Transposable genetic elements, spontaneous mutations and the doubling-dose method of radiation genetic risk evaluation in man

The principal aspects of the 'doubling-dose method' currently used by the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) and the Committee on the Biological Effects of Ionizing Radiation (BEIR) of the U.S. National Academy of Sciences, for the evaluation of genetic radiation hazards in man are briefly reviewed. With this method, which is primarily applicable to autosomal dominant and X-linked disorders, the expected increase in risk from radiation is expressed as a fraction of the current prevalence of these disorders, and thus in relation to an understandable frame of reference. Since the doubling dose is estimated as a ratio of spontaneous to induction rates of mutations, its magnitude is susceptible to changes in either the numerator (spontaneous rate) or the denominator (induction rate). Studies during the past 20 years or so with a number of experimental systems have demonstrated the existence of mobile DNA sequences in the genome and their causal role in the origin of spontaneous mutations, although the proportion of the latter among all spontaneous mutations is not known for any species. If a major proportion of spontaneous mutations in man is mediated by these mobile DNA sequences, and if their mobility is unaltered by radiation exposures, the calculation of the doubling dose in the manner mentioned above, and its use in risk evaluations becomes questionable. However, considerations based on the organization of the human genome would suggest that it is unlikely that a major fraction of spontaneous mutations that lead to disease states in man is due to mobile genetic elements. Consequently, the use of the doubling-dose method for the evaluation of genetic radiation hazards in man would appear to be valid at the present time.     

Genetic efficacy of low doses of ionizing radiation in chronically-irradiated small mammals

Earlier we have established the genetic effects of low dose chronic irradiation in bank vole (somatic and germ cells, embryos), in pond carp (fertilized eggs, embryos, fry) and in laboratory mice (somatic and germ cells) in the range of doses from near-background to 10 cGy. These low dose effects observed in mammals and fish are not expected from extrapolation of high dose experiments. For understanding reasons this discrepancy the comparative analysis of genetic efficiency of low dose chronic irradiation and the higher doses of acute irradiation was carried out with natural populations of bank vole which inhabited the two sites differing in ground of radionuclide deposition. For comparing efficiency the linear regression model of dose-effect curve was used. Dose-effect equations were obtained for animals from two chronically irradiated bank vole populations. The mean population absorbed doses were in the range 0.04-0.68 cGy, the main part of absorbed doses consisted of external radiation of animals exposed to 137Cs gamma-rays. Dose-effect equations for acute irradiation to 137Cs gamma-rays (10-100 cGy) were determined for the same populations. Comparison of genetic efficiency was made by extrapolation, using regression coefficient beta and doubling dose estimation. For chronic exposure the doubling doses calculated from low-dose experiments are 0.1-2 cGy and the doubling doses determined from high-dose experiments are in the range of 5-20 cGy. Our hypothesis that the doubling dose estimate is calculated in higher-dose ionizing radiation experiments should be much higher than the deduced from the low dose line regression equation was verified. The doubling dose estimates for somatic cells of bank vole and those for germ cells of laboratory mice are in close agreement. The radiosensitivity of bank vole chromosomes were shown is practically the same as that for human lymphocytes since doubling dose estimates for acute irradiation close to each other. For low LET radiation a higher genetic efficiency of chronic low doses in comparison with the higher doses of acute gamma-irradiation (137Cs source) was proved by three methods.     

Further studies on effects of X-irradiation on prespermatid stages of the Northern vole Microtus oeconomus: Low induction of sex-chromosomal nondisjunction and very high induction of diploid spermatids

Microtus males have been irradiated with X-ray doses of 25, 50, 100 and 200 rad and early spermatids were then analyzed for evidence of induction of sex-chromosomal nondisjunction and diploid spermatids at 1, 4, 6, 7, 9 and 12 days after treatment. In contrast to earlier findings, there was no induction of nondisjunction above control levels. A possible explanation for the differences in results of old and new experiments might be that genetic changes have taken place in the Microtus colony that was initiated with animals trapped in the wild, but which has now become highly inbred.In the present experiment, diploid spermatids were frequently induced. The dose—effect relationships at the different time intervals were linear, but the slopes were different, indicating stage-specific differences in sensitivity. The average doubling dose is of the order of 12 rad with a range of 5–30 rad for the individual time intervals.When diploid spermatozoa in man are also inducible by such low doses of X-rays, the consequence would be an increase of triploid abortions which would constitute and undesirable form of personal or family harddhip.     

