Clastogenic effects of ftorafur. II. Cytogenetic analyses of bone marrow in mice

Our previous work has demonstrated that whereas near-UV radiation is not a mutagen for Haemophilus influenzae cells, it does induce mutations in purified transforming DNA. In order to test various hypotheses concerning this difference, we have irradiated cells at 334 and 365 nm, then lysed them and assayed the DNA for induced mutations and for inactivation of transforming ability. The inactivation was only a little lower than observed with highly purified transforming DNA. The DNA irradiated in vivo was mutated at both wave-lengths, but with considerably lower efficiency than was purified DNA. Neither incubation of the cells after irradiation and before lysis nor freezing and thawing the cells significantly changed the amount of mutation. It is concluded that there is some protection of the DNA against premutational lesions by the in vivo environment, but that it is not enough to account for the total lack of mutation of the cells. A probable explanation of this lack of cell mutation is that lethal lesions in the cells are induced much more readily than premutational lesions.     

Action spectrum for lethality of near-UV light on Haemophilus influenzae and lack of mutation.

Mutation and inactivation of H. influenzae have been measured following irradiation at various near-UV wavelengths. Inactivation takes place most readily at 334 nm (but is unaffected by absence of excision or postreplication repair), and decreases markedly at longer wavelengths. No induced mutations to resistance to novobiocin or streptomycin or to ability to utilize protoporphyrin instead of hemin were detected at any of the wavelengths used. There were also no detectable induced mutations in an excision-defective strain after 334-nm irradiation. These results are in contrast to the in vitro mutation of purified transforming DNA we previously observed.     

Action spectra for killing and mutation of Chinese hamster cells exposed to mid- and near-ultraviolet monochromatic light

We have examined the response of Chinese hamster V79 cells to monochromatic light of selected wavelengths in the mid- to near-UV region, using cell survival and induction of mutants resistant to 6-thioguanine (6-TG) or ouabain (OUA) as end points. As the wavelength increased from 313 to 405 nm, the induction of mutants resistant to 6-TG and to OUA decreased to a greater degree than did cell survival. Cells resistant to OUA were induced with considerably lesser efficiency at wavelengths of 313 and 334 nm than cells resistant to 6-TG. No mutants resistant to either 6-TG or OUA were induced by 405-nm light, and no mutants resistant to OUA were induced by 365-nm light. Thus, cell killing and mutation induction have different action spectra, and furthermore, action spectra for mutation induction at the HGPRT and Na+/K+-ATPase loci are different from each other. These observations imply important differences in the cellular mechanisms, and/or lesions, for cell inactivation, induction of 6-TG and OUA resistance for V79 cells exposed to near-UV monochromatic light.     

Mutagenesis and cytotoxicity in human epithelial cells by far- and near-ultraviolet radiations: action spectra

Action spectra were determined for cell killing and mutation by monochromatic ultraviolet and visible radiations (254-434 nm) in cultured human epithelial P3 cells. Cell killing was more efficient following radiation at the shorter wavelengths (254-434 nm) than at longer wavelengths (365-434 nm). At 254 nm, for example, a fluence of 11 Jm-2 gave 37% cell survival, while at 365 nm, 17 X 10(5) Jm-2 gave equivalent survival. At 434 nm little killing was observed with fluences up to 3 X 10(6) Jm-2. Mutant induction, determined at the hypoxanthine-guanine phosphoribosyltransferase locus, was caused by radiation at 254, 313, and 365 nm. There was no mutant induction at 334 nm although this wavelength was highly cytotoxic. Mutagenesis was not induced by 434 nm radiation, either. There was a weak response at 405 nm; the mutant frequencies were only slightly increased above background levels. For the mutagenic wavelengths, log-log plots of the mutation frequency against fluence showed linear regressions with positive slopes of 2.5, consistent with data from a previous study using Escherichia coli. The data points of the action spectra for lethality and mutagenesis were similar to the spectrum for DNA damage at wavelengths shorter than 313 nm, whereas at longer wavelengths the lethality spectrum had a shoulder, and the mutagenesis spectrum had a secondary peak at 365 nm. No correlation was observed for the P3 cells between the spectra for cell killing and mutagenesis caused by wavelengths longer than 313 nm and the induction of DNA breakage or the formation of DNA-to-protein covalent bonds in these cells.     

