Hypersensitivity of human lymphocytes to UV-B and solar irradiation.

T-lymphocytes from three normal human donors, irradiated with broad-spectrum UV-B (peak emission, 312 nm), are 20-fold more sensitive than fibroblasts from four normal donors in a clonogenic assay. We have compared the formation of thymine cyclobutane dimers and pyrimidine-(6-4)-pyrimidone photoproducts following irradiation by UV-C (254 nm) and UV-B and studied killing at doses giving equal dimer formation. UV-B killing of fibroblasts appears to be associated with dipyrimidine photoproduct formation, whereas UV-B killing of lymphocytes is mediated by nondimer damage. Strand breakage following UV-B irradiation measured using the "Comet" assay (single cell gel electrophoresis) reflects this nondimer damage and has kinetics consistent with excisable damage. Lymphocytes from three excision-deficient xeroderma pigmentosum donors show reduced strand breakage and increased killing following UV-B irradiation, compared with lymphocytes from normal donors. We therefore suggest that UV-B kills human lymphocytes by excisable nondimer damage and that xeroderma pigmentosum lymphocytes are defective in its repair. The putative nondimer damage does not appear to be associated with radical attack, and the strand breakage is not a manifestation of apoptosis. A 1-min exposure of human lymphocytes in vitro to natural sunlight is sufficient to produce damage measurable by the Comet assay.

Immune function, mutant frequency, and cancer risk in the DNA repair defective genodermatoses xeroderma pigmentosum, Cockayne's syndrome, and trichothiodystrophy.

There is evidence for defective DNA repair in xeroderma pigmentosum, Cockayne's syndrome, and trichothiodystrophy, but for increased cancer risk only in xeroderma pigmentosum. Natural and adaptive immune surveillance and mutant frequency to 6-thioguanine resistance in circulating T-lymphocytes were studied in five patients with xeroderma pigmentosum, two with Cockayne's syndrome, and one with trichothiodystrophy. Forty-eight-hour cutaneous hypersensitivity responses to recall antigens excluded anergy and circulating CD3+, CD4+, CD8+, and CD16+ cell numbers were within normal limits in all patients tested, as were proliferative lymphocyte responses to PHA, except in the trichothiodystrophy patient. Proliferative responses to recall antigens (PPD, SKSD, and Candida) showed that all patients responded to one or more antigens. Direct natural killer cytotoxicity measured against the human erythromyeloid leukaemia cell line K562 using a 4-h 51Cr release assay was significantly reduced in xeroderma pigmentosum (specific cytotoxicity less than mean +/- SD greater than 17.4 +/- 9.4 per cent, with effector:target cell ratio of 50:1) compared to normal controls (45.8 +/- 17.8), but normal in Cockayne's syndrome and trichothiodystrophy. Generation of lymphokine activated killer cell activity was normal in the two xeroderma pigmentosum lines tested. The mutant frequency in the xeroderma pigmentosum donors was significantly increased (p less than 0.01) and was elevated in the two Cockayne's syndrome donors, taking age into account. No mutants were observed from the single trichothiodystrophy donor. These findings suggest that reduced natural killer cell activity may contribute to the greatly increased susceptibility to skin cancer in xeroderma pigmentosum.

Lack of evidence for an association between the frequency of mutants or translocations in circulating lymphocytes and exposure to radon gas in the home.

Radon measurements in the living room and main bedroom of 41 houses in the town of Street, Somerset, England have been made. Exposure levels, weighted using the formula of the UK National Radiological Protection Board, of 19-484 Bq m-3 (about half > 100 Bq m-3) were found. Blood samples were obtained from a total of 66 occupants in these homes, and the frequency of genetic alterations in lymphocytes was estimated using two different end points. Gene mutations at the hypoxanthine guanine phosphoribosyl transferase locus were determined in T lymphocytes for 65 subjects using a clonal assay, and the frequency of the BCL-2 t(14;18) translocation, a chromosomal event associated with leukemia/lymphoma, was estimated in lymphocytes using a polymerase chain reaction-based technique for 64 subjects. In neither case was a significant correlation with radon levels in the home found, in contrast to our earlier observation with a smaller series.