Procarbazine-induced specific-locus mutations in male mice.

Procarbazine is used in drug-combination treatment of Hodgkin's disease. The specific locus method was used to test and confirm the ability of procarbazine to induce gene mutations in pre- and post-meiotic germ cells of male mice. The lowest dose of procarbazine that significantly increased the mutation frequency in As spermatogonia over the control frequency was 400 mg/kg (P = 0.003). The corresponding dose for the post-spermatogonial germ-cell stages was 600 mg/kg (P = 0.009). The dose--response was linear for the point estimates of the mutation frequencies after treatment of As spermatogonia with 0, 200, 400 and 600 mg/kg. The point estimate of the mutation frequency at the 800 mg/kg level was one-third of that expected from a linear extrapolation. Variation in mutation rates among the 7 loci between the lowest (a locus) and the highest (p locus) was 12-fold. Only 24% of procarbazine-induced specific locus mutations in As spermatogonia were lethal in the homozygous condition. From the mutation spectra and the viability tests, it is concluded that procarbazine-induced mutations may be mainly due to base-pair changes. Procarbazine-induced specific-locus mutations fulfilled the criteria for the estimation of the doubling dose, the dose necessary to induce as many mutations as occur spontaneously. The doubling dose of procarbazine in As spermatogonia of mice was 114 mg/kg. The therapeutic dose for procarbazine is about 215 mg/kg. If man and mouse were equally sensitive, this dose would induce 1.9 times as many mutations as arise spontaneously. From the incidence of patients with Hodgkin's disease (1 : 42 000) the calculated population dose of procarbazine is 5.12 micrograms/kg. Assuming equal sensitivity between the sexes we can calculate, for an estimated number of 30 000 genes, the induction of about 22 mutations per million children due to procarbazine treatment. The same number of induced mutations can be calculated if the risk of patients is used for the estimation of the genetic hazard.     

Ionizing radiation and genetic risks. XI. The doubling dose estimates from the mid-1950s to the present and the conceptual change to the use of human data on spontaneous mutation rates and mouse data on induced mutation rates for doubling dose calculations

This paper provides an overview of the concept of doubling dose, changes in the database employed for calculating it over the past 30 years and recent advances in this area. The doubling dose is estimated as a ratio of the average rates of spontaneous and induced mutations in a defined set of genes. The reciprocal of the doubling dose is the relative mutation risk per unit dose and is one of the quantities used in estimating genetic risks of radiation exposures. Most of the doubling dose estimates used thus far have been based on mouse data on spontaneous and induced rates of mutations. Initially restricted to mutations in defined genes (with particular focus on the seven genes at which induced recessive mutations were studied in different laboratories), the doubling dose concept was subsequently expanded to include other endpoints of genetic damage. At least during the past 20 years, the magnitude of the doubling dose has remained unchanged at approximately 1 Gy for chronic low LET radiation exposures.One of the assumptions underlying the use of the doubling dose based on mouse data for predicting genetic risks in humans, namely, that the spontaneous rates of mutations in mouse and human genes are similar, is incorrect; this is because of the fact that, unlike in the mouse, the mutation rate in humans differs between the two sexes (being higher in males than in females) and increases with paternal age. Further, an additional source of uncertainty in spontaneous mutation rate estimates in mice has been uncovered. This is related to the non-inclusion of mutations which arise as germinal mosaics and which result in clusters of identical mutations in the following generation. In view of these reasons, it is suggested that a prudent way forward is to revert to the use of human data on spontaneous mutation rates and mouse data on induced mutation rates for doubling dose calculations as was first done in the 1972 BEIR report of the US National Academy of Sciences. The advantages of this procedure are the following: (i) estimates of spontaneous mutation rates in humans, which are usually presented as sex-averaged rates, automatically include sex differences and paternal age-effects; (ii) since human geneticists count all mutations that arise anew irrespective of whether they are part of a cluster or not, had clusters occurred, they would have been included in mutation rate calculations and (iii) one stays close to the aim of risk estimation, namely, estimation of the risk of genetic diseases in humans.On the basis of detailed analyses of the pertinent data, it is now estimated that the average spontaneous mutation rate of human genes (n=135 genes) is: (2.95+/-0.64)x10(-6) per gene and the average induced mutation rate of mouse genes (n=34) is: (0.36+/-0.10)x10(-5) per gene per Gy for chronic low LET radiation. The resultant doubling dose is (0.82+/-0.29) Gy. The standard error of the doubling dose estimate incorporates sampling variability across loci for estimates of spontaneous and induced mutation rates as well as variability in induced mutation rates in individual mouse experiments on radiation-induced mutations. We suggest the use of a rounded doubling dose value of 1 Gy for estimating genetic risks of radiation. Although this value is the same as that used previously, its conceptual basis is different and the present estimate is based on more extensive data than has so far been the case.     

