Search

We have compared the induction of apoptosis and cytokine release by UVB and γ-radiation in primary (untransformed) and in two immortalized human epithelial/keratinocyte cell lines, HaCaT and KB (KB is now known to be a subline of the ubiquitous keratin-forming tumour cell line HeLa and we therefore designate it HeLa-KB). In both the primary and the immortalized cell lines apoptosis and release of the inflammatory cytokine interleukin-6 are induced rapidly following UVB irradiation. In contrast, only the immortalized cells undergo apoptosis and release interleukin-6 after γ-irradiation and here the onset of apoptosis and cytokine release are delayed. The same distinction between primary and immortalized cells was observed when double-strand breaks were induced with the anticancer drug mitoxantrone, which stabilizes topoisomerase II-cleavable complexes. We suggest that immortalization may sensitize keratinocytes to the apoptogenic effect of ionizing radiation or mitoxantrone by deregulating normal cell cycle checkpoints. In both human keratinocytes and fibroblasts, cell killing, as assayed by loss of colony-forming ability, is not coupled to apoptosis. Immortalization increases resistance to γ-radiation killing but sensitizes to apoptosis. In contrast, although immortalization also sensitizes to UVB-induced apoptosis, it does not affect UVB-induced cell killing. Apoptosis unambiguously indicates death at the single cell level but clonal cell survival integrates all the cellular and genetic processes which prevent or permit a scorable clone to develop.

In January 1993 the oil tanker Braer ran aground in the Shetland Islands, Scotland. Approximately 80,000 tons of crude oil were released. Exceptionally high winds caused extensive pollution and exposure of the local population to crude oil. We describe the study which was immediately set in place to examine the exposed population for evidence of genotoxic exposure. Blood samples were taken and primary DNA damage was measured in the mono-nuclear cell fraction by the butanol modification of the ³²P-postlabelling method. Mutation was measured at the hprt locus in T lymphocytes. No evidence of genotoxicity was obtained for either end point, but nevertheless, we believe that useful lessons were learnt, which should be incorporated into the design of future studies: (1) A rapid response is essential, and even if sufficient funds are not immediately available, it is still worth attempting to obtain samples quickly and use cryopreservation, also to attempt to estimate exposure. (2) Adequate numbers of volunteers must be sought, together with enough controls, not just to allow meaningful analysis but to overcome loss of samples and failure of things to go according to plan. (3) Points concerning laboratory practice include: (i) samples should be coded, (ii) clearly defined and proven protocols should be used, (iii) irreplaceable samples should not be used for method development, (iv) should a problem become apparent during the study, work on such samples should cease immediately until the problem is solved, (v) all critical experimental components should be pretested against a laboratory standard. (4) The study design should include replicate experiments to monitor experimental variability and reproducibility, as well as internal standards and cryopreserved “in house” samples. Care must be taken that samples from any one exposure group are spread between a number of independent experiments and that each experiment includes samples from a number of exposure groups. (5) A computerised data base should be maintained with full details of experimental variables, donor attributes, and raw data so that any contribution of experimental artefacts to “outlier” results can be monitored. (6) Because of the nature of the statistical variation for many environmental genotoxicity end points, only a large-scale study is likely to be capable of yielding useful information. Environ. Mol. Mutagen. 30:97–111, 1997. © 1997 Wiley-Liss, Inc.

A method for the solution of second and higher order systems which may contain non-linear or time-variant elements is presented. These elements may be included in the forward or feedback paths of multi-loop systems as suggested in Part 1 of this contribution. No advanced mathematics are required.

