Search

Present models of carcinogenesis imply that apart from the tissue microenvironment the initiation of cancers involves the induction of lesions in DNA, error-prone or lack of DNA repair, the induction of mutations and genomic instability. The present review summarizes recent knowledge on the induction and repair of DNA damage, DNA damage response (DDR) signaling, the main repair pathways and their role in the maintenance of genomic stability and, in case of failure, the relationship to cancer development. Because of the multiplicity and variation of damage inflicted on human cells from endogenous and exogenous sources (normal cell metabolism and mutagenic chemical and physical agents), cells have developed an effective strategy to cope with such internal and external stresses: (1) rapid signaling of the damage, (2) activation of prominent effector proteins by phosphokinases such as ATM, ATR, and DNA-PKcs, (3) induction of cell cycle arrest leaving time for the mobilization of lesion and metabolic state-adapted DNA repair pathway(s). DDR and DNA repair are highly dependent on genetic and epigenetic control mechanisms including modifications in chromatin structure. Complex lesions such as DNA double-strand breaks (DSBs) arising from stalled replication forks, repair intermediates or directly induced chemical or radiation-induced damage are the most refractory to accurate repair. Single-strand breaks, base modifications, and some adducts are more easily repaired than complex lesions such as DNA DSBs or interstrand crosslinks. Inaccurate repair leads to mutations, chromosomal and genomic instability and is cancer prone. The repair pathways (mismatch repair, Base excision repair, nucleotide excision repair, homologous recombination) are very accurate, however, nonhomologous endjoining (NHEJ) and its variant, alternative NHEJ (Alt-NHEJ) are inaccurate. The latter shows that cells tend to use components from other repair systems when certain lesions cannot be completely repaired by one of the main pathways because functional repair proteins are absent (e.g., proteins such as KU, DNA-PKcs, and LigIV are missing). In that case, although acting sequentially in a concerted manner, the fidelity of DNA repair decreases, whereas genomic instability and susceptibility to cancer increases. Detailed analyses of the different repair systems show that they are very closely embedded in normal cellular metabolic networks. Future systems biology analysis of these networks should allow further insights into the relationship between failures in DNA repair and carcinogenic outcomes to the benefit of a better understanding of the mechanisms involved, predictions of carcinogenic risks and the development of new anticancer treatment modalities.

Resume La radiotherapie guidee par l’image (IGRT) et la radiotherapie conformationnelle avec modulation d’intensite (RCMI) sont utilisees de plus en plus frequemment. Certains auteurs recommandent meme que les irradiations par RCMI soient toujours guidees par l’image. Le controle de l’alignement, du realignement du patient et de la position des cibles en radiotherapie externe sont effectues de plus en plus par une imagerie de qualite radiologique. Toutefois ces nouvelles procedures provoquent des irradiations a faibles doses dans des volumes qui peuvent etre importants. Les doses physiques dues a cette imagerie sont connues, quantifiables et tracables. Lors de l’evaluation et la comparaison de ces nouvelles techniques, comparativement aux techniques classiques (radiotherapie tridimensionnelle ou classique), la prise en compte des volumes recevant de faibles doses ainsi que des doses relativement elevees (10–50 Gy) doit etre consideree. En effet, les effets secondaires des faibles doses ainsi que le risque de cancers secondaires avec ces nouvelles techniques sont mal connus. A faible niveau de dose (

Cells of higher eukaryotes possess several very efficient systems for the repair of radiation-induced lesions in DNA. Different strategies have been adopted at the cellular level to remove or even tolerate various types of lesions in order to assure survival and limit the mutagenic consequences. In mammalian cells, the main DNA repair systems comprise direct reversion of damage, excision of damage and exchange mechanisms with intact DNA. Among these, the direct ligation of single strand breaks (SSB) by a DNA ligase and the multi-enzymatic repair systems of mismatch repair, base and nucleotide excision repair as well as the repair of double strand breaks (DSB) by homologous recombination or non homologous end-joining are the most important systems. Most of these processes are error-free except the non homologous end-joining pathway used mainly for the repair of DSB. Moreover, certain lesions can be tolerated by more or less accurately acting polymerases capable of performing translesional DNA syntheses. The DNA repair systems are intimately integrated in the network of cellular regulation. Some of their components are DNA damage inducible. Radiation-induced mutagenesis is largely due to unrepaired DNA damage but also involves error-prone repair processes like the repair of DSB by non-homologous end-joining. Generally, mammalian cells are well prepared to repair radiation-induced lesions. However, some questions remain to be asked about mechanistic details and efficiencies of the systems for removing certain types of radiation-damage and about their order and timing of action. The answers to these questions would be important for radioprotection as well as radiotherapy.