Formation of a thymine photoproduct in transforming DNA by near ultraviolet irradiation.

Irradiation at 334 and 365 nm of a highly purified preparation of thymine-labeled transforming DNA from Haemophilus influenzae produced a photo product containing label from thymine but different from the cyclobutane dimer. The photoproduct is soluble in water and in ethanol and Rf values in a number of solvents are presented. The photoproduct has properties similar in a number of respects to those of the spore photoproduct, 5-thyminyl-5,6-dihydrothymine. The near ultraviolet photoproduct is more likely to affect the oxygen independent inactivation of transforming DNA rather than its mutagenesis, as judged by the quantitative relationship between amount of photboproduct and inactivation and mutagenesis.     

Influence of mutations at the rep gene on survival of Escherichia coli following ultraviolet light irradiation or 8-methoxypsoralen photosensitization: evidence for a recA+ rep+-dependent pathway for repair of DNA crosslinks

The mutagenic interaction between near-ultraviolet (365 nm) radiation and the alkylating agents ethyl methanesulphonate (EMS) and methyl methanesulphonate (MMS) was studied in a repair-competent and an excision-deficient strain of Escherichia coli. Near-UV radiation modified the metabolic response of exposure to these chemicals and either reduced or increased their mutagenic efficiency. Based on these results, an experimental model was formulated to explain the mutagenic interactions that occur between near-UV and various agents that induce prototrophic revertants via error-prone repair of DNA. According to this model, low doses of near-UV provoke conditions for mutation frequency decline (MFD) and lead to a mutagenic antagonism. With increasing near-UV doses, damage to constitutive error-free repair systems increases, favouring the error-prone system and inhibiting the MFD. Under these conditions there will be a progressive decrease in antagonism until at high doses an enhancement of mutation frequency (positive interaction) will occur.     

Similar ultraviolet action spectra for the protection by glycerol of transforming DNA against single-strand breaks and inactivation of biological activity

An action spectrum for the protection of purified DNA by glycerol against the induction of single-strand breaks in the DNA by ultraviolet (uv) light is described. Protection was not observed below 300 nm, was maximal between 334 and 365 nm, and decreased at 405 nm. This spectrum closely matched the spectrum for the protection by glycerol against the inactivation of biological transforming activity by near uv, described previously. Also, deviations from the reciprocity rule are similar for inactivation of transforming activity and for induction of DNA breaks by 365-nm radiation. That is, the deviations for the two end points are quantitatively the same, such that high fluence rates are less effective than low fluence rates.     

Mutagenic interactions between near-ultraviolet (365 nm) radiation and alkylating agents in Escherichia coli

The mutagenic interaction between near-ultraviolet (365 nm) radiation and the alkylating agents ethyl methanesulphonate (EMS) and methyl methanesulphonate (MMS) was studied in a repair-competent and an excision-deficient strain of Escherichia coli. Near-UV radiation modified the metabolic response of exposure to these chemicals and either reduced or increased their mutagenic efficiency. Based on these results, an experimental model was formulated to explain the mutagenic interactions that occur between near-UV and various agents that induce prototrophic revertants via error-prone repair of DNA. According to this model, low doses of near-UV provoke conditions for mutation frequency decline (MFD) and lead to a mutagenic antagonism. With increasing near-UV doses, damage to constitutive error-free repair systems increases, favouring the error-prone system and inhibiting the MFD. Under these conditions there will be a progressive decrease in antagonism until at high doses an enhancement of mutation frequency (positive interaction) will occur.     

Mutagenic interaction between near-(365 nm) and far-(254 nm)ultraviolet radiation in repair-proficient and excision-deficient strains of Escherichia coli.

