Life shortening in BALB/c mice following brief, protracted, or fractionated exposures to neutrons

The effects of acute, protracted, or fractionated exposures to fission neutrons on survival times of female BALB/c mice were examined and compared. Mice were given single, brief exposures or exposures given in equal fractions at either 1- or 30-day intervals to doses of 0, 2.5, 5, 10, 20, 50, and 200 rad at the Health Physics Research Reactor (HPRR) or protracted exposures at rates ranging from 0.1 to 10 rad/day using a moderated 252Cf source to doses of 0, 2.5, 5, 10, 20, and 40 rad. The 252Cf source was moderated to have a similar spectron to that of the HPRR facility. After single or fractionated exposures the extent of life shortening increased rapidly over the 0-50 rad range and then began the plateau. No simple model adequately described the dose response over this entire dose range. Over the 0-50 rad dose range for exposures at the HPRR and over the 0-40 rad dose range for protracted exposures the dose response could be adequately described by either a linear model or a square root of the dose regression model except when the dose was fractionated using a 30-day interval. In this instance a linear model provided an adequate fit while a square root of the dose model could be rejected. No increase in effectiveness after fractionation or protraction was observed for neutron-induced life shortening at doses below 50 rad, while at 50 and 200 rad an increase in effectiveness was observed in this and in previous studies. These data were interpreted to suggest that in the dose range below 20-40 rad the dose-effect curve for life shortening may be linear and begins to flatten at higher doses rather than continuously bending at low doses.     

Effect of dose rate and exposure time on the stimulation effect of tube growth ofPinus Silvestris pollen

The stimulating effect of ionizing radiation in respect to dose rate and exposure time was studied using the tube growth of Pinus silvestris pollen. Stimulation was registered with a small dose (50 rad) supplied at low dose rates (0.5; 1.0; 3.0 and 5.0 rad/sec) and with higher doses (300, 800 and 1400 rad) supplied at higher dose rates (10; 40 and 50 rad/sec). This suggests that only the exposure time is of importance for radiation-induced stimulation provided that the exposure time does not exceed 100 sec.     

Study of dose-rate effects of neutron radiation in a wild-type and a repair-deficient yeasts saccharomyces

We report here a comparative analysis of RBE for lethality of a single pulse (duration 65 micros) of fast neutron with ultra high dose rates (up to 6 x 10(6) Gy/s) and continuous neutron radiation (3.6 x 10(3) s) of the pulse reactor BARS-6. Three diploid strains, one haploid strain and three diploid repair-deficient strains (rad52-1/rad52-1; rad54/rad54; rad2/rad2) were used. The RBE values (D(0gamma)/1D(0n)) of a single pulse and continuous neutron irradiation were equal (1.7-1.8) with maximum RBE (4.1-3.1) in region of low doses (shoulder region). Haploid cells were found to be more (3 times) sensitive to both gamma-rays and neutrons than the wild type. There was no obvious decrease in the RBE of 1.9 in highly sensitive haploid cells as compared with highly resistant diploid cells. The repair-deficient strains (rad52-1/rad52-1; rad54/rad54) were more (up to 10 fold) sensitive to both neutrons and gamma-rays as compared with their parent line. The RBE values of 1.5-1.7 of neutrons for these mutants (independent by of the mode of irradiation) were found. The repair-deficient mutant rad2/rad2 had similar sensitivity as a wild type and a RBE value was 2.0. We have concluded that biological effectiveness of the neutrons of pulse reactor BARS-6 was independent of the dose-rate, differing up to 10(8) fold. The RBE didn't vary significantly with the capacity of cells to repair DNA damages.     

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.     

Effects of mixed neutron-gamma total-body irradiation on physical activity performance of rhesus monkeys

Behavioral incapacitation for a physical activity task and its relationship to emesis and survival time following exposure to ionizing radiation were evaluated in 39 male rhesus monkeys (Macaca mulatta). Subjects were trained to perform a shock avoidance activity task for 6 hr on a 10-min work/5-min rest schedule in a nonmotorized physical activity wheel. Following stabilization of performance, each subject received a single, pulsed dose of mixed neutron-gamma, whole-body radiation (n/gamma = 3.0) ranging between 1274 and 4862 rad. Performance testing was started 45 sec after exposure. A dose-response function for early transient incapacitation (ETI) during the first 2 hr after irradiation was fitted, and the median effective dose (ED50) was calculated to be 1982 rad. More subjects experienced both incapacitation and emesis in this study than has been reported for other behavioral tasks in similar radiation fields. Analysis done on the relationship of dose to ETI, emesis, and survival time found (a) a significant relationship between the radiation dose and the number and duration of ETIs; (b) no correlation between emesis and dose, survival time, or ETI; (c) no relation between survival time and ETI at any dose; and (d) no significant difference in survival time for dose groups between 1766 +/- 9 (SEM) and 2308 +/- 23 rad.     

