Development of a 32P-postlabeling assay for 7-methylguanines in human DNA.

The application of a 32P-postlabeling assay for 7-methylguanines in DNA was studied either by labeling the imidazole ring-opened dinucleotide derivatives or by using strong-anion-exchange column chromatography for the adduct enrichment from normal nucleotides. Data showed that 7-methylguanines can be efficiently labeled as dinucleotides when in vitro methylated DNA was first imidazole ring-opened and then digested to the dinucleotide level with deoxyribonuclease I, snake venom phosphodiesterase, and prostatic acid phosphatase. When using ion exchange chromatography for the adduct enrichment, DNA was digested with micrococcal nuclease and spleen phosphodiesterase. Anion exchange chromatography was applied for 7-methylguanine measurements in white blood cell DNA of healthy nonsmokers (n = 17) and patients (n = 4) treated with the methylating drugs procarbazine and decarbazine. We found that the mean level of 7-methylguanine residues in nonsmokers was 2.5 per 10(7) nucleotides. The corresponding level in the patient samples immediately after the drug treatment was 57 per 10(7) nucleotides.

DNA adducts in experimental cancer research.

The role of DNA adducts in the initiation of cancer is scrutinized in this presentation. Work on the activation of oncogenes, particularly on ras, has provided new evidence to link DNA adducts and tumour formation. Polycyclic hydrocarbons and nitroso compounds can cause activation of ras oncogenes through a defined point mutation. The properties of two DNA binding agents, styrene oxide and cisplatin are discussed. Styrene oxide is a versatile electrophile causing numerous adducts; cisplatin has a high specificity towards guanine-N-7 and cross-link formation. Finally, the relation of specific adducts to carcinogenic potency as defined by TD50 values is investigated. For small alkyl groups, O-alkylation of bases correlates with potency but among others, particularly the bulky carcinogens, 7-alkylguanines appear as correlates of potency. Most such potent agents forming 7-alkylguanines induce depurination and imidazole ring-opening.

Do recorded doses overestimate true doses received by Chernobyl cleanup workers? Results of cytogenetic analyses of Estonian workers by fluorescence in situ hybridization.

Studies of workers who were sent to Chernobyl after the 1986 reactor accident are being conducted to provide a better understanding of the effects of chronic low-dose radiation exposures. A crucial component to these investigations is an accurate assessment of the radiation doses received during the cleanup activities. To provide information on biological measurements of dose, fluorescence in situ hybridization (FISH) with whole-chromosome painting probes has been applied to quantify stable chromosome aberrations (translocations and insertions) among a defined cohort of 4,833 cleanup workers from Estonia. Cytogenetic analysis of 48-h lymphocyte cultures from 118 Estonian cleanup workers (10.3 cGy mean recorded dose; 25 cGy maximum), 29 Estonian population controls and 21 American controls was conducted by three laboratories. More than 258,000 painted metaphases were evaluated. Overall, we observed lower translocation frequencies than has been reported in previous studies using FISH among Chernobyl cleanup workers. In our data, a clear association with increased levels of translocations was seen with increasing age at blood drawing. There was no correlation, however, between aberration frequency and recorded measurements of physical dose or any category of potential high-dose and high-dose-rate exposure such as being sent to Chernobyl in 1986, working on the roof near the damaged nuclear reactor, working in special zones or having multiple tours. In fact, the translocation frequency was lower among the exposed workers than the controls, though not significantly so. To estimate the level of effect that would have been expected in a population of men having an average dose of approximately 10 cGy, blood from six donors was exposed to low-LET radiation, and more than 32,000 metaphases were scored to estimate dose-response coefficients for radiation-induced translocations in chromosome pairs 1, 2 and 4. Based on these results, we estimate that had this group of 118 men received an average whole-body dose of 10-11 cGy, as chronic or acute exposures, an increase in the mean frequency of chromosome translocations of more than 40-65% would have been observed in their lymphocytes compared to findings in nonirradiated controls. In spite of evaluating more than a quarter of a million metaphases, we were unable to detect any increase in the mean, median or range in chromosome aberrations in lymphocyte cultures from a group of Estonian men who took part in the cleanup of the Chernobyl nuclear power site and those who did not. We conclude that it is likely that recorded doses for these cleanup workers overestimate their average bone marrow doses, perhaps substantially. These results are consistent with several negative studies of cancer incidence in Chernobyl cleanup workers and, if borne out, suggest that future studies may not be sufficiently powerful to detect increases in leukemia or cancer, much less distinguish differences between the effects of chronic compared to brief radiation exposures.

