Damage and repair in mammalian cells after exposure to non-ionizing radiations : I. Ultraviolet and visible light irradiation of cells of the rat kangaroo (Potorous tridactylus) and determination of photorepairable damage in vitro

Cornea cells of the rat kangaroo or “potoroo” (Potorous tridactylus) were exposed to far-UV (254 or 302 nm) radiation, with or without subsequent illumination by near-UV or visible light. The DNA of these cells was extracted and tested for the presence of photoproducts binding yeast photoreactivating enzyme (PRE). The criterion for the latter was competitive inhibition of an in vitro photorepair system, consisting of UV-irradiated transforming DNA of Haemophilus influenzae and an extract containing yeast PRE. The effects on repair kinetics of the transforming DNA indicate that in UV-irradiated potoroo cornea cells up to approximately 90% of photorepairable DNA damage can be photorepaired within 15 min. However, the extent of cellular photorepair, assessed by the reduction in competitive inhibition of the in vitro repair system depends appreciably on experimental parameters during photoreactivating treatment. Control experiments with non-UV-irradiated cells indicated that, depending on specific conditions, the photoreactivating treatment itself produces a varying amount of DNA damage, which reacts with the PRE in vitro. To avoid most of this kind of damage, cells are nitrogen-gassed and kept at 5°C during illumination, and the photoreactivating light must not contain wavelengths shorter than 380–400 nm. Our results show that wavelengths >470 nm are still very effective, whereas wavelengths >555 nm are ineffective in photorepairing potoroo DNA. For unknown reasons, one particular strain of potoroo cornea cells lost its potential for photorepair. Treatment of unirradiated potoroo cells, or their extracted DNA, with hydrogen peroxide also results in competitive inhibition of photorepair in vitro, resembling that observed after near-UV illumination. Because of the occurrence of synergistic effects it is not clear whether the damage only interacts with PRE or can actually be photorepaired under appropriate conditions.

The results presented in this paper suggest that the expression of photorepair in mammalian cells, unlike that in prokaryotes, greatly depends on a number of experimental parameters, including the spectral composition of photoreactivating light. Apparently superposition of damage by the photoreactivating treatment itself is the critical factor. This may explain experimental discrepancies existing in different laboratories studying photorepair in UV-irradiated cells of placental mammals.     

Damage and repair in mammalian cells after exposure to non-ionizing radiations. II. Photoreactivation and killing of rat kangaroo cells (Potorous tridactylus) and Herpes simplex virus-1 by exposure to fluorescent "white" light or sunlight

Photoreactivation (PR) of ultraviolet (254 nm)-inactivated cornea cells of the potoroo (or rat kangaroo; Potorous tridacylus) has been studied at wavelengths greater than 375 nm from either fluorescent "white" light or sunlight. In both cases the PR kinetics curves pass through maxima, which most likely result from the superposition of concomitant inactivation by the photoreactivating light. The inactivating effect of light was directly demonstrated for non-UV-irradiated cells, permitting correction of the PR curves. Wavelengths greater than 475 nm, and even greater than 560 nm, which do not noticeably damage cells, still photoreactivate, though less effectively than shorter wavelengths. Light treatment of UV-inactivated Herpes simplex Virus-1 (HSV-1) after infection leads to PR effects resembling those observed for cells, while light treatment of unirradiated virus after infection likewise causes inactivation. The "fluence-reduction factor" of PR, which is greater than 3 for the virus, exceeds that for the cells, where it decreases with increasing UV fluence. In vitro tests have indicated that sunlight greater than 375 nm causes photorepairable DNA lesions which are virtually fully repaired by the same light. Thus cell inactivation resulting from these solar wavelengths must be due to non-photorepairable damage.     

In vitro fertilization, development, and implantation after exposure of mature mouse oocytes to visible light.

Mature mouse oocytes were exposed prior to in vitro fertilization to visible light during 1, 2, or 4 hr at an intensity of 4,000 lux. Compared to controls cultured under identical conditions but protected from light, exposed eggs did not show any significant modification of cleavage speed and rate. After transfer of blastocysts obtained in vitro in uteri of pseudopregnant females, the implantation rate and the proportion of normal fetuses were not found to be different in relation to preliminary light exposure of oocytes fertilized and cultured in vitro.     