In January 1993 the oil tanker Braerran aground in the Shetland Islands, Scotland. Approximately 80,000 tons of crude oil were released. Exceptionally high winds caused extensive pollution and exposure of the local population to crude oil. We describe the study which was immediately set in place to examine the exposed population for evidence of genotoxic exposure. Blood samples were taken and primary DNA damage was measured in the mono‐nuclear cell fraction by the butanol modification of the 32P‐postlabelling method. Mutation was measured at the hprtlocus in T lymphocytes. No evidence of genotoxicity was obtained for either end point, but nevertheless, we believe that useful lessons were learnt, which should be incorporated into the design of future studies: (1) A rapid response is essential, and even if sufficient funds are not immediately available, it is still worth attempting to obtain samples quickly and use cryopreservation, also to attempt to estimate exposure. (2) Adequate numbers of volunteers must be sought, together with enough controls, not just to allow meaningful analysis but to overcome loss of samples and failure of things to go according to plan. (3) Points concerning laboratory practice include: (i) samples should be coded, (ii) clearly defined and proven protocols should be used, (iii) irreplaceable samples should not be used for method development, (iv) should a problem become apparent during the study, work on such samples should cease immediately until the problem is solved, (v) all critical experimental components should be pretested against a laboratory standard. (4) The study design should include replicate experiments to monitor experimental variability and reproducibility, as well as internal standards and cryopreserved “in house” samples. Care must be taken that samples from any one exposure group are spread between a number of independent experiments and that each experiment includes samples from a number of exposure groups. (5) A computerised data base should be maintained with full details of experimental variables, donor attributes, and raw data so that any contribution of experimental artefacts to “outlier” results can be monitored. (6) Because of the nature of the statistical variation for many environmental genotoxicity end points, only a large‐scale study is likely to be capable of yielding useful information. Environ. Mol. Mutagen. 30:97–111, 1997. © 1997 Wiley‐Liss, Inc.

SummaryAtaxia-telangiectasia (A-T) cells are hypersensitive to the lethal effects of ionizing radiation and fail to inhibit DNA synthesis following radiation exposure. A cell line derived from an A-T line following DNA-mediated gene transfer has normal radiation sensitivity, but the kinetics of DNA synthesis after γγγ-irradiation are similar to those of A-T cells.

We have compared cell killing following 60Co gamma irradiation in 22 primary human fibroblast strains, nine SV40-immortalized human fibroblast lines and seven SV40-transformed pre-crisis human fibroblast cultures. We have examined material from normal individuals, from ataxia-telangiectasia (A-T) patients and from A-T heterozygotes. We have confirmed the greater sensitivity of A-T derived cells to gamma radiation. The distinction between A-T and normal cells is maintained in cells immortalized by SV40 virus but the immortal cells are more gamma radiation resistant than the corresponding primary fibroblasts. Cells transformed by plasmids (pSV3gpt and pSV3neo) expressing SV40 T-antigen, both pre- and post-crisis, show this increased resistance, indicating that it is expression of SV40 T-antigen, rather than immortalization per se which is responsible for the change. We use D0, obtained from a straight line fit, and [image omitted], estimated from a multitarget curve, as parameters to compare radiosensitivity. We suggest that both have their advantages; D0 is perhaps more reproducible, but [image omitted] is more realistic when comparing shouldered and non-shouldered data.

A method is described for determining the radiation sensitivity of dividing human T-lymphocytes in long-term culture. The results are fitted to a single-hit multi-target model. For cobalt-60 gamma radiation D0 values of cells from five normal individuals range from 0·99 to 1·37 Gy with an overall D0 of 1·09 Gy, and the extrapolation numbers range from 1·20 to 1·71 with an overall extrapolation number of 1·42.

The repair of potentially lethal damage following treatment by gamma radiation was investigated in human fibroblasts held in a non-cycling state by maintenance in a medium containing 0·5 per cent foetal calf serum. Variation in their capacity to repair PLD was noted between three normal cell strains. A failure to repair PLD in ataxia-telangiectasia cells (AT5BI) was confirmed. In three cell strains which were intermediate between normals and A-T cells in their sensitivity, XP3BR, 46BR and GM1142, a limited capacity for the repair of PLD was observed. Two other cell strains, 47BR and 67BR, which showed little if any hypersensitivity could be clearly distinguished from normals after a 24 hour period for the repair of PLD. Thus the technique might permit better discrimination between cell strains. One other cell strain, H15617, could be distinguished from normals by proving hypersensitive under all conditions. Here, however, the repair of PLD appeared to be normal

The use of the graphical technique for the solution of complex and non-linear feedback problems has previously been explained. This contribution further extends the method to the solution of systems containing transport delays. No advanced mathematics is required and examples of systems with transport delays in forward and feedback paths are included.

Trichothiodystrophy is a genetic disease which in the majority of cases studied is associated with a deficiency in the ability to repair UV damage in cellular DNA. Three categories of UV response have been identified. In type 1 the response is completely normal, whereas type 2 cells are deficient in excision-repair, with properties indistinguishable from those of XP complementation group D. Type 3 cells have normal survival following UV-irradiation and normal rates of removal of cyclobutane pyrimidine dimer sites. Nevertheless repair synthesis is reduced by 50% in these cell strains and this is associated with a marked reduction in the repair of 6-4 photoproducts from cellular DNA. The present results show that 50% or more of repair synthesis at early times after irradiation of normal primary human fibroblasts is attributable to repair of 6-4 products. They also suggest that repair of cyclobutane dimers is crucial for cell survival.