The response of spermatogonia and spermatocytes of the Northern vole Microtus oeconomus to the induction of sex-chromosome nondisjunction, diploidy and chromosome breakage by X-rays and fast fission neutrons

Microtus males were exposed to different doses of 250 kV X-rays or fast fission neutrons of 1 MeV mean energy. Early (= round) spermatids were analyzed for the presence of extra sex chromosomes, diploidy and micronuclei at different time intervals corresponding with treated differentiating spermatogonia and spermatocytes. Induction of nondisjunction of sex chromosomes could not be detected. In contrast, induction of diploids by both types of radiation was statistically significant at all sampling times. Dose-effect relationships for most of the sampling times were linear and sometimes linear-quadratic concave upward or downward. There were pronounced stage-specific differences in sensitivity as reflected by differences in doubling doses that ranged from 4 to 22 cGy for X-rays and from 0.4 to 4 cGy for neutrons. Spermatocytes at pachytene were the most sensitive cells and proliferating spermatogonia the least sensitive ones. The relative biological effectiveness (RBE) of neutrons depended on the cell stage treated and fluctuated between 1.4 and 9.2. Evidence for radiation-induced chromosomal breakage events was obtained via detection of micronuclei. Induction of micronuclei by X-rays or neutrons was statistically significant at all spermatocyte stages tested. There was no effect in spermatogonia. With a few exceptions dose-effect relationships were linear. Differences in stage sensitivity were clearly present as evidenced by doubling dose which ranged from 5 to 29 cGy for X-rays and from 1 to 3 cGy for neutrons. RBE values varied from 5.2 to 12.7. Maximum sensitivity was detected in spermatocytes at diakinesis, MI and MII. Resting primary spermatocytes (G1 and S phase) were somewhat less sensitive and actively proliferating spermatogonia were the least sensitive cells. The pattern of stage sensitivity for induction of diploids was distinctly different from that for induction of chromosomal breakage.     

Abnormal balance between proliferation and apoptotic cell death in fibroblasts derived from keloid lesions

A new culture model was developed to study the role of proliferation and apoptosis in the etiology of keloids. Fibroblasts were isolated from the superficial, central, and basal regions of six different keloid lesions by using Dulbecco's Modified Eagle Medium containing 10% fetal calf serum as a culture medium. The growth behavior of each fibroblast fraction was examined in short-term and long-term cultures, and the percentage of apoptotic cells was assessed by in situ end labeling of fragmented DNA. The fibroblasts obtained from the superficial and basal regions of keloid tissue showed population doubling times and saturation densities that were similar to those of age-matched normal fibroblasts. In contrast, the fibroblasts from the center of the keloid lesions showed significantly reduced doubling times (25.9 +/- 6.3 hours versus 43.5 +/- 6.3 hours for normal fibroblasts) and reached higher cell densities. In long-term culture, central keloid fibroblasts formed a stratified three-dimensional structure, contracted the self-produced extracellular matrix, and gave rise to nodular cell aggregates, mimicking the formation of keloid tissue. Apoptotic cells were detected in both normal and keloid-derived fibroblasts, but their numbers were twofold higher in normal cells compared with all keloid fibroblasts. To examine whether apoptosis mediates the therapeutic effect of ionizing radiation on keloids, the cells were exposed to gamma rays at a dose of 8 Gy. Under these conditions, a twofold increase in the population of apoptotic cells was detected. These results indicate that the balance between proliferation and apoptosis is impaired in keloid fibroblasts, which could be responsible for the formation of keloid tumors. The results also suggest that keloids contain at least two different fibroblast fractions that vary in growth behavior and extracellular matrix metabolism.     

