Cellular reprogramming in the F3 mouse with paternal F0 radiation history.

PURPOSE: In the mouse, paternal F0 acute irradiation of Type B spermatogonia produces biological effects in offspring, including altered signalling kinase activities and protein levels. It was hypothesized that these effects represented cellular reprogramming that would alter the response of somatic cells in these offspring to an acute ionizing radiation exposure. MATERIALS AND METHODS: Nineteen-day-old third generation (F3) CD1 mice with and without an acute 1.0 Gy paternal F0 radiation history each received an acute dose of 1.0 Gy from attenuated 137C n-rays. Kidney PKC and MAPK activities, and p53 protein levels were evaluated immediately following F3 irradiation. The same endpoints and DNA damage were evaluated in kidney-derived fibroblast primary cell cultures 3 weeks post-irradiation. RESULTS: Kidneys had significantly decreased PKC and MAPK activities and p53 protein levels related to F0 irradiation and increased PKC and MAPK activities following F3 irradiation irrespective of F0 radiation history. Kidney-derived fibroblasts had significant changes or strong trends for all selected endpoints based upon cross-interaction of F0 radiation history with F3 irradiation. Comet assays demonstrated significantly increased DNA damage in fibroblasts related to F0 irradiation and increased DNA damage following F3 irradiation. However, significantly decreased F3 irradiation damage was demonstrated based upon cross-interaction of F0 radiation. CONCLUSIONS: The data suggest that irradiation of paternal F0 Type B spermatogonia resulted in cellular reprogramming causing offspring with this radiation history to have altered responses to acute somatic n-irradiation.

Heritable effects of paternal irradiation in mice on signaling protein kinase activities in F3 offspring.

We evaluated F3 mouse offspring from paternal F0 attenuated 137Cs gamma-irradiation (1.0 Gy) for heritable effects on gene products that can modulate cell proliferation rate and that may be markers for genomic instability. The F3 generation was selected for evaluation as a stringent test for heritability of effects from paternal F0 germline irradiation. Male CD1 mice were bred 6 weeks after irradiation so that the fertilizing sperm were type B spermatogonia at the time of irradiation. The resulting F1 males were bred to CD1 females to produce F2 four-cell embryos. The F2 embryos with a radiation history were paired with 'control' CD1 four-cell embryos that were heterozygous for the neo transgene. These F2 XY-XY chimeras, consisting of cells derived from both an embryo with a paternal F0 radiation history and a control embryo, were transferred to foster mothers, raised to adulthood and bred to produce F3 offspring. F3 offspring were evaluated for hepatic activities of receptor tyrosine kinase, protein kinase C and MAP kinase and for protein levels of nuclear p53 and p21(waf1). All three protein kinase activities were altered and nuclear levels of p53 and p21(waf1) protein were higher in the group of offspring that included F3 offspring with a paternal F0 radiation history than in littermates in the neo-positive control group. To our knowledge, this is the first observation in the descendants of paternal germline irradiation of effects on signal protein kinase activities and downstream nuclear target proteins that can influence cell proliferation rates.

The stability of arterial blood gases during transportation of patients using the RespirTech PRO.

The transportation of critically ill patients requiring mechanical ventilation is recognized as a high-risk and expensive procedure. Approaches have included using manual bag-type valve resuscitators and expensive portable transport ventilators. This study evaluated the effectiveness of the inexpensive portable RespirTech PRO (RTP) gas-powered automatic resuscitator during intrahospital transport of critically ill mechanically ventilated patients. Twenty medical intensive care patients on stable mechanical ventilator settings had arterial blood gas and vital sign determination before routine transport out of the intensive care unit (ICU). Repeat measurements were made during transport approximately 30 minutes after being placed on the RTP portable pressure-cycled automatic resuscitator using an FiO2 of 100%. During use of the RTP for transport, there were no statistically significant variations observed in mean arterial blood pressure [82 +/- 11 SD (range 65 to 112) mm Hg before transport versus 85 +/- 14 SD (range 59 to 110) mm Hg during transport], heart rate [94 +/- 16 SD (range 74 to 127) beats/min) before versus 96 +/- 17 SD (range 69 to 132) beats/min during], arterial pH [7.41 +/- 0.07 SD (range 7.31 to 7.58) before versus 7.42 +/- 0.05 SD (range 7.37 to 7.52) during], and PaCO2 [43 +/- 10 SD (range 26 to 65) mm Hg before versus 43 +/- 10 SD (range 27 to 61 mm Hg) during]. Because the FiO2 before transport was 63 +/- 26 SD (range 30% to 100%) versus 100% during transport using the RTP, the mean PaO2 was significantly increased from 124 +/- 86 SD (range 57 to 367) mm Hg before transport to 297 +/- 168 SD (range 65 to 537) mm Hg during (P< .001). No transportation associated clinical adverse events were noted. Several previous investigations have shown that portable ventilators are safe and effective in intrahospital transport of mechanically ventilated patients. This study showed that the portable pressure-cycled RTP also allows safe transportation of mechanically ventilated ICU patients. By analogy, the RTP is potentially useful as an automatic resuscitator for emergency medical care. This RTP is a disposable resuscitator/ventilator device that provides an inexpensive alternative for transporting ventilator-dependent patients.