We have measured clonal survival following gamma-irradiation of unstimulated (G0) T-lymphocytes from 35 donors, of 11 T-lymphocyte cell lines, of six lymphoblastoid cell lines, and of nine primary fibroblast strains for which we have G0 T-lymphocyte material from the same donor. Amongst the G0 lymphocytes we have results from nine normal donors, from eight cord bloods, from seven ataxia-telangiectasia (A-T) patients and from nine A-T heterozygotes. Although there is some variation between samples, G0 T-lymphocytes from normal donors appear to be slightly more radioresistant than T-lymphocyte lines, with a more shouldered survival curve. From our limited sample, lymphoblastoid cell lines appear to be slightly more radiosensitive than T-lymphocytes. The overall radiosensitivity of primary fibroblasts appears to be broadly similar to that of G0 T-lymphocytes. In nine instances, five A-Ts and four A-T heterozygotes, both G0 T-lymphocytes and primary fibroblasts from the same donor were tested. In five cases there was closely similar radiosensitivity in the two cell types, but in four cases there was some discrepancy. Further work, especially with normal donors, will be required in order to establish how reliably radiosensitivity in other cell types can be predicted from that of G0 T-lymphocytes. In all cell types the hypersensitivity of A-T cells was confirmed. Furthermore, the marginally greater sensitivity of A-T heterozygotes, when compared as a group with normals, was confirmed with G0 T-lymphocytes. Our results also suggest a slightly increased radiosensitivity in G0 T-lymphocytes from some, but not all, cord blood samples.

A predictive assay of normal tissue radiosensitivity could benefit 'treatment tailoring' of radiotherapy for certain categories of tumour. The use of present clonogenic cell survival assays for this purpose would be impractical in routine clinical practice because of the lengthy assay time. Fluorescence in situ hybridization (FISH) using whole chromosome probes on metaphases was investigated as a potential substitute. Eight human fibroblast cell strains with a range of radiosensitivities were tested. For each strain, cell survival curves were determined and correlated with chromosome aberrations detected by FISH performed on metaphase cells collected 52 h after irradiation. A whole chromosome probe for chromosome 4 was used for all cell strains. The results revealed an increase in the percentage of metaphases with aberrant chromosomes (translocations and/or breaks) with increasing radiation dose for all strains. For the more radiosensitive cell strains there were relatively more aberrant metaphases for a given radiation dose when compared with fibroblasts from a normal donor. The relationship between surviving fraction and chromosome aberrations showed some variation between strains, but a linear regression for all data showed a highly statistically significant correlation (r = 0·89, p < 0·0005). These results suggest that an assay of chromosome damage using FISH could substitute for the clonogenic assay to predict the radiation sensitivity of human fibroblasts.

The mutant frequency at the hprt locus in circulating T-lymphocytes has been determined in 16 ataxia-telangiectasia (A-T) patients, 19 A-T heterozygotes and 12 A-T sibs. Mutant frequency in the A-T patients is highly significantly elevated as a group, even when the relatively poor cloning efficiency of many of the A-T lymphocyte samples is taken into account. However, within the A-T set, considerable variation in both cloning efficiency and mutant frequency is seen. Cellular radiation sensitivity, measured by clonal survival assays and chromosome breakage, also varies, as do the clinical symptoms of the patients, including the age at which they become wheelchair bound and the severity of their telangiectasia. Here all the information available to us on this group of patients is presented in an attempt to discern if there is any relationship between those cellular characteristics we have observed and the severity of the symptoms and progression of the disease in the patients. Although we feel that it may be relevant that the adult patients in our study all have mutant frequencies that are not highly elevated, insufficient data are available at present to resolve any relationship between the heterogeneous clinical symptoms and cellular responses seen in the A-T patients as a group.