To determine how radiation-induced arrest in G2 affects the response of mammalian cells to a challenging dose of radiation or to antitumour drugs producing DNA double-strand breaks.V79 fibroblast survival to 5 Gy gamma-rays followed at intervals by 3 Gy irradiation or by contact with an equitoxic dose of neocarzinostatin or etoposide, was measured by clonogenic assays. The pattern of radiation-induced DNA double-strand breaks was determined by filter elution and CFGE (continuous field gel electrophoresis) or PFGE (pulsed-field gel electrophoresis) in G2-arrested cells as well as in nonpre-irradiated asynchronous or synchronized cells. The cell-cycle phase specificity of drug susceptibility was determined in synchronized HeLa cells.Cell kill by radiation-drug combined treatment varied markedly with the time elapsed after priming irradiation. Pre-irradiated, G2-arrested V79 fibroblasts demonstrated excess double-stranded DNA cleavage upon re-irradiation and hypersensitivity to drugs and radiation, although maximum resistance to both neocarzinostatin and etoposide in synchronized HeLa cells was in G2. This effect occurred in the megabase range only, with a peak around 4 Mbp; no change in the electrophoretic migration profile of DNA was observed below 1 Mbp. Moreover, the DNA migration profile and the yield of DNA cleavage in G2-arrested cells were close to those expected from S-phase cells.The available data suggest that mechanisms operating within the radiation-induced G2 block promote susceptibility to DNA double-strand break inducers at this stage. It is also proposed that the conformation of DNA in cells accumulated in G2 following irradiation bears resemblance to that for cells in S phase, due either to active repair mechanisms or to inhibition of chromosome disentanglement at the S-G2 transition.

Repair pathways of DNA are now better defined, and some important findings have been discovered in the last few years. DNA non-homologous end-joining (NEHJ) is a crucial process in the repair of radiation-induced double-strand breaks (DSBs). NHEJ implies at least three steps: the DNA free-ends must get closer, preparation of the free-ends by exonucleases and then a transient hybridisation in a region of DNA with weak homology. DNA-dependent protein kinase (DNA-PK) is the key enzyme in this process. DNA-PK is a nuclear serine/threonine kinase that comprises three components: a catlytic subunit (DNA-PKCS) and two regulatory subunits, DNA-binding proteins, Ku80 and Ku70. The severe combined immunodeficient (scid) mice are deficient in DNA-PKCS: this protein is involved both in DNA repair and in the V(D)J recombination of immunoglobulin and T-cell receptor genes. It is a protein-kinase of the P13-kinase family and which can phosphorylates Ku proteins, p53 and probably some other proteins still unknown. DNA-PK is an important actor of DSBs repair (induced by ionising radiations or by drugs like etoposide), but obviously it is not the only mechanism existing in the cell for this function. Some others, like homologous recombination, seem also to have a great importance for cell survival.

The increase in the incidence of cancers in the general population, in particular the rapid increase of certain types of skin cancers in recent years , represents an alarming threat for human health and a permanent challenge for scientists. In fact, a doubling of the frequence of melanoma during the last fifteen years and a doubling of non melanoma skin cancers have been observed in the last ten years in France; already in the He of France one counts approximately 8000 new cases per year (Dore et al. 1990, Cesarini , personal communicat ion). The worldwide reported rapid rise in the incidence of these cutaneous malignancies (Glass and Hoover 1989) is almost entirely attributed to increased cutaneous exposure to solar UV radiation mostly related to present social behaviours including skin tanning and increased outdoor leisure activities (Mukhtar and Elmets 1996). Increased UV exposure due to stratospheric ozone depletion has also become a factor of concern (Henriksen et al. 1990) as well as indoor sunbed exposures (Autieret al. 1994. 1995). A 10% depletion in the total-column of ozone is expected to cause , if sustained over several decades , a 2 0 % increase in basa celt carcinoma and an about 4 0 % increase in squamous cell carcinoma (Van der Leun, 1995). However , at the present time the decrease in ozone layers observed cannot be taken as the main factor for the increase in skin cancer. The recent development of molecular biology has helped to get a better understanding of the mechanisms involved and to enlarge the classical concept of carcinogenesis including as principal steps the initiation and promotion of somatic cells. Actually, carcinogenesis is a multistep phenomenon (Figure 1). ln particular, one of the presently favoured hypotheses describes cancer induction as a consequence of the induction of somatic mutations following the induction of DNA damage , error prone or incomplete enzymatic repair of the lesions induced leading to altered gene expression , i.e. the inactivation of certain tumour suppressor genes and/or the activation of oncogenes which favour the progression of transformed cells and tumor development. Indeed, it is thought that the activation of genes which control cell proliferation facilitates the expansion and clonal selection of initiated and genetically instable cells showing an acceleration in cell cycle progression. If such cells escape the immunologic survey system, tumor cells may built foci and, depending on their nature (see models for the induction of melanoma (Albino and Fountain 1996) and squamous cell carcinoma (Ziegler et al. 1996)), may start spreading over the whole body in form of metastasis. That the individual repair capacities play an important role in carcinogenesis has become evident from studies on human cancer-prone diseases such as Xeroderma pigmentosum (XP) (Sarasin 1994, Kraemer 1996, Grossman and Wei 1995), Fanconi ' s Anemia (FA) (Moustacchi 1994) and Ataxia telangectasia (AT) (Aurias 1994). For example , X P patients are more than 1000 fold more susceptible to the induction of skin cancers by sunlight than normal individuals (Kraemer 1996). Ultraviolet radiation can be distinguished from low density ionizing radiation such as X-rays by differences in efficacy and specificity of the cancer induced. It appears that at comparable cytotoxic level ultraviolet radiation exerts a higher mutagenic and carcinogenic potential than ionizing radiation. This is probably due to differences in energy deposition in cellular targets and in the frequency and type of lesions induced by these agents . In physics and many industrial and medical applications, lasers are employed emitting strong monochromatic light in different regions of the electromagnetic spectrum including ultraviolet, visible and infrared radiation as well as soft X-rays and very short wave ultraviolet radiation < 200 nm . Despite the fact that these highenergy monochromatic light sources are highly