Expression of cellular fibronectin and tenascin in the rabbit cornea after excimer laser photorefractive keratectomy: a 12 month study.

An indirect immunohistochemical technique was used to monitor the expression of cellular fibronectin (cFN) and tenascin (TN) in the rabbit cornea after photorefractive keratectomy (PRK) in a 1 year follow up study. Rabbits received a 5.0 D myopic PRK, and were killed 3 days, 1, 3, 6, or 12 months after the operation. In most corneas, secondary epithelial defects appeared after the primary healing (mean 6.3 (SD 1.2) days). Corneal haze appeared a few weeks after PRK and was observed throughout the follow up. Three days after wounding an immunoreaction for cFN was observed as a bright narrow subepithelial line, but no immunoreaction for TN could be seen in the anterior third of the corneal stroma. However, at 1-6 months a similar location of immunoreactions for both cFN and TN was observed. Both were found in the anterior stroma at depths of 30-50 microns. At 12 months, only a trace of cFN immunoreaction but no TN immunoreaction could be discerned. Our results suggest that subepithelial scar tissue contains both cFN and TN up to 12 months.

Distribution of radiation-induced exchange aberrations in human chromosomes 1, 2 and 4.

PURPOSE: To examine the distribution of radiation-induced breakpoints in chromosomes 1, 2 and 4 both in relation to their DNA content and by localization of the breaks along each chromosome. MATERIAL AND METHODS: The work consisted of two studies, one with chromosomal aberrations found in persons after accidental exposure in Estonia in 1994 and another involving aberrations seen in in vitro-irradiated lymphocytes. Localization of breakpoints in painted chromosomes involved in complete exchange-type aberrations was conducted by applying a computerized measuring system on stored image-files. RESULTS AND CONCLUSIONS: The yield of exchanges in chromosomes 1, 2 and 4 in both studies was equal to that expected from their DNA content. In contrast, the breakpoint location of complete exchanges within these chromosomes was not random. Chromosomes 1 and 4 contained more breaks in the middle parts of the p and q arms, whereas breaks were observed more uniformly along chromosome 2. Complete exchanges, however, were very rare in the terminal regions of all three chromosomes, most probably resulting from limitations in the resolution of small painted pieces.

The incidence of cytogenetically abnormal rogue cells in peripheral blood.

PURPOSE: To compare the occurrence of cytogenetically abnormal rogue cells, characterized by a high frequency of chromosome-type aberrations, in people exposed to ionizing radiation and in non-exposed subjects. MATERIALS AND METHODS: Data on rogue cells from a total of nine cytogenetic studies on radiation-exposed populations and controls were collected from three laboratories in the United Kingdom, France and Finland. The studies were conducted on first-division metaphases of peripheral blood lymphocytes. Solid Giemsa-stained, G- or R-banded and FISH chromosome-painted material was included. RESULTS: Rogue cells were found both from controls and from exposed subjects. The highest incidence of these cells was observed in a control group of young trainees (1:400), whereas the lowest incidence of rogue cells (1:36 500) was demonstrated in a follow-up study of people accidentally exposed to high levels of ionizing radiation. Rogue cells were found to be distributed non-randomly among individuals; the highest individual frequency was 1 in 50 analysed metaphases. CONCLUSIONS: The origin of rogue cells is still unclear. The incidence of rogue cells showed a large variability between studies and individuals. No correlation between long-term radiation exposure and the occurrence of rogue cells was demonstrated. Although the presence of rogue cells in astronauts after a 6 month space flight may be attributable to high-LET radiation, the frequencies were not remarkable when compared with those in the other studies in this review.

Chromosomal instability in in vivo radiation exposed subjects.