Correlated mutagenesis of bcl2 and hprt loci in blood lymphocytes.

In vivo measurement of human somatic mutations may be a valuable biodosimeter of exposure to carcinogens and of cancer risk. We have surveyed translocations at the bcl2 locus in B lymphocytes, and mutations at hprt in T lymphocytes, in 120 individuals with varying exposure to radon and cigarette smoke. bcl2 t(14:18) translocation is the commonest chromosomal alteration observed in non-Hodgkins lymphoma (NHL). We observed a significantly larger range of bcl2 translocation frequency (range: 0-372 x 10(-6), median: 1.9 x 10(-6)) than of hprt mutation frequency (range: 0-76.4 x 10(-6), median: 11.1 x 10(-6)), which is likely the result of clonal proliferation of deathless B cell mutants. We observed that the frequencies of these two distinct lymphocytic mutations are significantly correlated. Although some of the correlated variation is explained by age, a significant correlation of bcl2 mutagenesis persists after age adjustment. Correlated mutagenesis at distinct loci in distinct cell types could be explained by the existence of a mutator phenotype or by variation in exposure to environmental mutagens. NHL is commoner in men than in women, and our data indicate a trend toward higher bcl2 mutagenesis in males than females. There is mounting epidemiological evidence for a worldwide increase in NHL, which may have an environmental basis; molecular epidemiological analysis of bcl2 mutagenesis in exposed populations might be especially relevant to the identification of putative environmental causes. Given the relative ease of the bcl2 assay versus the hprt assay, and the consistency with which data are reproduced from laboratory to laboratory, it is likely that the bcl2 assay will be soon added to the array of assays used in human mutational surveillance.

Biomonitoring of possible human exposure to environmental genotoxic chemicals: lessons from a study following the wreck of the oil tanker Braer.

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 mononuclear 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.

Correlation of UVC and UVB cytotoxicity with the induction of specific photoproducts in T-lymphocytes and fibroblasts from normal human donors.

By using specific monoclonal antibodies in situ and a computer-assisted image analysis system we have determined the relative induction of cyclobutane dimers, (6-4) photoproducts and Dewar isomers in human mononuclear cells and fibroblasts following irradiation with UVC, broad-spectrum UVB and narrow-spectrum UVB. The lamps produced these lesions in different proportions, with broad-spectrum UVB inducing a greater combined yield of (6-4) photoproducts and Dewar isomers per cyclobutane dimer than UVC or narrow-spectrum UVB. The relative induction ratios of (6-4) photoproducts compared to cyclobutane dimers were 0.15, 0.21 and 0.10 following irradiation with UVC, broad- or narrow-spectrum UVB, respectively. Although Dewar isomers were induced by UVC, their relative rate of formation compared to cyclobutane dimers was significantly greater after irradiation with either broad-spectrum or narrow-spectrum UVB. These values were 0.001, 0.07 and 0.07, respectively. With each lamp source, we have determined the survival of normal human T-lymphocytes and fibroblasts at fluences, which induce equivalent yields of cyclobutane dimers, (6-4) photoproducts or (6-4) photoproducts plus Dewar isomers. Killing of fibroblasts appears to be associated with (6-4) photoproduct formation, whereas killing of T-lymphocytes seems to be mediated by combined (6-4) plus Dewar yields. These results emphasize the need to study the biological effects of UVB because cellular responses may be different from those following UVC irradiation.

Relationship of cataract to radiation sensitivity.