Image-guided radiotherapy (IGRT) combined or not with intensity-modulated radiation therapy (IMRT) are new and very useful techniques. However, these new techniques are responsible of irradiation at low dose in large volumes. The control of alignment, realignment of the patient and target positioning in external beam radiotherapy are increasingly performed by radiological imaging devices. The management of this medical imaging depends on the practice of each radiotherapy centre. The physical doses due to the IGRT are however quantifiable and traceable. In one hand, these doses appear justified for a better targeting and could be considered negligible in the context of radiotherapy. On the other hand, the potential impact of these low doses should deserve the consideration of professionals. It appears important therefore to report and consider not only doses in target volumes and in "standard" organs at risk, but also the volume of all tissue receiving low doses of radiation. The recent development of IMRT launches the same issue concerning the effects of low doses of radiation. Indeed, IMRT increases the volume of healthy tissue exposed to radiation. At low dose (100mGy), many parameters have to be considered for health risk estimations: the induction of genes and activation of proteins, bystander effect, radio-adaptation, the specific low-dose radio-hypersensitivity and individual radiation sensitivity. With the exception of the latter, the contribution of these parameters is generally protective in terms of carcinogenesis. An analysis of secondary cancers arising out of field appears to confirm such notion. The risk of secondary tumours is not well known in these conditions of treatment associating IMRT and IGRT. It is therefore recommended that the dose due to imaging during therapeutic irradiation be reported.

Cells are permanently exposed to genotoxic insults from internal metabolism and from exposures to chemicals and environmental agents giving rise to lesions in deoxyribonucleic acid (DNA). If not repaired, these may lead to cell death or to mutations, chromosomal aberrations, and initiation of carcinogenesis. During evolution, efficient multienzymatic repair pathways have been developed capable to remove damage before DNA replication or cell proliferation. Repair depends on damage recognition and activation of DNA damage signaling. Genetically controlled DNA repair pathways cope with different types of lesions, that is, mismatch repair (MMR) with replication errors, O6-guanine methyltransferase or DNA ligase I with simple methylated bases or single-strand breaks (SSBs) arising from alkylations and/or cellular metabolism, base excision repair (BER) with single base damages and complex SSBs induced by oxidants, oxidative chemical radicals, photodynamic sensitizers or ionizing radiation and nucleotide excision repair (NER) with bulky, DNA distorting lesions arising from chemical addition, metabolically activated polyaromatic hydrocarbons (PAHs), photoaddition of photoreactive molecules, or ultraviolet (UV) light-induced photolesions (pyrimidine dimers, 6-4 photoproducts). Complex lesions, that is, DNA double-strand breaks (DSBs) and DNA interstrand cross-links, rely on homologous recombination (HR) or nonhomologous end-joining (NHEJ) repair or sequential processing. Identification of rare human syndromes such as xeroderma pigmentosum (XP), Ataxia-telangiectasia (A-T), Bloom syndrome (BS), and along with knockout animal models have demonstrated a direct link between DNA repair deficiency and predisposition for spontaneous and induced cancers. Also, genetic polymorphisms of DNA repair and other genes can influence cancer predisposition in the general population. Thus, DNA repair activities are imbedded in a network of highly dose-dependent cellular responses involving activation or repression of general cellular metabolism, intra- and intercellular signaling, and epigenetic control. The initial level of DNA damage induced and the individual DNA repair capacity, physiological state, and cellular environment determine the final outcome in terms of carcinogenesis.

Dans le but de mieux comprendre les mecanismes a la base des relations existant entre les doses de radiations ionisantes et les effets genetiques radio-induits, il convient de prendre en compte non seulement les doses physiques d'irradiation, mais surtout les dommages primaires engendres au niveau cellulaire. Sans doute du point de vue des dommages genetiques radio-induits, l 'ADN est la cible principale de l'irradiation (Painter, 1980). Parmi de nombreux types de dommages induits dans l'ADN, c'est-a-dire dommages de bases, cassures simple et double brin de l'ADN et pontages ADN/proteines (Hutchinson, 1987 ; Teoule, 1987 ; Von Sonntag, 1991 ; Dizdaroglu, 1992), les cassures double brin (CDB) constituent des lesions d'une importance majeure, car, non reparees, elles sont tres probablement a l'origine de la mort cellulaire (lethalite) (Frankenberg-Schwager, 1989 ; Ward, 1990 ; Iliakis, 1991) et impliquees dans l'induction d'aberrations chromosomiques, de mutations et de transformation in vitro (Obe et al., 1992).