PURPOSE: To investigate whether delayed chromosomal instability arises in human peripheral T lymphocytes exposed in vivo to gamma-irradiation. MATERIALS AND METHODS: Long-term cultures were established from lymphocytes obtained from subjects involved in the radiological accident in Estonia in 1994. Two individuals exposed to a high dose, one individual with low exposure and one apparently unexposed person were studied. Two Estonian controls not involved with the accident were also analysed. Cells were grown for 6-42 days and chromosomal aberrations were assessed from G-banded metaphases. In addition, FISH chromosome painting analysis was performed on short-term cultures established from whole blood. RESULTS: No obvious sign of chromosomal instability was observed in the in vivo follow-up of the frequencies of chromosomal aberrations in lymphocytes of radiation accident victims performed by the FISH technique (48 h cultures established at different time intervals after the exposure). However, when the lymphocytes were cultured long term in vitro, chromosomal instability was observed. There was no dose-response, and the appearance of chromosomally unstable cells in long-term cultures was also observed in a subject exposed to a dose of less than 0.1 Gy. Moreover, in contrast with previous findings, chromosomal instability was also observed in cells from non-exposed control individuals. The chromosomal changes observed in the controls were less complex than the aberrations in the cultures derived from individuals exposed to high doses. CONCLUSIONS: Chromosomal instability was observed in long-term cultures of donors with in vivo exposure to gamma-radiation. No dose-response was apparent. However, in contrast with previous findings, signs of chromosomal instability were observed also in long-term cultures from non-exposed controls. Further studies are needed to assess possible inter-individual differences in the induction of chromosomal instability.

32P-postlabelling of 7-methyl-dGMP ring-opened 7-methyl-dGMP and platinated dGpdG.

The 32P-postlabelling technique introduced by Randerath and coworkers was used to investigate the efficiency of the phosphorylation reaction by T4 polynucleotide kinase using three synthesized adducts: 7-methyl-dGMP, ring-opened 7-methyl-dGMP and platinated dGpdG. The methylated substrates were detected at sub-fmol sensitivities. 7-Methyl-dGMP was quantitatively phosphorylated at these low concentrations. The efficiency of phosphorylation of the ring-opened product was less (about one order of magnitude) and that of Pt(dGpdG) about three orders of magnitude less. These results show that T4 polynucleotide kinase phosphorylation is an efficient reaction with 7-methyl-dGMP and with ring-opened 7-methyl-dGMP, even though in the latter case longer incubation times may have to be used to boost the reaction towards completion. By contrast, the low level of phosphorylation with Pt(dGpdG) does not appear encouraging for quantitative determination requiring a high sensitivity.

Normal human corneal cell populations evaluated by in vivo scanning slit confocal microscopy.

PURPOSE: To analyze cellular populations in healthy human corneas. METHODS: The study group consisted of 58 eyes of 45 patients with normal corneas. The age distribution was 45 +/- 17 years (mean +/- SD; range, 20-84). Scanning slit confocal microscopy of the central corneas was performed. The images were analyzed visually for cell morphology, and the densities and areas of cells were measured. RESULTS: No statistically significant differences were measured in cell densities or cell areas of any corneal layer between female and male patients (p = 0.22-0.50) nor between right and left eyes (p = 0.16-0.45). The area of superficial epithelial cells was 913 +/- 326 microm2 (mean +/- SD; range, 518-2,112), and the superficial epithelial cell density was 1,213 +/- 370 cells/mm2 (mean +/- SD; range, 473-1,929). The area of basal epithelial cells was 177 +/- 19 microm2 (mean +/- SD; range, 138-242), and the basal epithelial cell density was 5,699 +/- 604 cells/mm2 (mean +/- SD; range, 4,135-7,267). The average apparent keratocyte density was 1,058 +/- 217 cells/mm2 (mean +/- SD; range, 604-1,599) in the anterior stroma, and 771 +/- 135 cells/mm2 (mean +/- SD; range, 493-1,145) in the posterior stroma. The difference in apparent keratocyte densities between the anterior and posterior stroma was statistically significant (p < 0.001). The average endothelial cell area was 334 +/- 51 microm2 (range, 273-553), and the cell density was 3,055 +/- 386 cells/mm2 (mean +/- SD; range, 1,809-3,668). The endothelial cell density had a negative, statistically significant correlation with age (r = -0.68, p < 0.001). The densities of the other corneal cell layers did not have a statistically significant correlation with age. CONCLUSION: In vivo scanning slit confocal microscopy is a useful tool for studying corneal cell populations. Central corneal cell densities were found to decrease significantly with age only in the endothelium. For the first time in human corneas using in vivo confocal microscopy, this study statistically confirms a higher apparent number of keratocytes in the anterior stroma than in the posterior stroma.