A 14-year-old boy received standard induction chemotherapy for acute lymphoblastic leukaemia followed by standard dose cranial radiation prophylaxis (18 Gy). Severe chemosensitivity and acute radiation reactions occurred and he died at 8 months from late radiation damage. In vitroradiobiological studies of the boy's fibroblasts in culture demonstrated an enhanced radiosensitivity indistinguishable from ataxia-telangiectasia (A-T) cells. However, unlike A-T cells, DNA synthesis following irradiation was inhibited in a normal manner. This patient represents yet another example of extreme radiosensitivity, and the possibility of clinical prediction in the future is discussed.

The linear-quadratic model describes cell killing by radiation as due to two processes defined by the linear (alpha) component and by the quadratic (beta) component. As alpha and beta are interdependent, it is difficult to evaluate accurately the alpha component (which characterizes the intrinsic radiosensitivity). It has been suggested that irradiation at low dose-rate (around 1cGy min) allows the disappearance of the beta component and thus gives a direct measure of alpha. The present results verify this hypothesis with plateau phase cells. The survival of five human fibroblast cell lines in exponentially growing and density-inhibited, confluent cultures maintained at 37 C following exposure to 60Co gamma-rays at dose rates of 0.33-100 cGy min followed by delayed plating (only for plateau phase cells) was monitored. Three of these cell lines are considered to be 'normal' and two are derived from hypersensitive individuals. The mean inactivation doses (D) of the five cell lines for acute doses with immediate plating were 173, 163, 136, 107 and 67 cGy. (D) increased with delayed plating recovery for 4 of the 5 cell lines and the survival of the 5 cell lines increased after low dose-rate exposure (1cGy min) without altering the ranking. The differences between cell lines (absolute values of (D) increased with decreasing the dose-rate. Analysis of the survival curves with the General Linear Quadratic (GLQ) model gave repair half-times for each cell line which were not correlated with the intrinsic radiosensitivities. Surprisingly, the alpha component decreased with decreasing dose-rate for all 5 cell lines (only in plateau phase). Thus low dose-rates do not allow direct measurement of the alpha component; the decrease in alpha could be interpreted as adaptive radioresistance.

A technique for the solution of non-linear feedback problems is presented. The method is graphical and involves no advanced mathematics. It is easily understood and the manual solution of many problems is feasible although the use of a digital computer must be resorted to for more complex systems.

Cells cultured from most patients suffering from the sunlight-sensitive hereditary disorder xeroderma pigmentosum are defective in the ability to excise ultraviolet light (UV)-induced pyrimidine dimers from their DNA. There is, however, one class of these patients whose cells are completely normal in this excision repair process. We have found that these cells have an abnormality in the manner in which DNA is synthesized after UV-irradiation. The time taken to convert initially low-molecular-weight DNA synthesized in UV-irradiated cells into high-molecular-weight DNA similar in size to that in untreated cells is much greater in these variants than in normal cells. Furthermore, this slow conversion of low to high-molecular-weight newly synthesized DNA is drastically inhibited by caffeine, which has no effect in normal cells. Two cell lines from classes of xeroderma pigmentosum that are defective in excision-repair show intermediate effects, with regard to both the time taken to convert newly synthesized DNA to high molecular weight and the inhibition of this process by caffeine.

The levels of either sister chromatid exchanges or chromosome breakage (as assayed by the frequency of micronuclei) in cord blood lymphocytes from a total of 62 babies were analysed. The exposure of these babies to ultrasound during gestation was determined retrospectively from medical records and the data reclassified accordingly. No correlation was seen between the levels of chromosome breakage, as measured by these two end-points, and exposure to ultrasound.

A fibroblast cell strain, 46BR, derived from an immunodeficient patient is hypersensitive to the lethal effects of a wide range of DNA-damaging agents. It is also defective in strand-break rejoining after treatment with dimethyl sulfate and UV light. The present study shows that the cells have a defect in joining Okazaki-type fragments during DNA replication, supporting the interpretation that the basic defect is in ligation of DNA strands. The baseline level of sister chromatid exchange is slightly higher than in normal cells but it does not approach that of Bloom's syndrome or dyskeratosis congenita cells. Sensitivity to the induction of sister chromatid exchange and the hypersensitivity to the lethal effects of a set of DNA-damaging agents are correlated, implying that the basic defect influences both end points in a similar manner. No 6-thioguanine-resistant mutants could be induced by either gamma- or UV-irradiation in these cells, suggesting that error-prone repair pathways for damage induced by these agents may contain a common ligation step in human cells.

