Thomas P. Coohill

ACTION SPECTRA AGAIN

Action spectroscopy has a long history and is of central importance to photobiological studies. Action spectra were among the first assays to point to chlorophyll as the molecule most responsible for plant growth and to DNA as the genetic material. It is useful to construct action spectra early in the investigation of new areas of photobiological research in an attempt to determine the wavelength limits of the radiation region causing the studied response. But due to the severe absorption of ultraviolet (UV) radiation by biological samples, UV action spectra were first limited to small cells (bacteria and fungi). Advances in techniques (e.g. single cell culture) and analysis allowed accurate action spectra to be reported even for mammalian cells. But precise analytical action spectra are often difficult to obtain when large, pigmented, or groups of cells are investigated. Here some action spectra are limited in interpretation and merely supply a wavelength vs effect curve. When polychromatic sources are employed, the interpretation of action spectra is even more complex and formidable. But such polychromatic action spectra can be more directly related to ambient responses. Since precise action spectra usually require the completion of a relatively large number of careful experiments using somewhat sophisticated equipment over a range of at least six wavelengths, they are often not persued. But they remain central to the elucidation of the effect being studied. The worldwide community has agreed that stratospheric ozone is depleting, with the possibility of a consequent rise in the amount of UV‐B (290–320nm) reaching the earths surface. It is therefore essential that new action spectra be completed for UV‐B effects on a large variety of responses of human, animal, and aquatic plant systems. Combining these action spectra with the known amounts of UV‐B reaching the biosphere can give rise to solar UV effectiveness spectra that, in turn, can give rise to estimates of effect. Preliminary estimates suggest that ozone layer depletion may seriously impact such important biological end‐points as skin cancer, cataracts, the immune system, crop yields, and oceanic phytoplankton. So action spectra continue to play a central role in important photobiological research.

The effects of the ultraviolet wavelengths of radiation present in sunlight on human cells in vitro.

This yearly review is intended to supplement and complement similar reviews appearing in the past two years (Kantor, 1985; Gange and Rosen, 1986; Peak and Peak, 1986a). Kantor’s review, “Effects of Sunlight on Mammalian Cells,” was limited to manuscripts that appeared in 1984. Gange and Rosen’s “UVA Effects on Mammalian Skin and Cells” was limited to work published in 1985; Peak and Peak’s “DNA-to-Protein Crosslinking and Backbone Breaks Caused by Farand Near-Ultraviolet and Visible Radiation in Mammalian Cells” considered DNA damages only. Although this review limits itself to papers known to us that either appeared or were in press from January 1986 through July 1987, necessary limited references to earlier work are included. Other reviews relating to this topic that have appeared since 1984 include “UV-Carcinogenesis” (van der Leun, 1984), “Effect of UV Light on Humans” (Grieter and Gschnait, 1984), and “Photoimmunology” (Morison, 1984). Our review includes only studies meeting specific qualifications: (1) The experimental radiation source contained at least some portion of the ultraviolet radiation (UV) present in the solar spectrum reaching the surface of the earth, i.e. 29WOO nm. (2) Only studies that involved human cells were considered. (3) Only in vitro work (cells in culture) was included, with the exception of a small section on studies of human epidermis. (4) Effects mediated by endogenous photosensitizers were considered. A few selected papers that deviated from the above conditions were reviewed, however, because they contributed directly to the objectives of the review. Much of the motivation for focusing on studies of the effects of the wavelength region referred to in this review as the “solar UV wavelength region,”* is provided by three observations. First, although the many studies concerning the wavelength region

Ultraviolet action spectra (280 to 380 nm) and solar effectiveness spectra for higher plants

Several ultraviolet (UV) action spectra that typify the responses of higher plants to irradiation by wavelengths between 280 nm and 380 nm are shown. An attempt is made to generate common spectra that may be used, at least temporarily, to represent the effects of UV on such important biological parameters as photosynthesis. The goal is to provide an estimate of plant response to solar UV and to the potential increase in ground level UV postulated for a depleted stratospheric ozone layer. Solar plant damage effectiveness curves are generated under “normal” solar UV conditions, and under an assumed UV increase corresponding to a 16% depletion in total ozone. Additional effects due to ozone depletion are concentrated in the UV‐B region, especially at wavelengths between about 297 nm and 315 nm. Common features of these effectiveness curves are noted, and limitations are pointed out. As expected, no common spectrum has been found that can substitute for any specific spectrum nor that is unique enough to provide more than a limited first approximation of a plant damage spectrum. Additional information must be generated to fulfill this need.

THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET INACTIVATION OF HOST CAPACITY IN A MAMMALIAN CELL‐VIRUS SYSTEM

Abstract. The ability of UV‐irradiated African green monkey kidney cells (CV‐1) to support the growth of unirradiated herpes simplex virus type 1 as measured by plaque forming ability has been investigated. The lowering of plaque formation by the virus when the host cell was irradiated was examined at thirteen different wavelengths. An action spectrum for this cellular parameter (capacity) was obtained in the wavelength region of 235–302 nm. This action spectrum points to nucleic acid as the critical target molecule for this effect.