Assessment of DNA damage and repair in Mycobacterium terrae after exposure to UV irradiation

AIM: Ultraviolet (UV) irradiation for drinking water treatment was examined for inactivation and subsequent dark and photo-repair of Mycobacterium terrae. METHODS AND RESULTS: UV sources tested were low pressure (monochromatic, 254 nm) and medium pressure (polychromatic UV output) Hg lamps. UV exposure resulted in inactivation, and was followed by dark or photo-repair experiments. Inactivation and repair were quantified utilizing a molecular-based endonuclease sensitive site (ESS) assay and conventional colony forming unit (CFU) viability assay. Mycobacterium terrae was more resistant to UV disinfection compared to many other bacteria, with approximately 2-log reduction at a UV fluence of 10 mJ cm(-2) ; similar to UV inactivation of M. tuberculosis. There was no difference in inactivation between monochromatic or polychromatic UV lamps. Mycobacterium terrae did not undergo detectable dark repair. Photo-repair resulted in recovery from inactivation by approximately 0.5-log in less than 30 min for both UV lamp systems. CONCLUSIONS: Mycobacterium terrae is able to photo-repair DNA damage within a short timeframe. The number of pyrimidine dimers induced by UV light were similar for Escherichia coli and M. terrae, however, this similarity did not hold true for viability results. SIGNIFICANCE AND IMPACT OF THE STUDY: There is no practical difference between UV sources for disinfection or prevention of DNA repair for M. terrae. The capability of M. terrae to photo-repair UV damage fairly quickly is important for wastewater treatment applications where disinfected effluent is exposed to sunlight. Finally, molecular based assay results should be evaluated with respect to differences in the nucleic acid content of the test micro-organism.     

Chromosome damage in mouse-human hybrid cells after BUdR treatment and light irradiation

Mouse cells grown in the presence of bromodeoxyuridine were fused with human cells in the presence of Sendai virus. The cells were then irradiated with blue light. Three days after fusion, the hybrid cell metaphases showed intact human chromosomes and many mouse chromatids with numerous abnormalities, indicating the great potentiality of the technique for genetic analysis and manipulations.     

Inducible cellular responses to ultraviolet light irradiation and other mediators of DNA damage in mammalian cells

Summary Both naturally occuring and carcinogen-induced tumors display not only point mutations in cellular oncogenes but also more complex changes in cellular oncogenes and other cellular genes. For this and other reasons, it seems likely that DNA damage in mammalian cells can induce alterations in gene expression that may have both short and long term consequences in the target cell. The purpose of this review is to summarize current available information on inducible responses to UV-irradiation and other mediators of DNA damage in mammalian cells, and to provide some working hypotheses. We have divided these responses into three time frames, immediate (0–12 hours), early (12–48) and late (beyond 48 hours). Immediate responses include the action of DNA repair enzymes, some of which are induced as a consequence of DNA damage, and transient inhibition of DNA synthesis. Within the past few years considerable evidence has accumulated that during this immediate period there is increased expression of certain cellular oncogenes, proteases and proteins whose functions remain to be identified. It is of interest that the expression of some of these genes is also induced by certain growth factors, tumor promoters and heat shock. Alterations in gene expression during the subsequent early period (12–48 hrs.) have not been studied in detail, but it is during this period that one can detect increased replication of several types of viruses in cells that harbor these viruses. We have examined in detail the induction of asynchronous polyoma DNA replication (APR) in a rat fibroblast cell line carrying integrated copies of this DNA. We have obtained evidence that UV-irradiation of these cells leads to the synthesis of a 40 kd protein, within the first 1–24 hrs after irradiation, that binds to a specific sequence TGACAACA in the regulatory region of polyoma DNA. We suggest that this protein acts together with other proteins to induce APR and that this serves as a useful model for understanding the mechanisms responsible for amplification of cellular genes, a phenomenon often seen in malignant tumors. Finally, we discuss how the events occurring during the immediate and early periods following DNA damage might lead to late effects in the target cell that are stable and contribute to the genotype and phenotype of some of the progeny of these cells that are destined to become tumor cells.     