Differential and stage dependent effects of retinoic acid on chondrogenesis and synthesis of extracellular matrix macromolecules in chick craniofacial mesenchyme in vitro

Retinoic acid (RA) is well known to be a potent teratogen and induces a variety of facial defects in vivo, but at concentration levels lower than those that cause facial defects, RA seems to play an important role in normal facial development. In a previous study, we demonstrated the ability of RA to stimulate chondrogenesis in vitro in HH stage 23/24 chick mandibular (MND) but not frontonasal (FNP) mesenchyme cultured in a serum-free medium. The present study furthers these results by examining the effects of RA on chondrogenesis of chick facial mesenchyme at earlier embryonic stages and the effects on cell proliferation and synthesis of specific extracellular matrix macromolecules at stage 23/24. MND and FNP cells were cultured as micromasses for 4 days in defined media. As described previously, chondrogenesis in stage 23/24 MND cells was significantly enhanced by concentrations of RA of 0.1-1 ng/ml; however, at all earlier stages examined (18 to 22) RA at these concentrations had no significant effect. Higher concentrations of the retinoid inhibited chondrogenesis in MND cultures from all stages tested. Cells of the FNP from all stages displayed no significant change in chondrogenesis below 1 ng/ml RA and a dose dependent inhibition at higher concentrations. Thus RA's promotional effects in the face are not only tissue specific (MND), but also stage-dependent (HH 23/24). The specific effects of RA on matrix production and cell proliferation of stage 23/24 MND and FNP cells was examined by analysis of 35S sulfate, 3H thymidine and 3H proline incorporation. Analysis of 35S sulfate incorporation into sulfated proteoglycans confirmed that concentrations of RA of 0.1-1 ng/ml stimulated cartilage matrix production in MND but not FNP cultures. Above this level of RA, 35S sulfate incorporation was reduced in both. Likewise, 3H proline incorporation into collagenous protein, and to a lesser extent non-collagenous proteins, was stimulated by low levels of RA in MND, but not FNP cultures. Higher concentrations of the retinoid in either MND or FNP cultures did not lower collagen production, undoubtedly due to stimulation of non-chondrogenic cells within the population. This indicates that levels of RA as high as 100 ng/ml cause phenotypic change rather than cell death. This last point is corroborated by the analysis of 3H thymidine uptake in the cultures which was only transiently modified in most. The data indicate that cell proliferation occurred even in the presence of high RA levels.     

Influence of X-ray dose fractionation on the frequency of somatic mutations induced in tradescantia stamen hairs

X-rays were used to investigate the influence of dose fractionation on the induction of pink and colorless somatic mutations in stamen hair cells of Tradescantia clone 02. Inflorescences were exposed to a single acute dose of 60 rad, two acute doses of 30 rad, or three acute doses of 20 rad. The dose rate in all cases was 30 rad/min. Intervals between dose fractions were varied from 35 sec to 48 h and the mutation frequency was compared with that resulting after the single dose of 60 rad. The data show a reduction in mutation frequency for fractionation intervals longer than 15 and 6 min for pink and colorless mutations, respectively, but not for shorter intervals.One interpretation of the data predicts that pink mutation frequencies are reduced by 11% for fraction intervals of from 30 min to 6 h, and that colorless mutation frequencies are reduced by 24% for intervals of from 15 min to 6 h. The corresponding sparing effect of dose fractionation is equal to 6 rad for pink mutations and 9 rad at the colorless mutation endpoint. A calculation has been made which indicates that the percentages of the total repairable (presumably two-hit) damage that is repaired during fraction intervals up to 6 h, are 16 and 35% for pink and colorless mutations respectively.     

Chromosome translocations in the sex cells of mice subjected to chronic low-dose gamma-irradiation

Translocation induction in mouse spermatogonia by continuous whole-body gamma irradiation (radium 226) was studied. Total doses, delivered at a rate of 13.0 +/- 1.3 X 10(-4) rad/min for various time intervals, were 97, 195, 294 and 442 rad. Cytological examination within 3 to 4 months after irradiation indicated the presence of translocations in 0.16, 0.30, 0.75 and 1.29 percent respectively, of primary spermatocytes at diakinesis metaphase I. Data on translocation induction (Y) as related to total irradiation dose (D) were best fitted to a second power parabola equation (Y=5.1 X 10(-6)D2 + 7.32 X 10(-4) X D). The results obtained confirm that chronic gamma irradiation is of low genetic efficiency, and support the suggestion that there exists a dose-rate threshold under which no more changes in exposure efficiency will occur.     