Cells of higher eukaryotes possess several very efficient systems for the repair of radiation-induced lesions in DNA. Different strategies have been adopted at the cellular level to remove or even tolerate various types of lesions in order to assure survival and limit the mutagenic consequences. In mammalian cells, the main DNA repair systems comprise direct reversion of damage, excision of damage and exchange mechanisms with intact DNA. Among these, the direct ligation of single strand breaks (SSB) by a DNA ligase and the multi-enzymatic repair systems of mismatch repair, base and nucleotide excision repair as well as the repair of double strand breaks (DSB) by homologous recombination or non homologous end-joining are the most important systems. Most of these processes are error-free except the non homologous end-joining pathway used mainly for the repair of DSB. Moreover, certain lesions can be tolerated by more or less accurately acting polymerases capable of performing translesional DNA syntheses. The DNA repair systems are intimately integrated in the network of cellular regulation. Some of their components are DNA damage inducible. Radiation-induced mutagenesis is largely due to unrepaired DNA damage but also involves error-prone repair processes like the repair of DSB by non-homologous end-joining. Generally, mammalian cells are well prepared to repair radiation-induced lesions. However, some questions remain to be asked about mechanistic details and efficiencies of the systems for removing certain types of radiation-damage and about their order and timing of action. The answers to these questions would be important for radioprotection as well as radiotherapy.

Repair pathways of DNA are now better defined, and some important findings have been discovered in the last few years. DNA non-homologous end-joining (NEHJ) is a crucial process in the repair of radiation-induced double-strand breaks (DSBs). NHEJ implies at least three steps: the DNA free-ends must get closer, preparation of the free-ends by exonucleases and then a transient hybridisation in a region of DNA with weak homology. DNA-dependent protein kinase (DNA-PK) is the key enzyme in this process. DNA-PK is a nuclear serine/threonine kinase that comprises three components: a catlytic subunit (DNA-PKCS) and two regulatory subunits, DNA-binding proteins, Ku80 and Ku70. The severe combined immunodeficient (scid) mice are deficient in DNA-PKCS: this protein is involved both in DNA repair and in the V(D)J recombination of immunoglobulin and T-cell receptor genes. It is a protein-kinase of the P13-kinase family and which can phosphorylates Ku proteins, p53 and probably some other proteins still unknown. DNA-PK is an important actor of DSBs repair (induced by ionising radiations or by drugs like etoposide), but obviously it is not the only mechanism existing in the cell for this function. Some others, like homologous recombination, seem also to have a great importance for cell survival.

The poly (ADP-ribose) polymerase is an ubiquitous nuclear protein capable of binding specifically to DNA strand breaks. It synthesizes ADP-ribose polymers proportionally to DNA breaks. The actual method of reference to determine DNA double strand breaks is pulsed-field gel electrophoresis, but this requires many cells. It thus appeared of interest to use poly (ADP-ribos)ylation to follow and estimate gamma-ray-induced DNA fragmentation at the level of isolated cells after gamma-irradiation in chinese hamster ovary cells (CHO-K1). The results obtained by the immunolabelling technique of ADP-ribose polymers were compared to those obtained by pulsed-field gel electrophoresis. They show that poly (ADP-ribos)ylation reflects the occurrence of radiation-induced DNA strand breaks. A clear relationship exists between the amount of ADP-ribose polymers detected and DNA double strand breaks after gamma-irradiation.

The dioxinocoumarin derivatives 5H-[2]benzopyrano-[3,4-g][1,4]benzodioxin-5-one (I), 5H-[2]benzopyrano-[3,4-g][2,3]-dihydro-[1,4]benzodioxin-5-on e II, 6H-[2]benzopyrano[3,4-f]-1,4-benzodioxin-6-one (III) and 6H-[2]benzopyrano[3,4-f]-2,3-dihydro-1,4-benzodioxin-6-one (IV) were synthesized. Their biological effect was studied in the presence and absence of UVA radiation, and compared with that of 8-methoxypsoralen (8-MOP) and angelicin derivatives on T7 phage, diploid yeast (Saccharomyces cerevisiae) and HeLa cells. The photobiological activities of compounds I and III were stronger than that of 8-MOP in phage inactivation and DNA synthesis inhibition in HeLa cells, whereas compounds II and IV, with a saturated dioxin ring, showed very poor activity. The photosensitizing activity of dioxinocoumarins on phage inactivation decreased by a factor of two to three in the absence of oxygen. Treatments with compound I and UVA in the presence of oxygen modified the helical structure and stability of phage DNA and proteins. Compounds I and II were more active than IV for photoinduced cell killing in yeast, although always less active than 8-MOP. At comparable photocytotoxic levels, compounds I and III were as strong inducers of cytoplasmic "petite" mutants in yeast as angelicin, suggesting a possible monofunctional mode of action with cellular DNA.

Bases for the elaboration of a standardized protocol are proposed for studying phototoxic effects of skin tanning preparations containing photosensitizing agents. The experimental procedure includes in vivo phototoxicity tests, evaluation of the photogenotoxic risk and determination of the photosensitizer concentration in plasma after topical application. This procedure was carried out with tanning preparations containing a well-known photosensitizer, 5-methoxypsoralen, as a component of bergamot oil. The whole study has been performed using topical application of the commercial suntan product, i.e. containing the sunscreens and all other components. Whereas the exposure to solar simulated radiation never triggered any phototoxic response, a photosensitizing effect was observed for skin type I volunteers exposed to high doses of ultraviolet A. The transepidermal penetration resulted in a 5-methoxypsoralen concentration of 1-4 ng/ml in the suction blister fluid. The photogenotoxicity of this suction blister fluid containing 5-methoxypsoralen and also other ingredients of the tanning preparation was assayed on yeast cells and was found to be rather low. 5-Methoxypsoralen was also detected in plasma after repeated applications but at low concentrations (about 1 ng/ml) which do not present a potential risk for systemic ocular effects.