VIRUS‐CELL INTERACTIONS AS PROBES FOR VACUUM‐ULTRAVIOLET RADIATION DAMAGE AND REPAIR

Irradiation of biological materials in the vacuum‐ultraviolet (VUV*‐wavelengths below 200nm) is inhibited by the substantial absorption of water, air, and of the material itself. We have made a rough estimate of the penetration of radiation to the center of three kinds of cells and three viruses of different sizes at three wavelengths in the VUV (155 nm, 170 nm, and 200 nm) and two wavelengths in the far‐UV (220 nm and 250 nm). With the exception of bacteria, absorption limits the penetration of wavelengths below 200 nm to the center of the cell, to less than one percent of the radiation striking the cell surface. Thus, effects on the genetic material when cells larger than bacteria are irradiated with VUV should be insignificant. However, even the largest viruses were relatively transparent to all the wavelengths that we used. Therefore viruses can be considered to be model organisms for VUV studies; we list some of the maj or advantages of using them as probes to study cellular responses to VUV biological damage. The use of viral probes to study membrane phenomena also is noted since damage by VUV often is limited to cell surfaces. We suggest that the use of viruses may make possible biological experiments in the VUV that were not feasible with cells alone.

THE EFFECT OF CHANGES IN CELL GEOMETRY ON THE SENSITIVITY TO ULTRAVIOLET RADIATION OF MAMMALIAN CELLULAR CAPACITY

Abstract— We have measured a calcium and magnesium dependent change in cell shape when mammalian cell monolayers are being prepared for irradiation by replacing their growth medium with certain buffers. In some cases, flattened cells (umbonate) assumed a spherical configuration. In order to assume a centrally located target molecule, we used a DNA‐dependent cellular function–pacity for herpes viral growth–as the parameter to measure ultraviolet (UV) sensitivity of cells irradiated while in either of the two shapes. Umbonate cells were more sensitive to UV than were spherical cells. Exposures to the cell that lowered the cellular capacity of umbonate cells to the 10% survival level only lowered spherical cells to the 50% level. Twenty‐seven per cent additional UV exposure to spherical cells was required to get the same effect as with umbonate cells. Included in the text are photographs of both cell types, survival curves for cellular capacity, a measure of the absorbance of cell homogenates, and a calculation of the relative number of photons absorbed by each cell nucleus.

Photodynamic therapy for herpes simplex: a critical review.

Abstract The purpose of this review is to summarize available experimental and clinical data concerning benefits and potential risks of photodynamic therapy for oral and genital herpes simplex virus infections. Since such infections are a source of discomfort and pain, and since recent evidence links the virus itself with carcinogenesis, clinicians have felt an increased need for more effective therapy for herpesvirus infections. In 1973 a human clinical therapeutic procedure based on photodynamic inactivation was developed. The treatment consists of applying a photosensitizing dye to herpesvirus lesions and then exposing them to visible light. A number of human clinical trials have been completed; some of these show reduction of herpesvirus infectivity, whereas others question the efficacy of the procedure. Data from a number of in vitro virus-host cell studies suggest the procedure may be potentially carcinogenic and therefore clinically hazardous. Thus a controversy exists concerning further use of the treatment. This review gives the reader a basis for understanding the controversy and included basic principles of photodynamic inactivation, medical aspects of herpesvirus infections, development of the therapeutic procedure, results of clinical studies, and results of in vitro studies which indicate potential long-term side effects.

Ultraviolet mutagenesis of radiation-sensitive (rad) mutants of the nematode Caenorhabditis elegans

A mutational tester strain (JP10) of the nematode C. elegans was used to capture recessive lethal mutations in a balanced 300 essential gene autosomal region. The probability of converting a radiation interaction into a lethal mutation was measured in young gravid adults after exposure to fluences of 254-nm ultraviolet radiation (UV) ranging from 0 to 300 Jm-2. Mutation frequencies as high as 5% were observed. In addition, three different radiation-hypersensitive mutations, rad-1, rad-3 and rad-7 were incorporated into the JP10 background genotype, which allowed us to measure mutation frequencies in radiation-sensitive animals. The strain homozygous for rad-3 was hypermutable to UV while strains homozygous for rad-1 and rad-7 were hypomutable. Data showing the effects of UV on larval development and fertility for the rad mutants is also shown and compared for wild-type and JP10 backgrounds.

THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET ENHANCED REACTIVATION IN A MAMMALIAN CELL-VIRUS SYSTEM

Abstract— The effect of UV radiation in the wavelength region 230 nm to 302 nm on the ability of an irradiated mammalian cell to reactivate UV‐irradiated mammalian virus was tested. An action spectrum for radiation enhanced reactivation (RER) is presented. The shape of the action spectrum points to a combined nucleic acid‐protein target for UV radiation effects on this cellular parameter. An analysis of the results of others involving the biochemical and photobiological events involved in RER does not allow us to distinguish which macromolecule is the major contributor to this effect. Studies involving an analogous phenomenon in bacteria (Weigle reactivation) imply that RER and WR may involve similar mechanisms.

A COMPARISON OF MAMMALIAN CELL SENSITIVITY TO EITHER 254 nm OR ARTIFICIAL “SOLAR” SIMULATED RADIATION

The sensitivity of six mammalian cell strains to either germicidal (254 nm) or artificial “solar” simulated radiation was tested. The solar simulator used had an output similar, in some respects, to natural sunlight. Cellular capacity for Herpes simplex virus production was used as the assay procedure. The tested cells were a strain of African green monkey kidney cells and five human skin fibroblast cell strains. The latter included a “normal” cell strain, and four photosensitive cell strains; three of which were strains of xeroderma pigmentosum cells, and one strain of Blooms syndrome cells. When comparing the D10 values, the different cell strains varied by a factor of six in response to germicidal radiation, but only by a factor of two to artificial “solar” simulated radiation. The relative sensitivity of the cells to either type of radiation also varied from 1.7 to 10.9. Large variations in response occurred even among the xeroderma pigmentosum cell strains. These responses suggest that mammalian cell sensitivity to 254 nm radiation may not be a true indicator of a cells responses to natural sunlight.