Paternal effects from methamidophos administration in mice.

In this study, the mouse was used to evaluate paternal germline exposure to the organophosphate methamidophos for its potential to produce adverse effects on spermatozoa and in the offspring. There have been reports that organophosphate exposure can increase abnormal sperm morphology in mice. However, effects transmitted to the offspring following paternal exposure have not been reported previously. The maximum tolerated dose (MTD) was 7.5 mg kg(-1) body weight and this dose resulted in no deaths, although blood plasma cholinesterase activity was still decreased. Males were euthanized 4 weeks after an acute intraperitoneal injection of methamidophos (0.5, 3.75, 5.0, and 7.5 mg kg(-1) body wt) and the number of spermatids per gram testes and sperm morphology were analyzed. In this study, abnormal sperm morphology on a per group basis exhibited a dose-response significantly related to increased methamidophos exposure as indicated by regression analysis and a nested ANOVA (p < 0.0001). Preimplantation embryos that were conceived 6 weeks after paternal methamidophos exposure (5 mg kg(-1) body wt) exhibited a significant increase in cleavage arrest. Fertility of males was also affected as shown by a decrease in the number of two- to four-cell embryos per male (postexposure week 6) and an increase in the number of degenerated embryos (postexposure weeks 4-6). We conclude that methamidophos may have the potential to produce transmissible adverse embryonic effects following an acute paternal germline exposure.

Continuous nebulization therapy for asthma with aerosols of beta2 agonists.

BACKGROUND: Various studies have demonstrated the benefits of continuous nebulization therapy for delivering aerosols of the beta2 agonists such as terbutaline sulfate or albuterol sulfate to patients with severe asthma and/or impending respiratory failure. OBJECTIVE: The purpose of this investigation was to explicate the operational factors associated with the use of nebulizers for extended aerosol respiratory therapy including those factors that affect the prescribed aerosol dosages and the relationship to actual delivery of prescribed drugs to the respiratory airways of the lungs of a patient under treatment conditions. METHODS: Operational characteristics and methods have been investigated for use of long-running nebulizers for continuous nebulization therapy. Factors considered were particle size distribution, setup conditions, aerosolization concentrations and rates, delivery fraction of aerosol reaching patient, and changes in medication concentration during extended operation. With a large volume nebulizer, aerosols can be delivered to the patient without dilution via a standard open mask for up to eight hours without refill. The pneumatic HEART nebulizer with 240 mL reservoir was evaluated. RESULTS: The nebulizer was operated from a single compressed air or oxygen source and found to provide from 10 to 15 L/min of aerosol with 38 to 50 microL of aerosolized medicine per liter of air (or oxygen) and utilize from 30 to 56 mL/hour of medicinal liquid. The mass median aerodynamic diameter of the aerosol droplets was found to be about 2.0 microm (sigma(g) = 2.7). Delivery efficiency to the patient mask was about 90%. The aerosolized medicine delivered to the patient can be increased by adjusting the flow rate of the gas source or changing the solution concentration of medicine. Typically, several milligrams of drug can be delivered to the patient as inhaled aerosol per hour of treatment of which about one-quarter can be expected to be deposited in the lungs. During eight hours of operation the concentration of medicinal solution increased by about a factor of two because of water evaporation. CONCLUSIONS: Continuous nebulization therapy is an important means of treating patients with severe asthma. Dosage criteria can be established based on the operating characteristics of the nebulizer system, drug solution concentration, and patient respiration.