Interaction between radiation and drug damage in mammalian cells. III. The effect of adriamycin and actinomycin-D on the repair of potentially lethal radiation damage.

We have studied the effects of actinomycin-D (AMD) and Adriamycin (ADRM) on the repair of radiation damage in Chinese hamster cells (V79) in plateau phase growth. Suppression of potentially lethal damage repair (PLDR) was observed in the presence of non-toxic levels of AMD and minimally toxic levels of ADRM. The suppression of PLDR by AMD persisted as long as the drug was present. Removal of AMD was followed by prompt repair of potentially lethal injury suggesting that suppression of PLDR by AMD was not accompanied by fixation of injury to a non-repairable state. On the other hand, irradiated cells exposed to ADRM eventually repair potentially lethal injury in the presence of drug after an initial delay. AMD, but not ADRM, inhibited repair of sublethal radiation damage.     

Damage to cellular DNA from particulate radiations,the efficacy of its processing and the radiosensitivity of mammalian cells

Summary For several years, it has been evident that cellular radiation biology is in a necessary period of consolidation and transition (Lett 1987, 1990; Lett et al. 1986, 1987). Both changes are moving apace, and have been stimulated by studies with heavy charged particles.From the standpoint of radiation chemistry, there is now a consensus of opinion that the DNA hydration shell must be distinguished from bulk water in the cell nucleus and treated as an integral part of DNA (chromatin) (Lett 1987). Concomitantly, sentiment is strengthening for the abandonment of the classical notions of direct and indirect action (Fielden and O'Neill 1991; O'Neill 1991; O'Neill et al. 1991; Schulte-Frohlinde and Bothe 1991 and references therein). A layer of water molecules outside, or in the outer edge of, the DNA (chromatin) hydration shell influences cellular radiosensitivity in ways not fully understood. Charge and energy transfer processes facilitated by, or involving, DNA hydration must be considered in rigorous theories of radiation action on cells. The induction and processing of double stand breaks (DSBs) in DNA (chromatin) seem to be the predominant determinants of the radiotoxicity of normally radioresistant mammalian cells, the survival curves of which reflect the patterns of damage induced and the damage present after processing ceases, and can be modelled in formal terms by the use of reaction (enzyme) kinetics. Incongruities such as sublethal damage are neither scientifically sound nor relevant to cellular radiation biology (Calkins 1991; Lett 1990; Lett et al. 1987a).Increases in linear energy transfer (LET) up to 100–200 keV µm^–1 cause increases in the extents of neighboring chemical and physical damage in DNA denoted by the general term DSB. Those changes are accompanied by decreasing abilities of cells normally radioresistant to sparsely ionizing radiations to process DSBs in DNA and chromatin and to recover from radiation exposure, so they make significant contributions to the relative biological effectiveness (RBE) of a given radiation. As the LET is raised above a few hundred keV µm^–1, the damage associated with DSBs continues to increase, but the efficiency of DSB induction declines to low values (0.1), as do RBE and the effective processing of DSBs and chromatin breaks, and the decline in RBE seems to mimic the overall decline in suitable processing of DSBs. Hence, the quality factor (Q) for a given radiation cannot be based solely upon the pattern of energy deposition, a fact attested to also by the quite different RBE responses exhibited by repair-deficient mutant (or variant) cells.Understanding of the biological effects of heavy ions is important not only for the welfare of astronauts who will undertake extended interplanetary missions in space but also for the facilitation of a rigorous scientific basis for conventional radiation therapy.Based on a review lecture delivered at the Fourth Workshop on Heavy Charged Particles in Biology and Medicine, GSl, Darmstadt, Germany, September 1991. A number of scientists, see Acknowledgements, also gave unreservedly of their time at the 40th Annual Meeting of the Radiation Research Society, Salt Lake City, Utah, March 1992, to discuss topics in this article with the author     

Induction and repair of DNA strand breaks in cultured mammalian cells following fast neutron irradiation.