The genetic effects of two mono-functional photosensitizing furocoumarins, 3-carbethoxypsoralen (3-CPs) and angelicin, were compared with those of two bi-functional furocoumarins, 8-methoxypsoralen and psoralen in Saccharomyces cerevisiae. A drug concentration of 5 × 10−5 M plus various doses of 365-nm irradiation at a dose rate of 1.2 kJ m−2 min−1 were used. Per dose of 365-nm irradiation, the frequency of induced nuclear events such as gene mutation and mitotic recombination (conversion and crossing-over) is higher for the bi-functional than for the mono-functional compounds. The higher efficiency of the bi-functional furocoumarins is also evident when the frequency of mutants is expressed as a function of survival. However, the photoaddition of the 4 furocoumarins studied leads to the same response for the induction of recombinational events per viable cell. Amongst genetically altered colonies induced in the diploid strains D5 and D7, the colonies corresponding to the induction of crossing-over are effectively produced by bi-functional furocoumarins, but are rare (D7) or even absent (D5) after treatment with monofunctional furocoumarins. This suggests a certain specificity of genetic alterations produced by the bi-functional agents. 3-CPs is the most effective inducer on the cytoplasmic “petite” mutation in stationary phase cells per unit irradiation dose or per viable cell.

Tow types of dose-rate effect that alter the survival response of haploid yeast cells to 8-methoxypsoralen (8-MOP) plus treatment with irradiation at 365 nm were studied. (1) When the concentration of 8-MOP was varied between 9.2 X 10(-5) and 2.3 X 10(-8) M and the dose rate of 365-nm irradiation kept constant, the efficiency of the irradiation for killing increased relatively to that of 8-MOP whe the concentration of 8-MOP decreased. This indicated that there was no strict reciprocity between radiation dose and concentration of drug. (2) When the dose rate of radiation was varied between 0.66 X 10(3) and 108 X 10(3) J m-2 h-1 and the concentration of 8-MOP was kept constant, the survival of wild-type cells increased strikingly at low dose rates of radiation as compared with high dose rates. Cells responded more to changes at low dose rates than to equal changes a high dose rates. The high resistance of wild-type cells to 8-MOP plus radiation delivered at low dose rates absent from rad 1-3 cells defective in excision-repair. This suggests that the dose-rate effect seen in wild-type cells depended at least in part on an active excision-repair function. At low dose rates of radiation, the shoulder of the survival curve for rad1-3 cells, i.e. the ability to accumulate sub-lethal damage, was increased by a factor of about 2 when compared with that seen at a high dose rate. Thus it is likely that at low dose rates a repair function other than excision-resynthesis may operate in rad1-3 cells.

The possible existence of thermal effects specific to microwaves at 2.45 GHz and not found with classical heating in a waterbath was studied by measuring cell survival (colony-forming ability) and cell viability (the ability to exclude trypan blue) in Chinese hamster V79 cells. The microwaves were employed at high power densities (125 to 175 mW/cm2) corresponding to specific absorption rates ranging between 62 and 87 mW/g. When matching the rises in temperature, the effects of microwave-induced hyperthermia at 125 mW/cm2 on cell survival were comparable to those of classical heating. However, they were statistically significantly different when using power densities of 150 and 175 mW/cm2. The response obtained in terms of cell viability appeared to be comparable. The conclusions are also valid when taking into account a correction factor for energy losses during microwave treatment. The apparent specific effect of microwaves appears to be associated with exposures at high power densities involving short treatment times and rapid rises in temperature.

Experiments were set up to investigate the effects of open space microwave irradiation of the millimeter (73 GHz) and the centimeter (17 GHz) range in Drosophila melanogaster. We used the wild type strain Paris and the strain δ carrying melanitic tumors in the 3rd larval stage, in the pupae and the adults. The power densities were up to lOOmW.cm-2 for 73 GHz and about 60 mW.cm-2 for microwaves at 17 GHz. After 2 h exposure to microwaves of 17 GHz or 73 GHz the hatching of the irradiated eggs and their development were normal. In a few cases there was a tendency towards a diminution of the survival of eggs treated at different stages, of larvae treated in the stages 1, 2 and 3 and of treated pupae. However, this was not always statistically significant. The microwave treatment did not induce tera- tological changes in the adults. A statistical analysis brought about slight diminutions in the incidence and multiplicity of tumors in adult flies. When wild type females were exposed to microwaves of 17 GHz for 16 or 21 h and crossed with untreated males we observed a marked increase in fertility as compared to untreated samples. The viability and tumor incidence in the offspring was not affected. Similar results were obtained when microwaves treated males were crossed with untreated females.