Aerosol delivery of lipid:DNA complexes to lungs of rhesus monkeys.

PURPOSE: The potential use of aerosol delivery for non-viral gene therapy was tested by nebulization of lipid:DNA complexes to the lungs of rhesus monkeys. METHODS: Four female rhesus monkeys were dosed with lipid:DNA formulations via aerosol inhalation, where the DNA coded for the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR) protein. Delivery of DNA was determined in lung samples by polymerase chain reaction (PCR) by qualitative and quantitative methods. Transgene specific messenger RNA was measured by reverse transcriptase PCR (RT-PCR) and protein expression and localization were evaluated by immunohistochemistry (IHC). RESULTS: Approximately four mg of DNA, complexed with cationic lipid 1.2-dimyristoyl-sn-glycero-3-ethylphosphatidylcholine (EDMPC) and cholesterol were delivered to the lungs of animals by airjet nebulizer. Three days after dosing, tissue samples from the lung were collected and shown to have vector specific DNA, RNA and the presence of CFFR protein. Specifically, the hCFTR protein was distributed widely, although non-uniformly, throughout airway epithelium being located on the apical surface of epithelial cells. Importantly, no adverse clinical effects were observed and the lungs showed no histological abnormalities or signs of acute inflammation. CONCLUSIONS: This study shows that lipid:DNA formulations based on EDMPC and cholesterol can be administered to primates by nebulization resulting in measurable expression of the hCFTR protein. The absence of inflammation is also encouraging and such systems may have utility for delivery of genes to the lungs for the treatment of a variety of pulmonary diseases including cystic fibrosis.

In vitro fertilization rate of mouse oocytes with spermatozoa from the F1 offspring of males irradiated with 1.0 Gy 137Cs gamma-rays.

Previous studies suggest that the spermatozoa from acutely irradiated male mice exhibit a reduced fertilization rate in vitro with the maximum decrease occurring for spermatozoa produced 6 weeks after irradiation (Y. Matsuda et al., Mutation Res. 142 (1985) 59-63). We have found that spermatozoa from unirradiated F1 males conceived 6 weeks after paternal F0 irradiation also exhibit a significantly reduced fertilization rate in vitro. After acute 137Cs gamma-irradiation yielding an absorbed dose of 1.0 Gy, adult CD1 F0 male mice were mated at weekly intervals with unirradiated female CD1 mice. Unirradiated adult males from F1 litters conceived 5 and 6 weeks after paternal F0 irradiation were allowed to mature. Their epididymal spermatozoa were evaluated for in vitro fertilization rates using oocytes from unirradiated 8-12-week-old CD1 females. The mean fertilization rate for spermatozoa from F1 males conceived 5 weeks after paternal F0 irradiation (80.74 +/- 15.74 SD %, n = 5) did not differ significantly from the control fertilization rate (89.40 +/- 10.94 SD %, n = 8). However, the fertilization rate for spermatozoa from F1 males conceived 6 weeks after paternal F0 irradiation (56.14 +/- 21.93 SD %, n = 5) was significantly less than the fertilization rate for control spermatozoa (p < 0.006) or for that of the F1 males conceived 5 weeks after paternal F0 irradiation (p < 0.04). These data suggest that spermatozoa obtained 6 weeks after paternal F0 irradiation can transmit a decrease in fertilization rate to the F1 generation males as well as exhibit decreased fertilization rate themselves when tested directly in vitro.

Impaired cell proliferation in mice that persists across at least two generations after paternal irradiation.

Irradiation of male F0 mice 6 to 7 weeks prior to mating causes significant changes in the proliferation of F1 and F2 embryonic cells. These changes are revealed as a competitive cell proliferation disadvantage in chimera assays when the affected embryo is paired with a normal embryo in an aggregation chimera. This effect has been observed previously to be transmitted to F1 embryos for absorbed doses from 0.01 to 1.0 Gy; 0.01 Gy is about 100-fold lower than detectable using conventional germline mutation assays. However, until now there has been no reported cross-generation heritability. We now report that this competitive cell proliferation disadvantage persists without degradation in the F2 generation of embryos when F0 males received 1.0 Gy from gamma irradiation 6 and 7 weeks prior to conception of F1 males.