Induction and repair of DNA breaks following irradiation with NIRS cyclotron neutrons were studied in cultured mammalian cells (L5178Y) in comparison to those following gamma-rays. The yield of the total single-strand breaks, 3'OH terminals and sites susceptible to S1 endonuclease following fast neutrons was found to be approximately 50 per cent of that following gamma-irradiation. On the other hand, the yield of double-strand breaks was slightly higher after fast neutrons than after gamma-rays. The percentage of the total single-strand breaks remaining unrejoined at 3 hours after post-irradiation incubation was found to be distinctly higher after the fast neutrons than after gamma-rays. The neutron-induced damage appears to carry a higher proportion of alkali-labile lesions compared to gamma-rays. It was concluded that the increase in the yield of double-strand breaks and of unrejoinable breaks is responsible for a high r.b.e. of the cyclotron neutrons.     

Escherichia coli Fpg protein and UvrABC endonuclease repair DNA damage induced by methylene blue plus visible light in vivo and in vitro.

pBR322 plasmid DNA was treated with methylene blue plus visible light (MB-light) and tested for transformation efficiency in Escherichia coli mutants defective in either formamidopyrimidine-DNA glycosylase (Fpg protein) and/or UvrABC endonuclease. The survival of pBR322 DNA treated with MB-light was not significantly reduced when transformed into either fpg-1 or uvrA single mutants compared with that in the wild-type strain. In contrast, the survival of MB-light-treated pBR322 DNA was greatly reduced in the fpg-1 uvrA double mutant. The synergistic effect of these two mutations was not observed in transformation experiments using pBR322 DNA treated with methyl methanesulfonate, UV light at 254 nm, or ionizing radiation. In vitro experiments showed that MB-light-treated pBR322 DNA is a substrate for the Fpg protein and UvrABC endonuclease. The number of sites sensitive to cleavage by either Fpg protein or UvrABC endonuclease was 10-fold greater than the number of apurinic-apyrimidinic sites indicated as Nfo protein (endonuclease IR)-sensitive sites. Seven Fpg protein-sensitive sites per PBR322 molecule were required to produce a lethal hit when transformed into the uvrA fpg-1 mutant. These results suggest that MB-light induces DNA base modifications which are lethal and that these modifications are repaired by Fpg protein and UvrABC endonuclease in vivo and in vitro. Therefore, one of the physiological functions of Fpg protein might be to repair DNA base damage induced by photosensitizers and light.     

Effect of protective agents on amount and repair of sublethal freeze--thaw damage in mammalian cells.

Glycerol, DMSO, and HES are able to reduce by a factor of 2 the sublethal damage produced in mammalian cells after one freeze-thaw cycle. When sublethal freeze-thaw damage is already present, DMSO and HES are able to prevent about half of this damage from becoming lethal when a second freeze-thaw cycle is applied. Glycerol is only able to do this if dilution shock is avoided by thawing the cells into medium containing glycerol. The cells can repair 100% of this sublethal damage and do so in 2–3 hr at 37 °C in suspension. The data imply that the sites protected by DMSO, HES, and glycerol are the same as the sites repaired by the cells. The results also suggest that cells stop progressing in the cell cycle while repairing sublethal freeze-thaw damage.     

DNA damage and repair in chick embryo cells following X-irradiation in vitro as compared to mammalian cells - biochemical and physico-chemical investigations

Summary Brain cells (b-cells) and liver cells (l-cells) of the chicken embryo and thymic cells (t-cells) of the rat were X-irradiated in vitro at doses of 1.25–50 Gy. When compared to t-cells, b- and l-cells exhibited1) a lower stimulation of poly (adenosine diphosphate-ribose) transferase and unscheduled DNA synthesis following X-irradiation,2) an almost fivefold higher inhibition of semiconservative DNA synthesis,3) a less condensed chromatin,4) about fourfold higher threshold doses with regard to significant effects on nucleoid sedimentation and viscometry of alkaline cellular lysates, and5) an apparently two- to threefold lower DNA repair during a 30 min post-exposure repair period. The results suggest that the lower radiation sensitivity of chicken embryo cells is attributable to an initial mechanism of DNA repair and/or DNA protection which may be closely connected to minor chromatin compactness and higher intrinsic activities of repair enzymes.