Recent microwave experiments have shown frequency dependent influences on the growth rate of bacteria. To determine whether microwaves are able to affect growth (or to induce lesions in cellular DNA of yeast cells), experiments were performed with millimeter microwaves at frequencies between 70 and 75 GHz. Saccharomyces cerevisiae cells were irradiated on millipore filter discs placed on agar plates in open petri dishes. A diploid strain of yeast (D5, Zimmerman), that is sensitive to genetic insult was used to study the effects of temperature and of microwave irradiation on cell survival, induction of mitotic recombination, and induction of cytoplasmic "petite" mutations. No evidence of altered survival, impaired function, or structural injury was seen at either frequency, even at power densities as high as 60 mW/cm2. Conventional heating had no deleterious effects until temperatures of specimens exceeded 50 degrees C. In addition, two haploid strains of yeast of opposite mating type were compared with respect to temperature and microwave treatment for formation of zygotes. The elevation of temperature due to the microwave treatment at 60 mW/cm2 and 2 mm distance was estimated to correspond to 3 degrees C.

Saccharomyces cerevisiae (wild-type strain 211) in a stationary phase was exposed to 12-MeV electrons over a dose range of 0. to 700 rads. In one set of experiments a first dose was given in air and a second 15 min later in nitrogen. In a second set of experiments, the first dose was in N/sub 2/ and the second in air. Survival curves were established by varying the size of the second dose. The shoulder of anaerobic curve was completely abolished by pre- irradiation with moderate doses in air. It is suggested that these results indicate a complete overlap of type O and N damage in both directions suggesting that both share a damage pathway in the cell, probably, but not necessarily acting on the same target molecule. (UK)

Three not closely linked genes (r1s, r2sand r3s), a mutation in each of which causes UV- and X-ray-sensitivity of the colony forming ability in haploid and homozygous diploid cells have been identified. In one gene, r3s, two mutations have occurred independently. The effect of the mutated genes on long time repair processes as well as the effect on reversion rates from isoleucine-valine dependence to independence has been investigated. A model to explain the mechanisms changed by the mutated genes is discussed.

Haploid wild-type and mutant cells of Saccharomyces carrying one of the single genes rad 2–20 or rad 9−4 and the double mutant rad 2–20 rad 9−4 were tested for their response to a treatment with 8-methoxypsoralen plus 365 nm light using immediate and delayed plating techniques. The mutant defective in the excision of ultraviolet-induced pyrimidine dimers ( rad 2–20 ) as well as that presumably deficient in a recombinational repair system ( rad 9−4 ) are more sensitive than wild type cells. The double mutant ( rad 2–20 rad 9−4 ) demonstrates a higher sensitivity than each of the single mutants, indicating that at least two pathways are involved in the repair of the 8-methoxypsoralen plus 365 nm induced damages. In all cases survival curves have shoulders. The survival of wild type and rad 9−4 cells is increased after dark holding whereas it remains constant for the rad 2–20 mutant and for the double mutant. These results show that the induced damages are reparable. Respiratory deficient mutant ( ϱ − ) were compared to the corresponding respiratory competent cells. It is shown that the respiratory function is required for the expression of the excision repair activity. The 8-methoxypsoralen plus 365 nm light treatment appears to be less effective than ultraviolet irradiation (254 nm) in the induction of the cytoplasmic ‘petite’ mutation at the same survival levels.

The induction of lethal effects and 6-thioguanine-resistant (6-TG r ) mutants were studied in Chinese hamster V79 cells after treatment with the two bifunctional furocoumarins 5- and 8-methoxypsoralens (5-MOP, 8-MOP) in the presence of 365-nm radiation (UVA). The in vivo DNA-photobinding capacity of these two compounds was measured and in parallel the cross-linking capacities of 5-MOP and 8-MOP were determined using the alkaline elution technique. The results show that 5-MOP plus UVA was about 2.5 times more effective than 8-MOP plus UVA for inhibiting cell survival and for inducing the same frequency of 6-TG r mutants (10 −4 ). The total number of photoinduced lesions by 5-MOP plus UVA was about 6 times higher than that induced by 8-MOP plus UVA. However, the cross-linking capacities of 5-MOP and 8-MOP were found to be within the same range at equal doses of UVA. At equal number of DNA photoadducts produced, the lesions induced by 5-MOP appeared to be less genetically active than those induced by 8-MOP. The apparently weaker genotoxicity of 5-MOP-induced lesions is likey to be due to the induction of a lower proportion of cross-links by 5-MOP at a given number of photoadducts.

The metabolism of 8-methoxypsoralen (8-MOP) by the yeast Saccharomyces cerevisiae has been investigated in order to determine if this cell system could provide a simple model to study the metabolism of new photosensitizing drugs in vitro. 8-MOP was found to be rapidly metabolized by S. cerevisiae in non growing conditions. A metabolite was detected which exhibits a structure similar to that of a metabolite previously isolated in dog and man. Ethanol showed a strong inducing effect on 8-MOP metabolization.

The study on the growth of E. coli cells under low power microwave irradiation the frequency of which can vary between 70 and 75 GHz, demonstrates that in the neighbourhood of 70.5 and 73 GHz a slowdown of growth is most frequently observed. On the contrary, the survival is not altered as long as the power of the irradiation is low. The irradiation does not induce lesions in DNA, whatever the frequency may be.

Furocoumarins such as psoralen and 8 methoxypsoralen (8-MOP) and some of their derivatives are photosensitizing agents. They are known to sensitize human and guinea pig skin to near ultraviolet (UV) light and sunlight (Musajo and Rodighiero, 1972a). They are used medically in cases of vitiligo and leukoderma (El-Mofty, 1968) and also in cases of psoriasis (Walter and Voorhees, 1973; Parrish et al., 197 4; Weber, 1974).