Considerable exposure to radiation always causes posterior subcapsular cataract (PSC). This investigation was conducted to ascertain whether cellular hypersensitivity to radiation may be identified as a possible cause of cataract in persons exposed to low levels of radiation. Patients were studied in whom PSC had followed probable exposure to low levels of radiation or in whom PSC had developed before the age of 60 without known exposure. The patients with cataract were compared with age and sex matched controls without cataract. Radiation sensitivity was estimated by measuring clonal growth of skin fibroblasts and peripheral blood lymphocytes after exposure to graded doses of radiation and by measuring postirradiation reconstruction of separated nuclear material from lymphocytes. The results show variations in the level of radiation sensitivity between the patients, without significant differences from the controls. It is concluded that radiation hypersensitivity, as tested by the methods used in this study, is not normally associated with the development of posterior subcapsular cataract.

Comparative human cellular radiosensitivity: IV. The increased sensitivity of human neonatal cord blood lymphocytes to gamma-irradiation compared with lymphocytes from children and adults.

We have compared the gamma-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 less than 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.

Comparative human cellular radiosensitivity: III. Gamma-radiation survival of cultured skin fibroblasts and resting T-lymphocytes from the peripheral blood of the same individual.

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.

Elevated hprt mutant frequency in circulating T-lymphocytes of xeroderma pigmentosum patients.

The mutant frequency to 6-thioguanine resistance in circulating T-lymphocytes from 10 xeroderma pigmentosum patients (including complementation groups D and G and XP variants) has been determined. A highly significantly elevated frequency was observed, compared to age-matched, non-smoking control donors (x 2.1-fold higher than the mutant frequency in normal control donors, adjusted for age and cloning efficiency, p less than 0.001). The mutant frequency of 5 XP heterozygotes was in the normal range, when age, smoking habit and log cloning efficiency were taken into account. A number of possible factors which may account for the elevated mutant frequency seen in the XP donors (including an elevated spontaneous mutation rate, UV mutagenesis of the T-cells as they pass through the skin, an effect of environmental mutagens such as tobacco smoke, or as a consequence of immune deficiency) are discussed.

Effect of deoxyribonucleosides on the hypersensitivity of human peripheral blood lymphocytes to UV-B and UV-C irradiation.

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(-5) M, 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.

Effect of diet and vitamin C on DNA strand breakage in freshly-isolated human white blood cells.

We have measured DNA strand breaks induced by ionising radiation in nucleated cells from freshly isolated whole blood from normal human subjects. Samples were taken after subjects had fasted overnight and again 1 h after they had eaten breakfast in combination with approximately 35 mg/kg vitamin C. Damage was measured by single cell gel electrophoresis (the 'comet' assay), in which DNA single strand breaks generate a comet tail streaming from the nucleus. In repeat experiments on 6 subjects a reduction in DNA damage, as indicated by a highly significant decrease in overall comet length, was observed following vitamin C ingestion, both in the unirradiated control blood samples and in the dose response to ionising radiation damage. In addition, consistent differences in dose response between individual subjects were found. The peak effect was 4 h after intake of food and vitamin C. An effect was also seen with vitamin C alone and after breakfast without additional vitamin C. Protection against strand breakage was also seen in Ficoll-separated mononuclear cells but evidence was not obtained for protection of separated, mitogen stimulated T-lymphocytes either against ionising radiation cell killing in a clonal assay, or against clastogenicity assessed by micronucleus formation following one cell division. Exposure of separated lymphocytes in vitro to vitamin C, at doses greater than 200 microM, did not offer protection but induced strand breakage. Our results raise the possibility that variation in normal diet may not only affect susceptibility to endogenous oxidative damage, but may affect some responses of the individual to radiation.

Protective effect of deoxyribonucleosides on UV-irradiated human peripheral blood T-lymphocytes: possibilities for the selective killing of either cycling or non-cycling cells.