Safety of airway gene transfer with Ad2/CFTR2: aerosol administration in the nonhuman primate.

In this study, the safety and efficacy of aerosol delivery to non-human primates of an adenoviral vector encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR) were evaluated. The technique of concurrent flow spirometry was used to determine the deposited dose of Ad2/CFTR-2, which ranged from 3 to 8 x 10(10) I.U. Transgene DNA was detected by the polymerase chain reaction (PCR) in lung tissue from all treated animals, and human CFTR mRNA was detected on days 3, 7, and 21 post-exposure. The treatment was well tolerated, with no evidence of respiratory distress. Histologic changes in the lungs from Ad2/CFTR-2-treated animals were mild and, overall, indistinguishable from animals exposed to aerosolized vehicle. One vector-treated animal demonstrated an increase in lavage lymphocyte numbers 3 days after treatment and another had an abnormal chest radiograph 14 days after treatment. A third vector-treated animal had histologic evidence of a bronchointerstitial pneumonia 7 days after aerosol treatment that resolved by day 21. This study demonstrated that Ad2/CFTR-2 can effectively be delivered to the lungs of nonhuman primates and result in minimal adverse effects.

Transmitted proliferation disadvantage from mouse oocytes labeled in vivo with [3H]thymidine: radiosensitive target considerations.

This study was conducted to test the hypothesis that a nuclear target is involved in the embryonic cell proliferation disadvantage transmitted by irradiated mouse oocytes and detected by the chimera assay. In this assay, the cells from the irradiated embryo exhibit a competitive cell proliferation disadvantage when they are challenged by direct cell-cell contact with cells from a normal embryo in an aggregation chimera. Here, six pregnant CD-1 mice received a total of 1.85 TBq tritiated thymidine (TdR) delivered by multiple intraperitoneal injections during days 13-15 postconception. Six more pregnant mice were sham-injected to provide control embryos. Sixty randomly selected female progeny were mated at 47 days of age and their 4-cell embryos tested in the chimera assay. The mean proliferation ratio (PR, number of cells from the experimental embryo divided by total cell number of the chimera) for experimental chimeras was 0.45 +/- 0.02 SE (n = 43), which was significantly less than the mean PR of 0.49 +/- 0.01 SE (n = 47; p = 0.02) for control chimeras. This entire experiment was repeated, with similar results. A comparison for TdR confined to the nucleus (i.e., mean beta-ray range is only 0.7 microm) with the relationship for uniform irradiation by 137Cs gamma-rays demonstrates that these two very different modes of dose delivery produce essentially identical PRs. These results in vivo suggest a nuclear DNA target for embryonic cell proliferation disadvantage consistent with our previous findings in vitro.

Mouse immature oocytes irradiated in vivo at 14-days of age and evaluated for transmitted effects using the aggregation embryo chimera assay.

A previous study using the mouse-preimplantation-embryo-chimera assay demonstrated a reproducible transmitted effect (proliferation disadvantage observed in early embryos) from females irradiated as 49-day-old adults using 0.15 Gy of gamma rays and then mated seven weeks later, i.e., embryos were from oocytes that were immature at time of irradiation. Because mouse immature oocytes are known to be much more radiosensitive to cell killing in juveniles than in adults, a follow-on study was performed here using 14-day-old juvenile mice. In contrast to adults, the exposure of juveniles to 0.15 Gy of gamma rays did not result in a detectable transmitted proliferation disadvantage when animals were mated 7 or 12 weeks later. This observation is discussed in light of previous studies on mouse immature oocytes and embryo chimeras.

Radionuclides in fishes and mussels from the Farallon Islands Nuclear Waste Dump Site, California.