Specific locus mutation rates after repeated small radiation doses to mouse oocytes

When female mice were given a dose of 20 × 10 rad X-rays, the specific locus mutation rate among offspring conceived up to 7 weeks after the end of treatment was 1/39887 or 0.18·10⁻⁷/rad/locus, whereas when the same total dose of 200 rad was given in a single exposure the mutation rate was 9/34813 or 1.85·10¹⁰⁻⁷/rad/locus. The lower mutation rate after the 20 × 10 rad dose was obtained whether the total of 200 rad was given over a period of 5 days or 4 weeks, and if only young conceived in the first 20 days, rather than 7 weeks, were considered. It is suggested that each 10 rad fraction had the same small effect, and hence that these results confirm and extend Russell's previous finding that the dose-response relationship for specific locus mutations in females is curved.     

Chromosome rearrangements from spermatogonial chronic neutron irradiation in mice.

Adult male mice were given a range of neutron doses at 80 +/- 20 mrad/h from a plutonium-beryllium source. Cytogenetic analysis indicated that chronic spermatogonial exposure to a mean total dose of 10, 30, 52, 98 or 150 rad produced translocations, sampled in spermatocytes four months later, amounting to 0.32, 0.99. 1.69, 1.91 and 1.65%, respectively. The dose response for the 0-52 rad range was linear. For higher doses, a better fit to the data was an expression with dose exponent above unity.     

Spreading EEG depression elicited by controlled deformation of the rat cerebral cortex.

The elicitation of cortical spreading depression (SD) by electronically generated mechanical stimuli was studied in 52 pentobarbital-anaesthetized rats. The stimuli were applied with a rod (target area 7.065 mm2) immersed at a constant velocity (3 X 10(-6) to 2 X 10(-1) m/sec) to varying depths (0 to 2.5 mm) from the surface of the cerebral cortex at the level of the bregma. SD elicitation depended on the velocity as well as the amplitude of deformation. The relationship between the velocity, v (m/sec), and amplitude, A (m), of threshold stimuli for SD elicitation is described by the equation 6.4 log A + log v = -21.08 in the given measuring range. The mean threshold velocity at a depth of 0.9 mm was 2.85 X 10(-2) m/sec; at 2.2 mm it was 8.54 X 10(-5) m/sec. If the rod was immersed to a given depth at a subthreshold velocity, to elicit SD by plunging it in a further 0.5 mm it was necessary to use velocities of a higher order than the one needed to obtain the same total deformation with a single threshold pulse. In extensive deformation, this velocity difference disappeared. Comparison of the results obtained by a free fall and electronically controlled deformation showed that the threshold of the mechanical stimulus eliciting spreading EEG depression could be expressed by the deformation velocity and amplitude better than by the amount of kinetic energy absorbed.     

X-ray- and chemically induced germ-line mutation causing phenotypical anomalies in mice

A large and significant increase of phenotypical anomalies was observed in the progeny of ICR parent mice treated before mating with X-rays, urethane, 7,12-dimethylbenz[a]anthracene, ethylnitrosourea (ENU), and 4-nitroquinoline 1-oxide, but the increase was not significant with furylfuramide. Major types of induced anomalies were cleft palate, dwarf, open eyelid, tail anomalies, and exencephalus. Dwarf, open eyelid and tail anomalies were predominant types of viable anomalies and were inherited as if they were dominant mutations with varying expressivity or penetrance. Incidence of prenatal anomalies increased with treated doses of X-rays, urethan, or ENU for both spermatozoa and spermatogonia. Spermatogonia were less sensitive to X-rays and urethane than spermatozoa, while ENU induced a very high incidence of prenatal anomalies by the spermatogonial treatment. In contrast to the previous works with X-rays, there was a clear, almost linear increase of anomalies in the dose range from 0 to 216 rad after spermatogonial exposure. For treatment of oocytes, there was also a clear increase with doses of X-rays and urethane. Doubling doses of X-rays for prenatal anomalies were 12 rad for spermatozoa, 27 rad for spermatogonia, and 19 rad for mature oocytes. These values are similar to those for ordinary mouse mutations. However, the mean rate of prenatal anomalies per rad (1.2 X 10(-4), 6.6 X 10(-5) and 9.1 X 10(-5) for spermatozoa, spermatogonia and mature oocytes, respectively) and that for 1 micrograms/g of ENU (3.4 X 10(-4) for spermatogonia) were 4-40 times higher than that of ordinary mutation in mice, because overall phenotypical abnormalities were scored in this study. Information obtained from the work on phenotypical anomalies is valuable to assess genetic risk of radiation and chemicals, because a majority of human genetic diseases show this kind of irregular and uncertain inheritance and most of the induced anomalies are similar to those found in humans.     