Non-cycling human T-lymphocytes from normal subjects show a 10-fold greater sensitivity than fibroblasts to UV-B (280-315 nm) irradiation from a Westinghouse FS20 lamp, but only a 2.7-fold greater sensitivity to UV-C (254 nm) irradiation. Hypersensitivity is associated with a deficiency in the rejoining of excision breaks. Non-cycling T-lymphocytes have extremely low deoxyribonucleotide pools. Addition to the medium of the four deoxyribonucleosides, each at a concentration of 10(-5) M, substantially increases survival and reduces the persistence of excision-related strand breaks following UV-B or UV-C irradiation (Yew and Johnson (1979) Biochim. Biophys. Acta 562, 240-241; Green et al. (1994) Mutation Res., 315, 25-32). UV-resistance of T-lymphocytes is also increased by stimulating the cells into cycle. The addition of deoxyribonucleosides does not further enhance survival of cycling cells and they do not reach the level of resistance achieved by non-cycling cells in the presence of deoxyribonucleosides. We suggest that two opposing effects are in operation. Cells out of cycle can show increased resistance to DNA damage in the absence of division but they also have reduced deoxyribonucleotide pools, which may limit DNA repair. With UV-B irradiation, the exceptionally low dNTP pools in non-cycling T-lymphocytes cause this second effect to predominate. In contrast, with ionising radiation, which forms highly cytotoxic double-strand breaks, non-cycling human T-lymphocytes are slightly more resistant than fibroblasts. Non-cycling cells such as T-lymphocytes should be especially sensitive to agents which produce a high proportion of read excisable damage, but should show normal resistance to agents which highly toxic lesions. It may be possible by choice of DNA damaging agent and manipulation of cellular deoxyribonucleotide pools, to choose regimes which will selectively kill either cycling or non-cycling cells and to improve the efficacy of standard therapeutic procedures. Conditions favouring selective killing of non-dividing T-lymphocytes but sparing stem cells may be of value in bone marrow transplantation. Conditions favouring selective killing of dividing cancer cells but sparing non-dividing normal tissue may be of value in cancer therapy.

Correlation of immunoregulatory function with cell phenotype in cord blood lymphocytes.

The strong suppressor activity of cord T lymphocytes contrasts markedly with their mainly CD4 (helper) rather than CD8 (suppressor) phenotype. We studied the phenotype of cord CD3, CD4 and CD8 cells compared to adult cells using the monoclonal antibodies, 2H4, 4B4, and UCHL1. Almost all cord CD4 lymphocytes carried the suppressor-inducer marker 2H4, whereas 4B4+ UCHL1+ helper-inducer cells were virtually absent; CD8 cord cells were also of the 2H4+ 4B4- UCHL1- phenotype. In contrast in adult peripheral blood, half of the T cells, whether CD4 or CD8, were 2H4+ and half 4B4/UCHL1+. The suppressor-inducer phenotype of cord T cells was shown, in parallel functional experiments, to correlate with their enhanced proliferation to lectin and poor production of immunoglobulin and with the ability of cord mononuclear cells to suppress proliferation and immunoglobulin production by adult cells in co-culture experiments. These results indicate that the major imbalance in the cord CD4 subset in favour of 2H4 cells can explain many of the functional differences from adult cells. However, involvement of other cell types, in particular of the monocyte lineage, is necessary to explain other properties of immunocompetent cord cells.

Possible association between mutant frequency in peripheral lymphocytes and domestic radon concentrations.

To investigate whether previously found geographical correlations between leukaemia incidence and exposure to radon are reflected in a detectable mutagenic effect on individuals, the frequency of mutations in the hypoxanthine guanine phosphoribosyl transferase gene (hprt) in peripheral blood T lymphocytes was measured in subjects with known domestic radon concentrations. These concentrations were measured in December, 1989, in houses in Street, Somerset, UK, by passive alpha-track radon detectors. 20 non-smoking subjects aged 36-55 years were selected from the patient list at the local health centre on the basis of the radon concentrations in their homes--the range selected varied by a factor of ten. Blood samples for preparation of T lymphocytes were taken in July, 1990. There was a significant association between the log mutant frequency and radon concentration (t = 3.47, p less than 0.01). A second analysis of a further set of radon measurements (October, 1990, to January, 1991), in both living rooms and bedrooms, and repeated mutant frequency determinations also showed a significant relation, which remained significant even after exclusion of the highest frequency and adjustment for subject's age and cloning efficiency. These data must be regarded as preliminary and further more extensive studies should be done to determine whether the observed association is causal.