In vivo confocal microscopy of Fuchs' endothelial dystrophy.

PURPOSE: The purpose of this study is to analyze in vivo confocal microscopic findings of corneas with Fuchs' endothelial dystrophy. METHODS: Central corneas of 17 eyes of 11 patients aged 41-86 years were examined using in vivo scanning slit confocal microscopy after being diagnosed with Fuchs' endothelial dystrophy. The cellular structure of the corneas was analyzed morphologically and quantitatively and compared to control results from 22 healthy corneas. RESULTS: Bullae were detected in the basal epithelial layer of one eye. Eight of 17 eyes (47%) exhibited an abnormal Bowman's layer: diffuse bright reflection and absence of nerves. Eleven eyes (65%) exhibited abnormal anterior stroma: lacunae and diffuse increased light reflection due to edema. In 12 eyes (71%), lacunae or dark bands 5-20 microm wide against increased background reflection were noted in the posterior stroma. Descemet's membrane was thickened in all eyes. Dark bands were detected in six eyes (35%). Guttae (137-1,231/mm2) 20-40 microm in diameter were found in every endothelial cell layer. The mean endothelial cell count was 1,202 +/- 850 (cells/mm2 +/- SD; range, 0-2,735). There was a positive correlation between endothelial cell counts obtained by specular microscopy and those obtained by confocal microscopy (r = 0.95). CONCLUSION: In vivo confocal microscopic findings of Fuchs' endothelial dystrophy are described for the first time in a series of cases. Pathological changes in Fuchs' dystrophy were detected in all corneal layers, more frequently in the posterior layers. Endothelial cell counts obtained with confocal microscopy were statistically similar to those obtained with standard specular microscopy.

A comparison of gamma and neutron irradiation on Raji cells: effects on DNA damage, repair, cell cycle distribution and lethality.

The Comet assay (microgel electrophoresis) was used to study DNA damage in Raji cells, a B-lymphoblastoid cell line, after treatment with different doses of neutrons (0.5 to 16 Gy) or gamma rays (1.4 to 44.8 Gy). A better growth recovery was observed in cells after gamma-ray treatments compared with neutron treatments. The relative biological effectiveness (RBE) of neutron in cell killing was determined to be 2.5. Initially, the number of damaged cells per unit dose was approximately the same after neutron and gamma-ray irradiation. One hour after treatment, however, the number of normal cells per unit dose was much lower for neutrons than for gamma rays, suggesting a more efficient initial repair for gamma rays. Twenty-four hours after treatment, the numbers of damaged cells per unit dose of neutrons or gamma rays were again at comparable level. Cell cycle kinetic studies showed a strong G2/M arrest at equivalent unit dose (neutrons up to 8 Gy; gamma rays up to 5.6 Gy), suggesting a period in cell cycle for DNA repair. However, only cells treated with low doses (up to 2 Gy) seemed to be capable of returning into normal cell cycle within 4 days. For the highest dose of neutrons, decline in the number of normal cells seen at already 3 days after treatment was deeper compared with equivalent unit doses of gamma rays. Our present results support different mechanisms of action by these two irradiations and suggest the generation of locally multiply damaged sites (LMDS) for high linear energy transfer (LET) radiation which are known to be repaired at lower efficiency.

Chemical and radiological risk factors associated with waste from energy production.

We have tried to estimate the toxic potential of waste from nuclear power plants and from power plants burning fossil fuels. The potential risks have been expressed as 'risk potentials' or 'person equivalents.' These are purely theoretical units and represent only an attempt to quantify the potential impact of different sources and substances on human health. Existing concentration limits for effects on human health are used. The philosophy behind establishing limits for several carcinogenic chemicals is based on a linear dose-effect curve. That is, no lower concentration of no effect exists and one has to accept a certain small risk by accepting the concentration limit. This is in line with the establishment of limits for radiation. Waste products from coal combustion have the highest potential risk among the fossil fuel alternatives. The highest risk is caused by metals, and the fly ash represents the effluent stream giving the largest contribution to the potential risk. The waste from nuclear power production has a lower potential risk than coal if today's limit values re used. If one adjusts the limits for radiation dose and the concentration limit values so that a similar risk is accepted by the limits, nuclear waste seems to have a much higher potential risk than waste from fossil fuel. The possibility that such risk estimates may be used as arguments for safe storage of the different types of waste is discussed. In order to obtain the actual risk from the potential risk, the dispersion of the waste in the environment and its uptake and effects in man have to be taken into account.