Effect of a high systemic background level of vitamin A on the teratogenicity of all-trans-retinoic acid given either acutely or subacutely

Groups of 12 Charles River CD virgin female rats were either supplemented with 25,000 IU/kg of vitamin A palmitate or not during the first 8 days of pregnancy and in the first experiment given a single dose of either 5 or 10 mg/kg of all-trans-retinoic acid (RA) on day 9 of pregnancy. In a second experiment, similar groups were given either 4 or 8 mg/kg RA daily from day 6 through day 15 so that each treatment with RA was given to vitamin A supplemented rats or nonsupplemented rats. The high systemic background of vitamin A increased the teratogenicity of the 10 mg/kg dose of RA given on day 9 by 50%, but reduced the teratogenicity of the 8 mg/kg dose given on days 6-15. The reasons for this paradox are discussed and related to the human propensity to self-medicate with megadoses of vitamins.     

Control of alkaline phosphatase activity in C3H10T1/2 cells: role of retinoic acid and cell density.

The enzyme alkaline phosphatase (AP) has been shown to be lost or inappropriately expressed during carcinogenesis in some tissues. Because retinoic acid (RA) appears to play a role in the normal regulation of the enzyme (RA up-regulates AP in a variety of cell types) we have suggested that altered AP expression in some cancers may be caused by a defect in the ability of the cells to respond normally to retinoid. We have begun to use the chemically transformable mouse embryo fibroblast cell, C3H10T1/2, to investigate this possibility. In this initial study we characterized AP regulation in normal C3H10T1/2 cells and show that: (1) 10(-7) M RA increases AP activity within 3-4 h in serum-free medium; (2) serum inhibits short-term induction (0-8 h) in a concentration-dependent manner (10% serum causes complete inhibition); (3) during long-term RA exposure (24 h and 48 h), induction can be detected in serum-containing medium; (4) AP induction is dose related at RA concentrations from 10(-10) M to 10(-6) M in serum-free medium; (5) 10(-5) M RA is ineffective at inducing AP in serum-free medium during 8 h but is the most effective concentration in serum-containing medium during 24 h and 48 h exposures; (6) AP inducibility by RA requires near-confluent cell densities; and (7) when cultures become confluent, cells become constitutive for AP and no longer require RA for enzyme expression. The effects of serum and cell density on AP inducibility by RA and implications of the RA up-regulation of AP for teratogenesis are discussed.     

Perfusion strategy for rosmarinic acid production by Anchusa officinalis

The production of an intracellular secondary metabolite rosmarinic acid (RA) by plant cell suspensions of Anchusa officinalis cultivated with intermittent medium exchange is investigated. Initially, a two-stage perfusion culture method was employed. After being cultured in the batch mode for ca. 6 days in B5 medium plus 3% sucrose, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 mg/L kinetin (2,4-D B5 medium), Anchusa culture was cultivated to high cell density by perfusion during the growth stage using a hormone-free Gamborg B5 medium supplemented with 6% sucrose. This was followed by a production stage, in which a complete medium exchange into B5 medium plus 3% sucrose and 0.25 mg/L naphthleneacetic acid (NAA) was conducted. The two-stage perfusion culture had a higher maximum culture RA concentration but a lower RA content per cell than the batch stock culture maintained in the 2,4-D B5 medium. Higher culture RA concentration was due primarily to high cell density. The high packed cell volume, however, seemed to reduce the synergistic effect of NAA on RA synthesis. Subsequently, a single-stage perfusion culture method was investigated. The best result was obtained by growing the culture in the batch mode for ca. 10 days using B5 medium supplemented with 3% sucrose and 0.25 mg/L NAA, followed by perfusing the culture with B5 medium plus 6% sucrose and 0.25 mg/L NAA at a constant perfusion rate of 0.1/day. A maximum cell dry weight of 35 g/L and a RA concentration of almost 4 g/L were achieved. This is the highest RA concentration ever reported in the Anchusa culture. (c) 1993 John Wiley & Sons, Inc.     

Induction of lethal mutations in female mice by 9 generations of gamma-irradiation during foetal development.