The Farallon Islands Nuclear Waste Dump Site (FINWDS), approximately 30 miles west of San Francisco, California, received at least 500 TBq encapsulated in more than 47,500 containers from approximately 1945 to 1970. During several seasons in 1986/87 deep-sea bottom feeding fishes (Dover sole = Microstomus pacificus; sablefish = Anoplopoma fimbria; thornyheads = Sebastolobus spp.) and intertidal mussels (Mytilus californianus) were collected from the vicinity of the FINWDS and from comparable depths at a reference site near Point Arena, CA. Tissues were analyzed for several radionuclides (137Cs, 238Pu, 239+240Pu, and 241Am). Radionuclide concentrations for fish mussel tissue ranged from non-detectable to 4,340 mBq kg(-1) wet weight, with the following means for Farallon fishes: 137Cs = 1,110 mBq kg(-1); 238Pu = 390 mBq kg(-1); 239+240Pu = 130 mBq kg(-1); and 241Am = 1,350 mBq kg(-1). There were no statistically significant differences in the radionuclide concentrations observed in samples from the Farallon Islands compared to reference samples from Point Arena, CA. Concentrations of both 238Pu and 241Am in fish tissues (from both sites) were notably higher than those reported in literature from any other sites world-wide, including potentially contaminated sites. Concentrations of 239+24OPu from both sites were typical of low values found at some contaminated sites worldwide. These results show approximately 10 times higher concentrations of 239+240Pu and approximately 40-50 times higher concentrations of 238Pu than those values reported for identical fish species from 1977 collections at the FINWDS. Radionuclide concentrations were converted to a hypothetical per capita annual radionuclide intake for adults, yielding the following values of annual Committed Effective Dose Equivalent (CEDE) from ionizing radiation emitted from these radionuclides: 0.000 mSv y(-1) for 137Cs, 0.009 mSv Y(-1) for 228Pu, and 0.003 mSv y(-1) for 239+240Pu. For 241Am, projected CEDE for Dover sole, sablefish, and thornyheads were higher, averaging 0.03 mSv y(-1). The observed isotopic ratio of 238Pu/239+240Pu was about 4 (which is two orders of magnitude higher than the ratio of 0.03 associated with fallout from weapons tests and accidental releases in the north temperate zone of the earth), indicating a considerably higher environmental mobilization for 238Pu compared to 239+240Pu. Likewise, the observed ratio of 241Am/239+240Pu of about 30 was nearly two orders of magnitude higher than the fallout ratio of 0.43 in the north temperate zone of the earth. The projected ionizing radiation CEDE to people from the ingestion of fish with fallout radionuclides was three times higher for 241Am than from the plutonium isotopes.

Toxicokinetic modeling of dose-dependent formate elimination in rats: in vivo-in vitro correlations using the perfused rat liver.

The dose-dependent elimination of formate was investigated in the rat using both in vitro and in vivo systems. The in situ perfused liver was used to define the kinetics of hepatic metabolism and obtain initial in vitro estimates of the hepatic metabolism kinetic parameters. Formate was eliminated from the perfused rat liver following Michaelis-Menten kinetics. Estimates of the Michaelis-Menten parameters obtained from the perfused liver studies were used in a two-compartment pharmacokinetic model of the dose-dependent elimination of formate in vivo. This model consisted of a central, well-mixed compartment and a urine compartment. Other features of the model included (1) endogenous production of formate, (2) Michaelis-Menten hepatic metabolism of formate, and (3) renal excretion consisting primarily of glomerular filtration and saturable tubular reabsorption. A good fit of the model to the observed in vivo data was obtained (overall r2 = 0.978). AR dose dependencies of the data could be adequately fitted using a single set of model parameters. Initial estimates of the Michaelis-Menten parameters, Vmax and Km, obtained from the perfused liver system, were within 40% of the final fitted values of these parameters in the in vivo model, indicating the utility of the perfused liver system for performing in vitro-in vivo correlations.

Development of partition coefficients, Vmax and Km values, and allometric relationships.

A knowledge of the methods used to obtain partition coefficients, Vmax, and Km values, and the use of allometric relationships is essential to understanding their role in physiologically based pharmacokinetic (PBPK) models. Vial equilibration methods for obtaining the partition coefficients of volatile and nonvolatile compounds were presented using the results from studies with p-chlorobenzotrifluoride (PCBTF) and isofenphos, respectively. Partition coefficients for volatile and nonvolatile compounds from published studies were included. Several published in vivo inhalation (gas uptake) studies and in vitro enzyme studies were presented to demonstrate several methods for obtaining Vmax and Km values. Allometric equations used in PBPK models for body weight scaling of respiration and cardiac rates between species were presented along with equations for within species body weight scaling of Vmax.

Particle inhalability curves for humans and small laboratory animals.