Natural radiation--a perspective to radiological risk factors of nuclear energy production.

Radiation doses from natural radiation and from man-made modifications on natural radiation, and different natural radiological environments in the Nodic countries are summarized and used as a perspective for the radiological consequences of nuclear energy production. The significance of different radiation sources can be judged against the total collective effective dose equivalent from natural radiation in the Nordic countries, 92,000 manSv per year. The collective dose from nuclear energy production during normal operation is estimated to 20 manSv per year and from non-nuclear energy production to 80 manSv per year. The increase in collective dose due to the conservation of heating energy in Nordic dwellings is estimated to 23,000 manSv per year, from 1973 to 1984. An indirect radiological danger index is defined in order to be able to compare the significance of estimated future releases of radionuclides from a final repository of spent nuclear fuel to the consequences of natural radionuclides in different environments. The danger index of natural radiological environments will not be significantly increased by future releases of nuclear fuel radionuclides.

Natural radioactivity in and radon exhalation from Finnish building materials.

A total of 369 samples of Finnish building materials were tested for their 226Ra, 232Th and 40K concentrations. The rate of radon exhalation was measured from 19 samples of material and 34 dwellings were tested for their room air ventilation rate and radon concentration. The mean values of 226Ra, 232Th and 40K concentrations in ballast materials for concrete production were 34.2 Bq X kg-1, 39.0 Bq X kg-1 and 964 Bq X kg-1, with standard deviations of 18.7 Bq X kg-1, 19.5 Bq X kg-1 and 265 Bq X kg-1, respectively. The activity concentrations were higher in clay bricks than in concrete, the mean values being 79.8 Bq X kg-1, 61.6 Bq X kg-1 and 986 Bq X kg-1 for 226Ra, 232Th and 40K, respectively. The normalized radon exhalation rates from 15-cm-thick concrete, slag-aggregate concrete and by-product gypsum were 0.38 (Bq X m-2 X h-1)/(Bq X kg-1), 0.15 (Bq X m-2 X h-1)/(Bq X kg-1), and 0.06 (Bq X m-2 X h-1)/(Bq X kg-1), respectively. The ventilation rates in dwellings varied between 0.27 and 1.99 air changes per h, the mean value being 0.60 h-1, and the corresponding steady state radon concentrations in room air varied from 17.0 to 149 Bq X m-3 in blocks of flats made of concrete and from 11.2 to 61.9 Bq X m-3 in blocks of flats made of brick.

Radioactivity of size fractionated fly-ash emissions from a peat- and oil-fired power plant.

Concentrations of gamma-emitting natural radionuclides and 137Cs were analyzed in the size fractionated fly-ash emissions from a 100-MWt peat- and oil-fired power plant. The emitted fly ash was separated into five size fractions with a high-volume impactor with cut sizes of 1.3 micron, 2.1 micron, 4.2 micron and 10 micron. The greatest activity emissions were associated with the smallest size fraction, below 1.3 micron. The mass median aerodynamic diameter (MMAD) of the fly-ash particles after the electrostatic precipitator was 1.9 micron with a geometric standard deviation of 3.0 and the median of the 32 fly-ash emission samples was 8.73 mg MJ-1 with a geometric standard deviation of 1.3. Lead-210 gave the greatest particulate activity emission per input fuel energy, 18.7 mBq MJ-1, and showed a strong enrichment onto small fly-ash particles.

Artificial radioactivity in fuel peat and peat ash in Finland after the Chernobyl accident.