Plasmids containing the bacterial gpt gene under control of the simian virus 40 promoter were transfected into a simian virus 40-transformed human fibroblast line. Two transfectants, E2 and C10, which contain stably integrated single copies of the gpt gene, were isolated. These two lines produce Gpt- variants spontaneously with a frequency of about 10(-4). We carried out a detailed molecular analysis of the spectrum of alterations which gave rise to the Gpt- phenotype in these variants. DNA from 14 of 19 Gpt- derivatives of one of the cell lines (E2) contains deletions or rearrangements of gpt-containing sequences. In four of the remaining five lines, the Gpt- phenotype was correlated with reduced levels of expression rather than with changes in the gross structure of the gpt gene, and it was possible to reactivate the gpt gene. In one Gpt- line, gpt mRNA was present at normal levels, but no active enzyme was produced. Spontaneous Gpt- derivatives of the other cell line (C10) produced a completely different spectrum of alterations. Very few deletions were found, but several derivatives contained additional extrachromosomal gpt sequences, and, remarkably, in two other Gpt- lines, gpt-containing sequences were amplified more than 100-fold. The phenotypes of the majority of the Gpt- derivatives of C10 could be attributed to alterations in gene expression caused by methylation.

Genomic DNA from normal human or mouse cells was transfected together with the selectable marker gpt into the simian virus 40-transformed ataxia telangiectasia fibroblast line, AT5BIVA. From a series of experiments involving over 400000 clones selected for the gpt marker, one unambiguously radiation-resistant clone (clone 67) was recovered following selection with repeated cycles of gamma irradiation. The normal level of radiation resistance of clone 67 has been maintained for at least 11 months in the absence of further selection by radiation. The resistant clone contains one copy of the gpt gene. Its DNA synthesis following gamma-irradiation is inhibited to an extent intermediate between that of ataxia telangiectasia and normal cells. Three out of four thioguanine-resistant derivatives of clone 67 have either lost or do not express the gpt sequence and show almost the same sensitivity to gamma irradiation as the original AT5BIVA line. This suggests that the radiation resistance of clone 67 may be linked to the gpt sequence and may have arisen as a consequence of the transfection, rather than as the result of an independent mutation to radiation resistance.

We have compared the γ-irradiation survival of G0 peripheral blood lymphocytes from 18 neonatal cord blood samples in a cloning assay with results from 21 controls (age range 1-65 years and consisting of 20 adults and one child). Using mean inactivation dose as the discriminating parameter, the cord blood cells showed a significantly greater radiosensitivity (mean inactivation dose for pooled data = 1·54 Gy) than the normal controls (mean inactivation dose for pooled data = 1·90 Gy, p < 0·001). These results confirm and extend earlier work suggesting that T-lymphocytes in newborn children are more radiosensitive than normal controls, and this may have implications for the radiation protection of the unborn child.

Skin and blood samples were obtained from 34 donors, for whom there was no indication of abnormal radiosensitivity. From these, in 33 cases both fibroblast and T-lymphocyte cultures were obtained and in 26 cases at least three fibroblast and at least two G0 (resting) T-lymphocyte survival assays were possible. Within this set of results, differences in radiosensitivity between donors were significant for fibroblasts but not T-lymphocytes, although the range of radiosensitivity was similar for the two cell types (D¯0·90-1·68 Gy for fibroblasts; 1·26-2·15 Gy for T-lymphocytes). Furthermore, there was little evidence for a correlation in radiosensitivity between the two cell types. These results suggest limitations in the predictive value of conventional measurement of cell survival.

Gamma radiation sensitivities of continuous cell lines from nine human tumours were measured, comparing four derived from transitional cell carcinomas of the bladder with five from non-seminomatous germ cell tumours of the testis. The testicular cells were significantly more radiosensitive than the bladder cells, corresponding to the response to therapy of these tumour types in patients. These observations indicate that radiosensitivity is retained in vitro and is an inherent property of the testicular tumour cells. These gamma radiation sensitivities were compared with those of SV40-transformed fibroblasts derived from a normal individual and one with the heritable disease, ataxia-telangiectasia (A-T). The bladder cells had gamma radiation sensitivities similar to that of the SV40-transformed normal line. The testicular cells were hypersensitive to gamma radiation, although not as sensitive as the SV40-transformed A-T line. A-T cells, unlike those derived from normal individuals, continue to synthesize DNA at a normal rate following radiation exposure, prompting a comparison of the kinetics of DNA synthesis in three bladder and three testicular tumour cell lines. One of the bladder and two testicular lines showed a reduced inhibition when compared to the other tumour cell lines and the SV40-transformed normal line. Thus there was no clear association between DNA synthesis inhibition and radiosensitivity.