Several inhalability curves for nose breathing in humans have been developed. No studies have been designed specifically to develop inhalability functions for animals, although it has been shown that pulmonary deposition of large particles (> 4-5 microns) via inhalation is minimal in laboratory animals [Raabe et al., Inhaled Particles VI, pp. 53-63. Pergamon Press, Oxford (1988)]. The logistic function was fitted to these animal deposition data of Raabe et al. (1988) to estimate an inhalability curve for laboratory animals. The logistic function was also fitted to the human data of Breysse and Swift [Aerosol Sci. Technol. 13, 459-464 (1990)] for comparison. The results suggest that ambient concentration is a good predictor (inhalability > 95%) of inhaled concentration for humans for particles < 11 microns dae. In small laboratory animals, however, the inhalable portion of the ambient concentration is predicted to be 95% for 0.7 microns dae particles but declines to 45% for 10 microns dae particles. It is, therefore, important to consider the effects of inhalability when estimating dose delivered to the target tissue in animals. In comparing delivered doses between animals and humans, adjusting for inhalability may change not only the magnitude of the difference but also which species is predicted to receive a greater delivered dose.

Radiosensitive target in the early mouse embryo exposed to very low doses of ionizing radiation.

We exposed mouse preimplantation embryos in vitro to either tritiated water (HTO) or tritiated thymidine (TdR) to determine whether the radiosensitive target was nuclear or extranuclear for embryonic cell proliferation disadvantage in the mouse embryo chimera assay. 8-cell embryos were incubated in either HTO or TdR for 2 h and paired with non-irradiated control embryos to form chimeras. Chimeras were cultured for an average of 20.2 h to allow for 2-3 cell cycles and then partially dissociated to obtain the number of progeny cells contributed by the two partner embryos for each chimera. These values were expressed as a "proliferation ratio" (number of cells from the irradiated embryo: total number of cells in the chimera). A ratio significantly less than 0.50 indicates that the experimental embryo expressed an embryonic cell proliferation disadvantage, which is the endpoint of this assay. The activity concentrations of HTO and TdR were adjusted so that both would deliver comparable mean absorbed nuclear doses during the combined initial 2-h irradiation incubation and subsequent 20.2 h chimera incubation periods. Although nuclear doses were comparable under these conditions, the extranuclear dose delivered by the uniformly distributed HTO was about 100 times greater than the extranuclear dose delivered by TdR for each given nuclear dose. Consequently, obtaining mean TdR proliferation ratios < or = mean HTO proliferation ratios would be evidence for a nuclear target while obtaining mean HTO proliferation ratios < mean TdR proliferation ratios would be evidence for an extranuclear target. TdR consistently produced lower mean proliferation ratios over a range of doses from 0.14 Gy to 0.43 Gy. Therefore, we conclude that the radiosensitive target for this endpoint is nuclear.

Embryonic effects transmitted by male mice irradiated with 512 MeV/u 56Fe nuclei.

High-energy, high-charge nuclei may contribute substantially to the yearly equivalent dose in space flight from galactic cosmic radiation (GCR) at solar minimum. The largest single heavy-ion component is 56Fe. We used the mouse embryo chimera assay to test 512 MeV/u 56Fe nuclei for effects on the rate of proliferation of embryonic cells transmitted by sperm from irradiated mice. Male CD1 mice were acutely irradiated with 0.01, 0.05 or 0.1 Gy (LET, 184 keV/micron; fluence, 3.5 x 10(4)-3.3 x 10(5) nuclei/cm2; average dose rate, 0.02 Gy/min) at the Lawrence Berkeley Laboratory BEVATRON/BEVALAC Facility in Berkeley, CA. Irradiated males were bred weekly for 7 weeks to nonirradiated females and their four-cell embryos were paired with control embryos, forming aggregation chimeras. After 30-35 h of culture, chimeras were dissociated to obtain "proliferation ratios" (number of cells contributed by the embryo from the irradiated male/total number of cells in the chimera). Significant dose-dependent decreases in proliferation ratios were obtained across all three dose groups for postirradiation week 2 (P < 0.05 to P < 0.003). The 0.01- and 0.05-Gy dose groups also produced significant decreases in proliferation ratios for postirradiation week 1 (P < 0.05 to P < 0.01) and the 0.05-Gy dose group produced significant decreases in proliferation ratios for postirradiation week 6 (P < 0.05). Postirradiation weeks 1, 2 and 6 correspond to irradiation of epididymal sperm, testicular spermatids and spermatogonia, respectively. We calculate that only about 5% of sperm in the 0.1-Gy, 2.5% in the 0.05-Gy and 0.5% in the 0.01-Gy dose groups sustained direct hits from 56Fe nuclei. However, up to 47% of sperm during postirradiation weeks 1 and 2 transmitted proliferation ratios that were at or below one standard deviation from control mean proliferation ratios. Morphometry on sectioned testes showed a significant log-linear dose response for cell killing of type B spermatogonia, which are the most radiosensitive stage of spermatogenesis and which would have been tested as mature sperm during postirradiation week 6. We conclude that amplification from secondary radiation produced in the mouse and/or from diffusible chemical products arising from hit sperm and adjacent cells contributed to the high incidence of transmitted effects on proliferation of embryonic cells.

