 NM

Abstract— Action spectra for lethality of E. coli WP2s under aerobic and anaerobic conditions. based on final slopes of the survival curves, reveal the absence of oxygen dependence at 313 nm and shorter wavelengths and a strong oxygen dependence (OER of 12 at 334 nm and 16 at 365 nm) at wavelengths longer than 313 nm. Shoulders or small peaks at340, 365, 410 and 500 nm suggest the participation of non‐DNA chromophores in aerobic lethality at these wavelength ranges.

DESTRUCTION OF PHOTOREACTIVATING ENZYME BY 365 nm RADIATION

Abstract— Following the observation that in vivo photoreactivation of 365‐nm‐induced pyrimidine dimers could not be observed chemically, a study was made of the inactivation of photoreactivating enzyme activity by this near‐ultraviolet wavelength. It was observed that: (1) Dimers induced in extracted bacterial DNA by 365 nm radiation are completely photoreactivable and are monomerized as an exponential function of the photoreactivation time. (2) Photoreactivability of 254‐nm‐induced damage in Escherichia coli B/r Hcr is progressively destroyed in vivo as a function of the dose of 365 nm radiation. (3) The ability of the yeast photoreactivating enzyme to monomerize dimers induced at 365 nm in bacterial DNA is destroyed in vitro as a function of the dose of 365 nm radiation, and at a rate comparable to killing of E. coli. These results are consistent with biological measurements which indicate that photoreactivability of ultraviolet (near and far) lethal damage is reduced by exposure of the bacteria to 365 nm radiation.

[Action spectra for lethality in recombination-less strains of Salmonella typhimurium and Escherichia coli].

Abstract— Action spectra for lethality of both stationary and exponentially growing cells of recombinationless (recA) mutants of Salmonella typhimurium and Escherichia coli were obtained. Maximum sensitivity was observed at 260nm which corresponds to the maximum absorbance of DNA. However, a shoulder occurred in the 280–300 nm range that departed significantly from the absorption spectrum of DNA. At wavelengths longer than 320nm, the shapes of inactivation curves departed significantly from those at wavelengths shorter than 320nm and survival curves at wavelengths longer than 320nm had a large shoulder. A small peak or shoulder occurred in the 330–340nm region of the action spectra. The special sensitivity of recA mutants to broad spectrum near‐UV radiation may be due to synergistic effects of different wavelengths. Parallels between the inactivation of recA mutants and the induction of a photoproduct of l‐tryptophan toxic for recA mutants (now known to be H2O2) suggest that H2O2 photoproduct from endogenous tryptophan may be involved in the high sensitivity of these strains to broad spectrum near‐UV radiation.

MUTAGENIC EFFECTS OF NEAR ULTRAVIOLET AND VISIBLE RADIANT ENERGY ON CONTINUOUS CULTURES OF ESCHERICHIA COLI

Abstract— –The induction of mutation to phage T5 resistance by near ultraviolet (u.v.) and visible light was studied in chemostat cultures of Escherichia coli strains B/r and B/r/1, trp. The visible light mutation rate to phage T5 resistance was independent of growth rate over the range studied. This result is consistent with a photochemical mechanism of mutagenesis. Changeovers, in which a faster growing subpopulation takes over the culture, usually causing the mutant frequency to decline sharply, occur more frequently in chemostat cultures irradiated with visible light than in cultures treated with far u.v. or caffeine. A preliminary action spectrum was obtained with aerated chemostats that revealed effective wavelengths to be between 330 nm and 500 nm. Wavelengths longer than 500 nm were not effective. Wavelengths longer than 340 nm were not mutagenic in anaerobic chemostats. This oxygen requirement for mutagenesis between 340 nm and 500 nm is consistent with a photodynamic mechanism of action. In aerated cultures, wavelengths between 400 nm and 500 nm were as effective as wavelengths between 330 nm and 400 nm. A number of naturally occurring compounds, including riboflavin and vitamin K, are consistent with the data as candidates for the chromophore responsible for near u.v. and visible light mutagenesis.