The accident at the Chernobyl nuclear power plant in April 1986 caused very uneven deposition of radionuclides in Finland. The deposited radionuclides were found in relatively high concentrations in fuel peat and especially in peat ash because a thin surface layer of peat-production bogs was extracted as fuel peat soon after the fallout occurred. Concentrations of artificial radionuclides in fuel peat and peat ash were measured at six peat-fired power plants in Finland throughout the heating season 1986-87. Concentrations of 137Cs in composite peat samples varied between 30 and 3600 Bq kg-1 dry weight and in ash samples between 600 and 68,000 Bq kg-1. High concentrations in peat ash caused some restrictions to the utilization of peat ash for various purposes.

Behavior of Chernobyl fallout radionuclides in peat combustion.

The fallout from the explosion and fire at the Chernobyl nuclear power plant concentrated levels of up to 10 kBq 137Cs kg-1 dry weight in the fuel peat harvested during the summer of 1986 in Finland. We investigated the behavior of fallout radionuclides 137Cs, 134Cs, 106Ru, 144Ce, 125Sb, 95Zr, and 110mAg together with naturally occurring 210Pb and 226Ra in the combustion of this contaminated peat in four different power plants. The elements antimony, ruthenium, lead, and cesium were enriched on the smallest particles, indicating that they were in a volatile chemical form, while cerium, zirconium, and radium were nonvolatile at the combustion temperatures. This result confirms the previous finding that ruthenium is volatile in combustion. Although metallic ruthenium requires 2,310 degrees C to melt, some of its oxides melt and evaporate at much lower temperatures.

7-Methylguanine levels in DNA of smokers' and non-smokers' total white blood cells, granulocytes and lymphocytes.

The effect of smoking was investigated on the formation of 7-methylguanines in human peripheral white blood cells. DNA was isolated from total white blood cells, granulocytes and lymphocytes from 10 smokers and 10 non-smokers. 32P-Postlabeling was performed by using anion-exchange chromatography enrichment of adducts. In smokers the mean DNA adduct levels were 6.9, 4.7 and 23.6 7-methylguanine residues/10(7) nucleotides in total white blood cells, granulocytes and lymphocytes respectively. The corresponding values in non-smokers were 3.4, 2.8 and 13.5 adducts/10(7) nucleotides. The mean adduct level was significantly higher in lymphocytes than in total white blood cells or granulocytes both in smokers and in non-smokers. The mean adduct levels differed significantly between smokers and non-smokers.

Retrolental fibroplasia. Cases seen at the Helsinki University Eye Hospital 1956-1974.

In the period 1956-1974, 31 new cases of retrolental fibroplasia (RLF) were diagnosed at the Helsinki University Eye Hospital. All were alive in 1974. 90% weighed less than 1500 g at birth and the gestational age of less than 30 weeks at birth was present in 85%. All patients had required supplementary oxygen during the first weeks of life. 84% of the eyes had visual acuity of finger counting 1 m or less and when classified according to the corrected visual acuity in the better eye, 71% of the patients were practically blind according to the classification by Rintelen. 81% of the eyes showed one of the cicatricial stages of RLF, III to V, and 45% showed the grade V of the disease. 11% of the eyes showed corneal opacities, 44% a flat or absent anterior chamber, 11% had intraocular pressure of greater than or equal to 30 mmHg and 21% showed lenticular opacities. When the period of the observation was surveyed, it was found that during the latter half of the observation period, twice as many new cases of RLF were born. At a rough estimate it was calculated that in 1956-1974 at least 50 new cases of RLF developed in Finland.

32P-postlabeling of 7-methylguanine adducts in DNA.

The 32P-postlabeling technique introduced by Randerath and co-workers has been particularly successful with stable and nonpolar DNA adducts, but the assay has not been used to any large extent to detect 7-alkylguanine derivatives. In the present communication, we have investigated the phosphorylation reaction by T4 polynucleotide kinase using 7-methyl-3'-dGMP, ring-opened 7-methyl-3'-dGMP and enzyme-digested methylated DNA as substrates. The methylated substrates were detected at femtomol (fmol) sensitivities. 7-methyl-3'-dGMP was quantitatively phosphorylated at these low concentrations. The efficiency of phosphorylation of the ring-opened product was less. It was shown that ring-opened 7-methyl-3'-dGMP was resistant to digestion with nuclease P1, making alkali-treatment and enzyme digestion of DNA possible approaches to the determination of 7-methylguanine in DNA.