The mutational interaction between radiation at 365 and 254 nm was studied in various strains of E. coli by a mutant assay based on reversion to amino-acid independence in full nutrient conditions. In the two repair-proficient strains (K12 AB 1157 and B/r), pre-treatment with radiation at 365 nm strongly suppressed the induction of mutations by far-UV, a phenomenon accompanied by a strong lethal interaction. The frequency of mutations induced by far-UV progressively declined with increasing dose of near-UV. Far-UV-induced mutagenesis to T5 resistance was almost unaltered by pre-treatment with near-UV. In AB 1886 uvrA there was no lethal interaction between the two wavelengths but the mutagenic interaction was synergistic. This synergism was maximal at a 365-nm dose of 8 X 10(5) J m-2. It is proposed that in the wild-type strain, cells containing potentially mutagenic lesions are selectively eliminated from the population because of abortive excision of an error-prone repair-inducing signal. In excisionless strains, 365-nm radiation may be less damaging to the error-prone than to the error-free post-replication repair system. Alternatively, mutation may be enhanced because of the occurrence of error-prone repair of 365-nm lesions by a system that is not induced in the absence of 254-nm radiation.     

Inactivation of transforming DNA by ultraviolet light. II. Protection by histidine against near-UV irradiation: action spectrum

Histadine, at concentrations greater than 0.075 M, quenches the inhibition of the transforming activity of DNA by near UV. This effect does not occur at wavelengths below about 300 nm and is maximal at about 380 nm. Since histidine at this concentration has no effect upon the inactivation by far UV, this observation shows that inactivation by near UV is clearly different, and suggests an indirect photosensitization mechanism. The histidine protection spectrum coincides closely with the near-UV DNA inactivation spectrum derived from the shoulder in the near UV.     

Lethal cellular changes induced by near ultraviolet radiation.

There is clear evidence that significant quantities of lesions are induced in DNA by near-UV radiation and that these lesions, although susceptible to repair, may lead to cell death because of the simultaneous disruption of DNA repair systems by the same wavelengths. No particular DNA lesion can be linked to cell death in wild type strains. However, there are good grounds for speculating that a type of near-UV lesion exists which is rapidly "fixed" as a lethal event in cells as a result of the oxygen-dependent disruption of repair. There is a strong indication that the relative ability of various near-UV wavelengths to sensitize cells to heat, chemicals or other radiations is directly related to their efficiency in disrupting DNA repair systems in general. Some important specific questions remain. For example, it is important to ask why breaks formed at 365 nm and 405 nm, although apparently requiring a pol dependent pathway for their repair, do not produce the predicted lethal biological action in the strains tested. In general terms it is hoped to provide more comprehensive physico-chemical data in support of, or contradicting, the proposed model.     

Interactions between uv radiation of different energies in the inactivation of bacteria.

A strong lethal interaction was observed between various monochromatic wavelengths (254, 334, 365, and 405 nm) in the repair-proficient E. coli K-12 strain AB 1157, except in the case of preexposure to 405-nm radiation which resulted in a protection against the inactivation resulting from subsequent exposure to 365-or 254-nm radiations. The results may be tentatively explained by assuming two classes of DNA lesions and two classes of damage to repair (reversible and inrreversible) whose proportions vary according to wavelength.     

Mutagenic and lethal action of polychromatic near-ultraviolet (325-400 nm) on Haemophilus influenzae in the presence of nitrogen

The lethal effect of polychromatic near-UV light (325-400 nm) on Haemophilus influenzae was 8 times higher under aerobic than anaerobic irradiation. This light increased the frequency of mutation to novobiocin resistance and ability to utilize protoporphyrin IX. The slope of mutagenic effect at low doses appeared greater for the aerobic than for the anaerobic group. We concluded that polychromatic near-UV mutation of H. influenzae under anaerobic irradiation was caused by direct oxygen-independent action on DNA.     