Female CBA mice were chronically gamma-irradiated in utero during either of two periods, the 10th to 14th days or the 14th to 18th days of gestation. The doses administered were 34 rad/generation in the earlier group and 160 rad/generation in the latter with dose rates of 0.3 rad/h and 1.7 rad/h, respectively. The doses were given through 9 generations. The effect of the irradiation was expressed as an increased frequency in the rate of recessive lethal equivalents by just above 4%. This corresponds to a mutation rate of 1.5 X 10(-4) mutation/rad/genome in the animals irradiated during the 10th to 14th gestational days and 0.3 X 10(-4) mutation/rad/genome in the 14th to 18th day group. As in earlier investigations, neither dominant mutations nor dominance effects of induced recessive lethal equivalents were found.     

Effect of low-dose acute X-irradiation on the frequencies of chromosomal aberrations in human peripheral lymphocytes in vitro

In a coordinated research programme sponsored by the International Atomic Energy Agency, the frequencies of chromosomal aberrations induced in peripheral blood lymphocytes (in vitro) by 250 kV X-rays at low doses (0.4, 1, 2, 3, 5, 10 and 30 rad) were determined. Blood from 2 donors was used to conduct one master experiment at these dose levels. The culture time used was 48 h and all samples including the controls were processed according to a standard protocol. The coded slides were scored by investigators from 10 participating laboratories. The main results are the following: (1) the frequencies of all types of chromosome aberrations at 0.4 rad are significantly lower than the control values; (2) there is no increase in the frequencies of dicentrics up to 2 rad and in those of terminal deletions up to 5 rad; (3) the mean frequencies of all aberrations considered together are not significantly different from one another at 1, 2 and 3 rad (P = 0.05); and (4) over the entire dose range the dose-effect relationship is clearly non-linear. A fit of these data to a linear quadratic model (E(D) = c + alpha D + beta D2) showed that the observed total aberration frequencies at doses 1, 2, 3 and 5 rad are below the curve defined by the model. The deviations can be explained by an altered kinetics of aberration production at very low doses probably due to DNA repair mechanisms operating these cells.     

Proinflammatory cytokines interact synergistically with epidermal growth factor to stimulate PGE2 production in amnion-derived cells.

Recent evidence has implicated cytokines and growth factors in the initiation of parturition in women. In the present study, the amnion-derived cell line WISH was used to determine whether proinflammatory cytokines (interleukins 1 beta, 6, and 8, tumor necrosis factor-alpha, and granulocyte/macrophage colony stimulating factor) could amplify epidermal growth factor-induced prostaglandin E2 production. WISH cells were preincubated with cytokines (0.0001-10 ng/ml) for 60 min and then challenged with EGF (10 ng/ml) for 4 hrs after which PGE2 production was measured by radioimmunoassay. EGF, IL-1 beta and TNF-alpha alone caused a dose-dependent increase in PGE2 production, while IL-6, IL-8 and GM-CSF were ineffective over the dose range tested. When cells were preincubated with IL-1 beta or TNF-alpha, there was a dose-dependent potentiation of EGF-induced PGE2 production that was greater than the sum of EGF alone and IL-1 beta or TNF-alpha alone. In each case, the minimum dose of IL-1 beta or TNF-alpha which amplified EGF-induced PGE2 production was 0.1 ng/ml (p less than 0.05, Student's t-test). These data show that low concentrations of IL-1 beta or TNF-alpha may serve to amplify EGF-mediated PGE2 biosynthesis in amnion-derived cells and suggest that cytokines may modulate EGF function in responsive cells.     

Collagen degradation by superoxide anion in pulse and gamma radiolysis

Delipidated collagen fibrils reconstituted from acid-soluble calf skin collagen, suspended in 50 mM phosphate buffer, pH 7.4, containing 100 mM sodium formate, were submitted to pulse radiolysis in Febetron devices or to gamma radiolysis in a 60Co irradiator. A collagen degradation process was found. The kinetics of this degradation was followed by evaluation of the amount of 4-hydroxyproline present in the small peptides liberated during the irradiation period. The yield of 4-hydroxyproline small peptides was low (0.1 mol/100 eV for an initial collagen concentration 3.2 microM). It increased linearly with the dose of irradiation and the concentration of collagen in suspension. The kinetic competition between O2-. dismutation and O2-. reaction with collagen was studied by pulse radiolysis at several concentrations of collagen. A value of the kinetic constant of k(O2-. + collagen) = 4.8 . 10(6) mol-1.l.s-1 was determined.