The enhancing effect of cell-wall aggressin on the virulence of Staphylococcus aureus in normal and irradiated mice and in man

Mice irradiated with lethal doses of total body X-irradiation are very susceptible to Staphylococcus aureus. A difference in virulence between a “mouse-virulent” strain and a “mouse-non-virulent” strain which lacks cell-wall aggressin could nevertheless be demonstrated. The virulence of the “mouse-non-virulent” strain could be increased by adding a cell-wall preparation with aggressin-activity (DOCR) from a “mouse-virulent” strain to the inoculum. When injected together with a dose of bacteria lower than the minimum pus-forming dose a lesion-enhancing effect of DOCR from a “mouse-virulent” strain was also demonstrated in man.     

Current problems of estimation of genetic risk of human exposure to radiation

The methodology of assessing the genetic risk of radiation exposure is based on the concept of "hitting the target" in development of which N.V. Timofeeff-Ressovsky has played and important role. To predict genetic risk posed by irradiation, the UN Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) has worked out direct and indirect methods of assessment, extrapolational, integral and populational criteria of risk analysis that together permit calculating the risk from human exposure on the basis of data obtained for mice. Laboratory mice are the main objects in studying radiation mutagenesis due to the fact that the data on the frequency of radiation-induced human mutations are rather scarce. The method of doubling dose based on the determination of a dose doubling the level of natural mutational process in humans is the main one used to predict the genetic risk. The evolution of views about the genetics risk of human exposure to radiation for last 40 years is considered. Till 1972 the main model for assessing the genetic risk was the "human/mouse" model (the use of data on the spontaneous human variability and data on the frequency of induced mutations in mice). In the period form 1972 till 1994 the "mouse/mouse" model was intensively elaborated in many laboratories. This model was also used in this period by UNSCEAR experts to analyze the genetic risk from human irradiation. Recent achievements associated with the study of the molecular nature of many hereditary human diseases as well as the criticism of number fundamental principles of the "mouse/mouse" model for estimating the genetic risk on a new basis. The estimates of risk for the different classes of genetic diseases have been obtained using the doubling-dose method. The estimate of doubling dose used in the calculations is 1 Gy for low dose/chronic low-LET radiation conditions.     

Shoot doubling time: A quantitative parameter for characterizing shoot cultures in vitro

Shoot doubling time is proposed as a suitable parameter for characterizing in vitro propagation rates in shoot cultures. Doubling times were estimated for cultures of black currant and apple; for black currant, doubling times ranging from 14 to 115 days were obtained, depending on the concentration of benzyladenine in the culture medium. These doubling times, which were constant for each culture, were maintained for periods of up to 76 days and could apparently be sustained indefinitely. For one subculture period only, a shoot doubling time as short as 5.6 days was obtained for black currant at high cytokinin concentration (3 × 10⁻⁵ M), but this rate could not be sustained. Shoot doubling time is a convenient parameter for use in optimizing proliferation rates in shoot cultures; its use may also facilitate investigations into the mechanisms of processes underlying shoot proliferation in vitro.     

Clastogenic effects of cis-diamminedichloroplatinum. I. Induction of chromosomal aberrations in somatic and germinal cells of mice