Mutagenesis and growth delay induced in Escherichia coli by near-ultraviolet radiations

The literature relating to genetic changes induced in Escherichia coli by near-ultraviolet radiations is reviewed and summarized: i) these radiations are much less mutagenic than would be expected from the known level of DNA damage, ii) pre-illumination with near-UV light antagonizes the mutagenic effect of UV (254 nm) light. In agreement with these findings, the SOS functions are not induced by near-UV radiations. Furthermore prior exposure of cells to near-UV light inhibits the subsequent 254 nm induction of the SOS response. Among the several hypothesis considered to explain these observations, one can be clearly favoured. Near-UV light triggers, at sublethal fluences, the growth delay effect. The target molecules, tRNAs, are photocrosslinked and some tRNA species become poor substrates in the acylation reaction. In vivo these tRNA molecules accumulate on the uncharged form, leading to a transient cessation of protein synthesis. The SOS response is inducible and as such requires protein synthesis. We therefore propose that near-ultraviolet radiations have a dual effect: i) they induce, mostly indirectly, DNA lesions which are potentially able to trigger the SOS response, ii) they prevent the expression of the SOS functions through the transient inhibition of protein synthesis (growth delay).     

Studies on the reactivation of bacteria photodamaged by psoralen

Summary In photoreaction with the pyrimidine bases (thymine, cytosine, uracil) as well as with nucleic acids (DNA, RNA) a C₄-cycloaddition of furocoumarins to the 5.6-double bond of pyrimidine bases takes place. The simple photoadduct furocoumarin-pyrimidine base can be split by reirradiation at wavelengths shorter than 334 nm. Reactivation of bacterial cells photodamaged by psoralen (365 nm) was tried experimentally. However, reirradiation at shorter wavelengths and with visible light of the psoralen-inactivated bacterial cells was without any effect. The inability of the shorter wavelengths to repair this photodamage was probably due to a filter effect of DNA for such wavelengths, as shown by experiments on a DNA-psoralen combination. On the other hand the observed ability of psoralen to form inter-strand cross-linkages in the photoreaction with DNA may be significant for explaining the absence of photoreactivation when the inactivated bacterial cells are irradiated with visible light.     

Studies on the reactivation of bacteria photodamaged by an angular furocoumarin: Angelicin

Summary The angelicin-thymine photoadduct formed by irradiation (365 nm) of an aqueous solution of angelicin and tritiated thymine was isolated by preparative paper chromatography. Reirradiation of this photoadduct at wavelengths shorter than 334 nm splits the adduct, forming again the two parent compounds. A DNA-angelicin combination (8.30 g angelicin per mg of DNA) was prepared by irradiating (365 nm) an aqueous solution of DNA with³H-angelicin. Reirradiation of this combination at wavelengths shorter than 312 nm releases³H-angelicin.The above mentioned conditions were employed to reactivate the photodamaged bacterial cells by angelicin. No reactivation was observed at shorter wavelengths; on the contrary, the lethality was higher after reirradiation. We conclude therefore, that the damage produced directly by the shorter wavelength radiations (formation of pyrimidine dimers) is greater than the small repair produced under our experimental conditions.Reirradiation of bacterial cells with visible light is a condition which activates the photoreactivating enzymes, which are able to provoke the cleavage of pyrimidine dimers. The inability to repair the photodamage caused by furocoumarins under these conditions suggests that this enzyme is highly specific for pyrimidine dimers. Though in both cases,i.e. pyrimidine-pyrimidine and pyrimidine-furocoumarine dimers a cyclo-butane ring is involved, the latter is not recognized by the photoreactivating enzyme.     

The light-dependent cytotoxicity of benzo[a]pyrene: effect on human erythrocytes, Escherichia coli cells, and Haemophilus influenzae transforming DNA

In vitro, the photodynamic compound benzo[a]pyrene (BAP) generates singlet oxygen efficiently when irradiated in organic solvents. It also photogenerates superoxide anion radical in water and can act as a photoreducing agent in the absence of oxygen. In vivo, the hemolysis of human erythrocytes, the inactivation of Escherichia coli cells representing a series of strains differing in excision repair and catalase proficiency, and the inactivation of Haemophilus influenzae transforming DNA activity were used to characterize the phototoxicity of BAP in the presence of near-UV light (290-400 nm). The results are consistent with BAP behaving as a photosensitizer that generates both superoxide and singlet oxygen, and that damages chiefly membranes. DNA does not seem to be a major target in the phototoxic reactions investigated.     