Bone sarcoma characteristics and distribution in beagles injected with radium-226.

A total of 155 primary bone sarcomas were found in 131 of the 246 beagles injected with 226Ra and 5 primary bone sarcomas were found in 4 of the 158 unexposed controls. Of these 155 bone sarcomas, 146 (94%) were osteosarcomas and 9 were non-osteosarcomas. An additional 31 primary bone sarcomas (28 osteosarcomas) developed in 44 dogs terminated from the main study because of limb amputation for bone sarcoma. Non-osteosarcomas predominated in both the controls and the second lowest of six logarithmically increasing dose levels (there were no bone sarcomas in the lowest dose group). Osteosarcomas predominated at the higher dose levels, and incidence tended to increase as dose increased. The 146 osteosarcomas were distributed quite evenly between males and females (72:74). Of the 9 non-osteosarcomas, 6 occurred in males and 3 in females. The ratio of bone sarcomas of the appendicular skeleton to those in the axial skeleton was 110:45, with osteosarcomas occurring more often in the appendicular skeleton (108:38). Cases of multiple primary bone sarcomas in dogs injected with 226Ra were found only in the four highest dose groups. Amputations were performed on 44 of the 96 dogs (94 injected and 2 unexposed) that developed appendicular bone sarcomas. A statistical study of the distribution of bone sarcomas among 16 separate bone groups showed a statistically significant correlation to cancellous skeletal surface, but the variability among bone groups was too large for this relationship to be of real predictive value. It is postulated that the distribution of bone sarcomas reflects primarily the relative cell division rates in the bone groups and secondarily the radiation dose distribution, with the highest occurrence of bone sarcoma in the humeri, pelvis, femora and tibiae/fibular tarsal, and no occurrence in the coccygeal vertebrae, sternum, forepaws or hindpaws.

Bone sarcoma characteristics and distribution in beagles fed strontium-90.

A total of 66 primary bone sarcomas were diagnosed in 47 beagles; 43 of these dogs were part of the 403 beagles fed 90Sr and 4 were part of the 162 controls. Multiple primary bone sarcomas were found in 15 of the 47 beagles (32%). The incidence of multiple primary bone sarcoma was restricted to the two highest dose groups, except for a single control dog which developed two bone sarcomas. A threshold-like radiation dose response was observed; no sarcomas were observed in the lowest three dose groups, but the number of primary bone sarcomas increased rapidly in the higher dose groups. Of the 66 primary sarcomas, 49 were osteosarcomas (74%). As the dose increased, the proportion of osteosarcomas increased sharply, 4/10 (40%), 26/29 (90%), and 16/18 (89%), in the three highest dose groups. Thirteen of the bone sarcomas of other types occurred in males, and 4 in females, whereas 21 osteosarcomas occurred in males, and 28 in females. The ratio of bone sarcomas of the appendicular skeleton to those in the axial skeleton was 40:26, with osteosarcomas occurring more often in the appendicular than the axial skeleton (32:17), whereas nonosteogenic tumors showed no predilection (8:9). A statistical study of the distribution of bone sarcomas among 16 separate bone groups showed a correlation only with the distribution of cancellous bone volume-to-surface ratio and not with either skeletal mass distribution or dose distribution. The highest occurrence of sarcomas was in the humeri, femora, and mandible, and no occurrence in the coccygeal vertebrae, paws, or sternum. It is postulated that the distribution of bone sarcomas reflects a critical combination of the osteosarcoma precursor cell population, their cell division rate, and the radiation dose absorbed by these cells.