The clastogenicity of cisplatin, cis-diamminedichloroplatinum(II), an extensively used antitumor drug, has been studied employing (101/E1 X C3H/E1)F1 mice, aged 12-14 weeks. Chromosomal aberrations were assessed in mitotic divisions of bone marrow cells and differentiating spermatogonia. The drug was tested at 3 doses, 0.5, 1.0 and 2.5 mg/kg and 1.0, 2.5 and 5.0 mg/kg, respectively, for bone marrow and spermatogonia. Cisplatin had a clastogenic effect which was dose-dependent in both cell types. The frequencies of aberrant cells increased non-linearly in bone marrow and the dose-response relationship could be best described by a linear-quadratic equation. At the highest dose the affected cells carried multiple aberrations. An average of 2.7 aberrations per aberrant cell was observed 12 h after treatment of the mice with 2.5 mg/kg of cisplatin. In differentiating spermatogonia the dose response for aberrant cells could be described by a linear equation. The damage to the individual affected cell was less dramatic than in bone marrow, averaging 1.4 aberrations per damaged cell at the highest dose tested. Gaps were excluded from these considerations but they generally also showed a dose-related increase. A quantitative comparison of the clastogenic response to cisplatin was based on the dose-response relationships using 2 criteria, the doubling dose and the dose of unit increase (DUI). For both comparisons the general conclusion was that bone marrow cells were twice as sensitive as differentiating spermatogonia to the clastogenic action of cisplatin.     

Schwann cell proliferation in vitro is under negative autocrine control

In healthy adult peripheral nerve, Schwann cells are believed to be generally quiescent. Similarly, cultures of isolated rat sciatic nerve Schwann cells hardly proliferate in serum-supplemented medium. The possibility that Schwann cells negatively regulate their own proliferation was supported by the demonstration that conditioned media from Schwann cell cultures inhibited the proliferation of mitogen- stimulated test cultures. The inhibition could be complete, was dose dependent, and was exhibited when the test Schwann cells were under the influence of different types of mitogens such as cholera toxin, laminin, and living neurons. The inhibition of proliferation was completely reversible and a rapid doubling of cell number resulted when treatment with conditioned medium was withdrawn from mitogen-stimulated Schwann cells. Conditioned medium from cholera toxin-stimulated and immortalized Schwann cell cultures contained less antiproliferative activity than that found in medium from quiescent Schwann cell cultures. However, media conditioned by two actively proliferating rat Schwannoma cell lines were rich sources of antiproliferative activity for Schwann cells. Unlike the mitogen-stimulated Schwann cells, whose proliferation could be inhibited completely, the immortalized and transformed Schwann cell types were nearly unresponsive to the antiproliferative activity. The antiproliferative activity in Schwann and Schwannoma cell conditioned media was submitted to gel filtration and SDS-PAGE. The activity exists in at least two distinct forms: (a) a high molecular weight complex with an apparent molecular mass greater than 1,000 kD, and (b) a lower molecular weight form having a molecular mass of 55 kD. The active 55-kD form could be derived from the high molecular weight form by gel filtration performed under dissociating conditions. The 55-kD form was further purified to electrophoretic homogeneity. These results suggest that Schwann cells produce an autocrine factor, which we designate as a "neural antiproliferative protein," which completely inhibits the in vitro proliferation of Schwann cells but not that of immortalized Schwann cells or Schwannoma lines.     

Models and assumptions underlying genetic risk assessment

Various methods employed for estimating the genetic risks of radiation are reviewed. With the doubling-dose method, genetic damage is expressed as an increase in cases of known genetic disease. The actual doubling dose is based on figures obtained with the mouse. There have been no recent data on induced mutation frequencies. Recent results suggest that the prevalence figure for multifactorial disease may be at least one order of magnitude higher than before. Various assumptions underlying the doubling-dose concept are discussed in the light of recent findings on: (1) spontaneous mutations resulting from insertion elements, and (2) the comparability between spontaneous and induced mutations. The so-called direct method makes use of figures for induction of dominant mutations affecting the skeleton and the lens of the eye in the mouse, and of translocation induction in monkeys. Induction rates are converted to overall rates of induced dominant effects in man by applying certain assumptions. The proportionality between dose and effect is the basis for all genetic risk assessments. The possible significance of data on human lymphocytes indicating a threshold below 4 rad and the induction of repair enzymes by low radiation doses is discussed. The parallelogram approach is based on the principle that estimates can be obtained on the amount of genetic damage that cannot always be assessed directly. Thus mutations in mouse germ cells can be predicted by using mutation frequencies in cultured mammalian cells and O6-ethylguanine adducts. Measurement of haemoglobin mutations in human and mouse erythrocytes, and of HPRT-deficient mutations in lymphocytes of man and mouse should make more precise estimates of mutation frequencies in human germ cells possible. The development of a database on mutations in somatic cells of the mouse, their induction frequencies and molecular nature are considered an important priority. Used in combination with mouse germ-cell mutation frequencies, they should enable more precise risk estimates on the basis of mutations in somatic cells of man.     