In order to study the relationship between certain photophysical and photochemical properties of furocoumarins and their photobiological activities, the quantum yields of the formation of excited triplet states, the capacity to generate singlet oxygen in vitro and the effect of oxygen on photoinduced cell killing and on the induction of cytoplasmic "petite" mutations in Saccharomyces cerevisiae were determined for a series of 4'-methylangelicins and 3-carbethoxypsoralen (3-CPs). The capacity to generate singlet oxygen, in good agreement with the photophysical data, correlates well with the oxygen effect observed on the lethality of Saccharomyces cerevisiae shown by the various compounds and followed the ranking order 3-CPs greater than 4',4-dimethylangelicin greater than 4'-methylangelicin greater than 4',5-dimethylangelicin. On the other hand the oxygen effect on the induction of cytoplasmic "petite" mutations on yeast appears much less pronounced than that shown on survival. Concerning other photobiological properties previously studied no correlation was observed between the capacity to generate singlet oxygen in vitro and the skin photosensitizing activity of the compounds. Moreover, the generation of singlet oxygen by furocoumarins does not appear as the main cause for the photoinduction of skin tumors in mice.

In conditions in which solely monoadducts (MA) are induced in DNA, i.e., treatment with 4,5',8-trimethylpsoralen and 405 nm radiation, Fanconi's anemia cells (FA) appear to be more sensitive than normal human fibroblasts (1 BR/3) to cytotoxicity. The repair of such induced MA is impaired in FA compared to normal cells. When increasing the proportion of DNA interstrand cross-links (CL) over MA using a reirradiation protocol, the differential sensitivity between FA and normal human cells increases. Moreover, for a constant number of total adducts or at different ratios of CL over MA, the repair of CL is systematically hampered in FA as compared to normal cells. Incision of CL being progressively diminished by increasing amounts of MA in normal cells (D. Papadopoulo, D. Averbeck, and E. Moustacchi, Photochem. Photobiol., 47:321-326, 1988), we show here that it is even more so for FA cells.

The mutagenic effects of monofunctional and bifunctional furocoumarins (psoralens) plus 365 nm radiation were analyzed in the yeast Saccharomyces cerevisiae. Per unit dose of 365-nm radiation, bifunctional compounds were more effective than were the monofunctional for the induction of reverse and forward mutations. The same was observed for the induction of 6-thioguanine-resistant mutants in V79 Chinese hamster cells when we compared the activity of 8-methoxypsoralen and 3-carbethoxypsoralen. An analysis of the kinetics of mutation induction indicated that DNA interstrand cross-links induced by bifunctional psoralens are more prone to error than are monoadducts induced by monofunctional psoralens. In yeast, 8-methoxypsoralen and 4,5'-dimethylangelicin were shown to photoinduce mitotic and nondisjunction. The implications of these findings for the use of psoralens in photochemotherapy and cosmetics are discussed.

In contrast to 8-MOP photoaddition on DNA, the renaturation capacity of heat or alkali denatured DNA treated with 3-CPs is absent. This shows that DNA interstrand cross-links are not produced by 3-CPs photoaddition even by a dose which leads to 10(-5) survivors. At equimolar concentrations of 3-CPs or 8-MOP (5 x 10(-4) M) and a dose of 10 kJ.m-2 of 365 nm radiation, 1 molecule of 3-CPs for 4 x 10(3) nucleotides and 1 molecules of 8-MOP for 9 x 10(3) nucleotides are fixed in vivo. Since wild type cells are about 4 times more sensitive to 8-MOP than to 3-CPs photoaddition, it implies that cross-links are important in genotoxicity and that monoadditions are more easily repaired than cross-links.

The carcinogenic risk of the photochemotherapy (PUVA) with bifunctional furocoumarins such as 8-methoxypsoralen (8-MOP) forming cross-links in cellular DNA has initiated a search for active but less dangerous psoralens. The 3-carbethoxypsoralen (3-CPs), studied in the yeast Saccharomyces cerevisiae, has been very photoactive on DNA forming only monoadditions to DNA. In Mice it was demonstrated that after local or intra-peritoneal administration, 3-CPs is non toxic, non erythematogenic and non carcinogenic in contrast to 8-MOP. A limited study on ten psoriatic patients has shown that after local application 3-CPs exhibits about the same therapeutic activity as 8-MOP.

Using monofunctional (Angelicin) and bifunctional furocoumarins (Psoralen and 8 Methoxypsoralen) plus 365 nm light it is shown that both damages, the induced monoadducts and/or crosslinks in DNA, provoke lethal and mutgenic effects in haploid and diploid cells of Saccharomyces cerevisiae. Bifunctional furocoumarins are about 20 times more effective in cell killing than Angelicin. Diploid cells are always more resistant than haploid cells. Dark repair (agar haolding) increases survival. This effect can be at least in part correlated to the release of bound material from DNA in dark repair conditions. Bifunctional psoralens (10 mug/ml) are at least 10-fold more effective in inducing nuclear gene black mutations (his- to HIS+) than Angelicin (10 mug/ml) plus 365 nm light or 254 nm ultraviolet light. In contrast cytoplasmic "petite" (delta-) mutations are about as frequently induced by Angelicin plus 365 nm light as by 254 nm UV light. Bifunctional furocoumarins are less effective. The frequency of cytoplasmic "petite" mutations per survivors decreases during dark repair conditions more efficiently after Angelicin than after Psoralen plus 365 nm light treatment.