Inactivation of adenosine 5''-triphosphate synthesis and reduced-form nicotinamide adenine dinucleotide dehydrogenase activity in Escherichia coli by near-ultraviolet and violet radiations.

Near-ultraviolet (near-UV) light (300 to 380 nm) is a significant component of sunlight and has a variety of effects on biological systems. The present work is an attempt to identify chromophores (molecular absorbers of light) and targets (critical damaged molecules) for inhibition of adenosine triphosphate (ATP) synthesis in Escherichia coli by near UV. The fluence of 334 nm required for 37% survival of net ATP synthesis (F37) in E. coli AB2463 in succinate medium is 140 kJ/m2. The action spectrum for this inactivation is almost structureless, exhibiting a smooth transition from high efficiency at 313 nm to low efficiency at 405 nm. The action spectrum for inhibition of net ATP synthesis is consistent with the chromophore being either ubiquinone Q-8 or vitamin K2. The fluence required is consistent with ubiquinone Q-8 also being a target molecule. The activity of reduced nicotinamide adenine dinucleotide dehydrogenase in extracts of E. coli B is also inactivated by near UV and shows an F37 of about 40 kJ/m2. The action spectrum for this effect is quite structureless; it shows high efficiency at 313 nm and low efficiency at 435 nm. The data do not suggest a target molecule for this action, although it is possible that ubiquinone Q-8 absorbs the near-UV energy and then passes it on to some other target molecule. The data further indicate that inactivation of the oxidative phosphorylation system is not a primary factor in near-UV-induced growth delay in E. coli.     

Biofouling control in water by various UVC wavelengths and doses

UV light irradiation is being increasingly applied as a primary process for water disinfection, effectively used for inactivation of suspended (planktonic) cells. In this study, the use of UV irradiation was evaluated as a pretreatment strategy to control biofouling. The objective of this research was to elucidate the relative effectiveness of various targeted UV wavelengths and a polychromatic spectrum on bacterial inactivation and biofilm control. In a model system using Pseudomonas aeruginosa, the inactivation spectra corresponded to the DNA absorption spectra for all wavelengths between 220 and 280 nm, while wavelengths between 254 nm and 270 nm were the most effective for bacterial inactivation. Similar wavelengths of 254-260-270 nm were also more effective for biofilm control in most cases than targeted 239 and 280 nm. In addition, the prevention of biofilm formation by P. aeruginosa with a full polychromatic lamp was UV dose-dependent. It appears that biofilm control is improved when larger UV doses are given, while higher levels of inactivation are obtained when using a full polychromatic MP lamp. However, no significant differences were found between biofilms produced by bacteria that survived UV irradiation and biofilms produced by control bacteria at the same microbial counts. Moreover, the experiments showed that biofilm prevention depends on the post-treatment incubation time and nutrient availability, in addition to targeted wavelengths, UV spectrum and UV dose.     

Control of sensitivity to inactivation by H2O2 and broad-spectrum near-UV radiation by the Escherichia coli katF locus.

Mutations in the Escherichia coli katF gene (hydroperoxidase II) result in sensitivity to inactivation by H2O2 and broad-spectrum near-UV (NUV; 300 to 400 nm) radiation. Another mutation, nur, originally described as conferring sensitivity to inactivation by broad-spectrum and monochromatic NUV, also confers sensitivity to inactivation by H2O2. Genetic analysis via transduction suggests that the nur mutation allele of the katF locus. As previously reported for broad-spectrum and monochromatic NUV wavelengths, the sensitivity of a particular strain to H2O2 inactivation is also independent of the recA and uvrA alleles. Extracts of nur and katF strains lack catalase (hydroperoxidase II) as revealed by polyacrylamide gels stained for such activity, which is consistent with the genetic results.