Aeromonas hydrophila exotoxin induces cytoplasmic vacuolation and cell death in VERO cells

Many organisms are able to cause cell vacuolation, but it is unclear if this can be considered a step of apoptosis or necrosis, or a distinct form of cell death. In this study VERO cells were used to evaluate the relationship between vacuolation and cell death pattern caused by exotoxins produced by environmental strains of A. hydrophila. Cell damage has been evaluated morphologically as well as biochemically. Cytotoxic and vacuolating titres were strictly correlated and the vacuolation has to be considered an early indicator of cytotoxicity that causes cell apoptosis or necrosis in relation to the dose. Signs of apoptosis (chromatin condensation and blebbing) were observed at low concentration and TGase activity, referable to apoptosis induction, confirms morphological observations. In fact, putrescine incorporation was related both to cytotoxin concentration and time of incubation. Moreover, the observed doubling cells with necrotic features permit us to suppose that cell sensitivity and death pattern could change during the different phases of cellular cycle.     

Biochemical and recessive visible specific locus responses of C3H/HeH to fractionated,acute radiation

The recessive visible specific locus test has been widely used for many years to investigate the genetic effects of radiation in mice. We devised an electrophoretic-specific locus test so that biochemical mutations leading to alterations in the activity or amount of four enzymes and proteins, as well as charge changes could be detected. We measured the yield of recessive visible and electrophoretic mutations in the same experiment so that a direct comparison of mutation incidence could be made. Dominant visible mutations were also scored. The recessive visible specific locus response of male C3H/HeH to a fractionated dose of 3 + 3 Gy X-irradiation separated by 24 h was similar to that previously reported for the F1 hybrid widely used in mutagenesis studies, and other strains. The response of C3H/HeH was significantly greater for the recessive visible mutations than for the biochemical mutations, supporting the contention that the recessive visible loci are more mutable than others. Mutational analysis of some of the mutants showed that the lesions ranged from a very deletion (30% of chromosome 14 deleted) to a point mutation. The number of loci scored in the electrophoretic test has been reassessed, and it is now considered that six, not four were scored, and this has implications for the calculation of the doubling dose.     

Add-on therapy options in asthma not adequately controlled by inhaled corticosteroids: a comprehensive review

Many patients with persistent asthma can be controlled with inhaled corticosteroids (ICS). However, a considerable proportion of patients remain symptomatic, despite the use of ICS. We present systematically evidence that supports the different treatment options. A literature search was made of Medline/PubMed to identify randomised and blinded trials. To demonstrate the benefit that can be obtained by increasing the dose of ICS, dose-response studies with at least three different ICS doses were identified. To demonstrate whether more benefit can be obtained by adding long-acting β₂-agonist (LABA), leukotriene antagonist (LTRA) or theophylline than by increasing the dose of ICS, studies comparing these options were identified. Thirdly, studies comparing the different "add-on" options were identified. The addition of a LABA is more effective than increasing the dose of ICS in improving asthma control. By increasing the dose of ICS, clinical improvement is likely to be of small magnitude. Addition of a LTRA or theophylline to the treatment regimen appears to be equivalent to doubling the dose of ICS. Addition of a LABA seems to be superior to an LTRA in improving lung function. However, addition of LABA and LTRA may be equal with respect to asthma exacerbations. However, more and longer studies are needed to better clarify the role of LTRAs and theophylline as add-on therapies.     

Evaluation of the level and radiosensitivity of the hematopoietic stem cell pool in man by the number of endocolonies of undifferentiated cells forming against a background of post-radiation bone marrow aplasia

The value and radiosensitivity of human haemopoietic stem pool may be assessed by the number of colonies of nondifferentiated cells (CFUnc) formed in situ during regeneration of the haemopoietic organ from the postirradiation aplasia. The time required for doubling the population, that constitutes nondifferentiated cell endocolonies, was reduced as the radiation dose increased.     

X-ray-induced mutants resistant to 8-azaguanine. II. Cell cycle dose response.