The photobiology of fucoroumarins has been covered by a number of reviews (1–4). In recent years we observed a rapid evolution in the knowledge on naturally occurring or synthetic furocoumarins which in the presence of near ultraviolet light or UVA, i.e. radia tion in the range of 320 to 380 nm, exhibit photosensitizing properties in various biological systems (1–4). The reaction scheme for furocoumarins with nucleic acids, especially with DNA, is well established (1, 4, 5, 6). It involves the formation of complexes between furocoumarins and DNA in the dark, and in the presence of 365-nm radiation the induction of cyclobutane type of mono-additions of pyrimidine bases in DNA involving the 3,4 or the 4’,5’ double bond of the furocoumarin molecule. Some of the 4’,5’ cycloadditions may photoreact further forming interStrand DNA cross-links (1, 4–7).

In order to demonstrate possible specific effects of microwaves at the cellular level V-79 Chinese hamster cells were exposed to 2.45-GHz radiation at power levels of 20-200 mW/cm2 and at specific absorption rates of 10-100 mW/g. Intracellular cytoplasmic changes were observed by fluorescence polarization using a method based on the intracellular enzymatic hydrolysis of nonfluorescent fluorescein diacetate (FDA). At levels of absorbed energy below 90 J/g, modifications of microviscosity and mitochondrial state were absent, but a slight stimulation of enzymatic hydrolysis of FDA was observed which may be explained by microwave-induced alterations of cellular membranes possibly due to differences in heating pattern of microwaves compared to water-bath heating. At levels of absorbed energy above 90 J/g, the decrease of enzymatic hydrolysis of FDA, increase in degree of polarization, and increase of permeation of the fluorescent marker correlated well with the decrease in cell viability as measured by the exclusion of trypan blue. At equal absorbed energy, microwaves were found to exert effects comparable to classical heating except that permeation was slightly more affected by microwave than by classical heating. This suggests that membrane alteration produced by microwaves might differ from those induced by classical heating or that microwaves may havemore » heated the membrane to higher temperatures than did classical heating.« less

—At an cquimolar concentration of 50 μM the bifunctional furocoumarin, 8-methoxypsoralen (8-MOP), is about 36 times more efficient in inhibiting the colony forming ability of CV-I monkey kidney cells than the monofunctional furocoumarin angelicin. In contrast 8-MOP is only 7.5 times more efficient than angelicin for the inhibition of herpes simplex virus (HSV) production in CV-1 cells. This latter factor seems to reflect differences in photoreactivity of the two compounds with host cell DNA. A substantial recovery of HSV production was seen when cells were infected at different time intervals after treatment with angelicin-plus-light, whereas recovery was very limited after 8-MOP plus light treatment. The recovery process was slow as compared to that observed after UV (254 nm)-irradiation. The repair capacities of treated normal and xeroderma pigmentosum (group A) skin fibroblasts were estimated by measuring HSV production and unscheduled DNA synthesis. XP-A cells repaired angelicin induced damage less efficiently than did normal cells. Neither cell type showed any repair activity after 8-MOP plus light treatment.

Psoralens (furocoumarins) of natural or synthetic origin show photosensitizing activity on biological systems in the presence of long-wave ultraviolet radiation (320–400 nm) (UVA) (Musajo and Rodighiero 1972, Scott et al. 1976, Ben-Hur and Song 1984, Hearst et al. 1984). Photosensitization by psoralens is believed to be mainly due to a photomediated covalent binding to pyrimidine bases in DNA. The DNA photobinding involves the following steps: formation of psoralen-DNA complexes in the dark and, in the presence of UVA, induction of monoadducts and diadducts (DNA interstrand cross-links). Mono-functional furocoumarins photoinduce only monoadducts, bifunctional furocoumarins mono-and diadducts (Ben-Hur and Song 1984).

The genetic effects of two mono-functional photosensitizing furocoumarins, 3-carbethoxypsoralen (3-CPs) and angelicin, were compared with those of two bi-functional furocoumarins, 8-methoxypsoralen and psoralen in Saccharomyces cerevisiae. A drug concentration of 5 X 10(-5) M plus various doses of 365-nm irradiation at a dose rate of 1.2 kJ m-2 min-1 were used. Per dose of 365-nm irradiation, the frequency of induced nuclear events such as gene mutation and mitotic recombination (conversion and crossing-over) is higher for the bi-functional than for the mono-functional compounds. The higher efficiency of the bi-functional furocoumarins is also evident when the frequency of mutants is expressed as a function of survival. However, the photo-addition of the 4 furocoumarins studied leads to the same response for the induction of recombinational events per viable cell. Amongst genetically altered colonies induced in the diploid strains D5 and D7, the colonies corresponding to the induction of crossing-over are effectively produced by bi-functional furocoumarins, but are rare (D7) or even absent (D5) after treatment with monofunctional furocoumarins. This suggests a certain specificity of genetic alterations produced by the bi-functional agents. 3-CPs is the most effective inducer on the cytoplasmic "petite" mutation in stationary phase cells per unit irradiation dose or per viable cell.