We have previously shown that non-cycling (unstimulated) human lymphocytes from normal donors show extreme hypersensitivity to UV-B irradiation, and are killed by an excisable lesion which is not a pyrimidine dimer or 6-4 photoproduct. In this paper we show that addition of the 4 deoxyribonucleosides to the medium, each at 10−5M, substantially increased the survival of non-cycling normal human T-lymphocytes following UV-B irradiation and substantially reduced the frequency of excision-related strand breaks in human mononuclear cells. Addition of ribonucleosides to the medium did not enhance excision-break rejoining. The survival of fibroblasts, of cycling T-lymphocytes and of unstimulated xeroderma pigmentosum T-lymphocytes was not enhanced by deoxyribonucleosides. This suggests that the hypersensitivity is due to reduced rejoining of excision breaks as a consequence of low intracellular deoxyribonucleotide pools and that it can be redressed by supplementation of the medium with deoxyribonucleosides or upregulation of ribonucleotide reductase following mitogen stimulation. We suggest that UV-B forms an additional DNA lesion which is not a pyrimidine dimer or 6-4 photoproduct, which is relatively common, and at which incision is particularly efficient. In fibroblasts, repair of this lesion is completed with high efficiency, whereas in normal unstimulated T-lymphocytes, rapid incision exacerbates the effects of the reduced rate of strand rejoining and leads to cell death.

The operation of a pumped storage plant immediately after it has come on load is complex. Here the analysis of a typical plant to steady state conditions following the loss of base load generation is detailed together with the method employed. The substantial non-linearities of the various elements of the system are accounted for and the importance of a shaping network leading to the guide vane actuator is underlined. The method chosen for the study is the much neglected graphical co-ordinated technique which has been developed to analyse multiloop systems which can include non-linearities, time-varying elements and transport delays to advantage over more conventional methods.

Interaction between ultra-violet and gamma irradiation for survival was observed with Aspergillus nidulans. It was suggested that this interaction provided a demonstration of repair mechanisms in this organism. In contrast to previous reports no interaction was observed in mutation induction for resistance to 8 aza-guanine or 6-methyl purine.

A cytoplasmic mutant of Aspergillus nidulans produced two classes of conidia, mutant and non-mutant. The mutant conidia were more resistant to u.v. than the non-mutant, and this difference was shown to be attributable entirely to the cytoplasm. There were no differences between the two classes on gamma-irradiation.It was not practicable to examine the possibility of differences in a cytoplasmic target in this system, and an attempt was made to test whether cytoplasmically determined differences in repair capacity might account for the different u.v. sensitivities. The chemical inhibitors of repair processes, acriflavine and caffeine, which are effective in some bacteria did not reduce but enhanced survival in the fungus. Some other treatments which enhanced survival also delayed germination, and an explanation based on a prolonged period for repair is suggested.

Mammalian cell populations, synchronized by the excess thymidine double block technique, were irradiated in the G2 phase of the mitotic cycle with u.v. or 60Co radiations then pulsed with 3H 5-bromo-2-deoxyuridine. Repair replication was demonstrated in each of the three cell lines investigated after u.v.-irradiation; but no evidence for repair was detected after 60Co-irradiation. The extent of repair did not increase with dose. A correlation between repair replication and unscheduled DNA synthesis was observed.

Chinese hamster cells were exposed to a variety of treatments after gamma-irradiation. When DNA or protein synthesis was inhibited, there was no effect on survival. There was also no effect on survival from a reduction of temperature or the addition of caffeine. Actinomycin D, acriflavine, and ethidium bromide, which have the property of binding to nucleic acids, brought about a reduction in survival. It is suggested that the activity of any enzymes which repair radiation lesions in nucleic acids is impeded in a steric manner by the resulting complexes.

No change in survival of two Chinese hamster cell lines was found when any of the following inhibitors were added in various concentrations to the culture medium after exposure to ultra-violet light: acriflavine, caffeine, theophylline, chloramphenicol, cycloheximide, actinomycin D and hydroxyurea. Acridine orange, puromycin and reduced temperature were tested with one of the cell-lines and were without effect.