Synchronous Chinese hamster ovary cells were irradiated in G1 or S phase. Colony survival in Alpha MEM medium with dialyzed serum was determined with or without 15 mug/ml 8-azaguanine (AG). An expression period of over three generations (multiplicity of 20) was utilized, with expression times ranging from 58 to 114 h. Both G1 and S phase were practically identical in sensitivity to X-ray-induced mutations, with mutant frequency/viable cell/rad ranging from 1 X 10(-7) (75-100 rad) to 8 X 10(-7) (1000 rad). The spontaneous mutation rate, shown by Luria-Delbruck fluctuation analysis, was 5 X 10(-7) per generation. Thirty-three mutants, isolated at random and grown for over 30 generations in the absence of AG, were analyzed for plating efficiency (PE) in different concentrations of AG or in hypoxanthine-aminopterin-thymidine (HAT) medium. Of these, 64% were resistant (PE greater than 0.1) to 7.5 mug/ml AG, 85% to 5.0 mug/ml, and 91% to 3.5 mug/ml. Only 42% showed possible hypoxanthine-phosphoribosyltransferase (hprtase) deficiency as evidenced by HAT sensitivity (PE less than 0.1). Wild type controls exhibited PE's in 3.5 mug/ml AG of less than 0.001 and in HAT of greater than 0.5. Of ten mutants studied, all demonstrated survival response to radiation similar to wild type cells (D0 of approx. 120 rad). For radiation protection standards, the radiation dose required to induce mutations at a rate equal to that occurring spontaneously is called the doubling dose. The doubling dose observed for acute irradiation was about 3 rad and was estimated to be 10-60 rad for chronic irradiation, similar to that often reported for in vivo studies.     

Evaluation of genetic radiation hazards in man: History,present status and perspectives

The evaluation of genetic radiation hazards in man is an ongoing scientific enterprise from about the mid-1950s. Since estimates of genetic risks are essential for providing a basis for protecting our genetical endowment and since strictly relevant human data are limited, there is no alternative at present but to use the data from mouse and certain non-human primates. This paper reviews the general principles and methods that have thus far been used, appraises the evolution of the conceptual framework, the data base and the assumptions involved, presents current estimates of genetic risks and provides some perspective of the advances that are likely to be made in the near future. Currently, risk estimates are made using the so-called “direct method” and the “doubling dose method”. Both these methods involve a number of assumptions and consequent uncertainties. With the direct method, it is now estimated that following low LET, low dose-rate or low-dose irradiation of males, there will be (i) about 10–20 cases of affected children per million births per rad of exposure, who will suffer from the effects of induced mutations having dominant effects and (ii) about 1 to 10 cases of congenitally malformed children (again per million births per rad), a consequence of the induction of reciprocal translocations. For irradiation of females under similar conditions, the estimated risks are 0–9 and 0–3 affected children per million births per rad, these being the consequence of induction of dominant mutations and of reciprocal translocations, respectively. The doubling dose method is used to estimate risks to a population under continuous irradiation. If the population is exposed to low LET irradiation at a rate of 1 rad/generation (1 generation = 30 years), the expected total increments in the frequencies of genetic diseases are about 20 cases per million births in the first generation and about 150 cases per million births at equilibrium. These expected increments are very small fractions of the spontaneous prevalence of genetic and partially genetic disorders, currently estimated to be about 10.6 %.     

Effect of Shaking on the Growth of Diluted Cultures of Tetrahymena

ABSTRACT. Cultures of Tetrahymena pyriformis, T. thermophila and T. pigmentosa have been studied with regard to growth rates in shaken and unshaken flasks. In the standard medium, a minimum doubling time of 170 min was obtained for T. pyriformis at 28° C in the unshaken cultures. If the depth of the medium was less than 1 cm, the gyratoric shaking increased the doubling time to 340 min. The effect of shaking could be reduced by the addition of dextrane. Cells subjected to shaking were observed in different media and at different growth temperatures. If cultures were inoculated with 10⁴ cell·ml⁻¹ or more, the effect of shaking was absent. However, with inoculates of 10³ or 10² cell·ml⁻¹, the doubling times for T. pyriformis increased to 240 and 275 min, respectively. Periods of 2 min shaking followed by rest for 60 min could not induce an effect.