Three-component-mediated serotype conversion in Pseudomonas aeruginosa by bacteriophage D3.

Bacteriophage D3 is capable of lysogenizing Pseudomonas aeruginosa PAO1 (serotype O5), converting the O-antigen from O5 to O16 and O-acetylating the N-acetylfucosamine moiety. To investigate the mechanism of lysogenic conversion, a 3.6 kb fragment from the D3 genome was isolated capable of mediating serotypic conversion identical to the D3 lysogen strain (AK1380). The PAO1 transformants containing this 3.6 kb of D3 DNA exhibited identical lipopolysaccharide (LPS) banding patterns to serotype O16 in silver-stained SDS-PAGE gels and displayed reactivity to an antibody specific for O-acetyl groups. Further analysis led to the identification of three open reading frames (ORFs) required for serotype conversion: an alpha-polymerase inhibitor (iap); an O-acetylase (oac); and a beta-polymerase (wzybeta). The alpha-polymerase inhibitor (Iap) is capable of inhibiting the assembly of the serotype-specific O5 B-band LPS and allows the phage-encoded beta-polymerase (Wzybeta) to form new beta-linked B-band LPS. The D3 phage also alters the LPS by the addition of O-acetyl groups to the FucNAc residue in the O-antigen repeat unit by the action of the D3 O-acetylase (Oac). These three components form a simple yet elegant system by which bacteriophage D3 is capable of altering the surface of P. aeruginosa PAO1.

Modular glovebox connector and associated good practices for control of radioactive and chemically toxic materials.

Design and associated good practices are described for a modular glovebox connector to improve control of radioactive and chemically toxic materials. The connector consists of an anodized aluminum circular port with a mating spacer, gaskets, and retaining rings for joining two parallel ends of commercially available or custom-manufactured glovebox enclosures. Use of the connector allows multiple gloveboxes to be quickly assembled or reconfigured in functional units. Connector dimensions can be scaled to meet operational requirements for access between gloveboxes. Options for construction materials are discussed, along with recommendations for installation of the connector in new or retrofitted systems. Associated good practices include application of surface coatings and caulking, use of disposable glovebags, and proper selection and protection of gasket and glove materials. Use of the connector at an inhalation toxicology research facility has reduced the time and expense required to reconfigure equipment for changing operational requirements, the dispersion of contamination during reconfigurations, and the need for decommissioning and disposal of contaminated enclosures.

Toxicity of inhaled 91YCl3 in dogs.

This study was conducted in dogs to determine the toxicity of inhaled 91YCl3, which is of interest because 91Y is a fission-product radionuclide that is abundant in a reactor inventory after sustained operation. Yttrium-91 has a short half-life, 59 days, and decays with the emission of beta particles and low-yield gamma rays. The study was conducted in 58 beagle dogs with equal numbers of males and females. Forty-six dogs inhaled the 91YCl3 aerosol, while 12 served as controls. Four exposure levels were used. To determine the long-term retained burden (LTRB) of 91Y, each dog was periodically whole-body counted and its excreta were analyzed radiochemically. Over time, the 91Y transferred from the lung primarily to the skeleton and liver. The dogs were observed over their life spans for biological effects. Fatal hematological dyscrasia occurred from 12 to 33 days after exposure in the dogs with the highest LTRBs. Bone-associated tumors of the nasal and oral mucosae occurred in 5 dogs from 2000 to 5800 days after they inhaled the 91YCl3 aerosols. Five dogs died with malignant lung tumors and 2 dogs with malignant liver tumors. The results of this study were compared to those from similar studies in beagles that inhaled 90SrCl2 or 144CeCl3 or were injected with 137CsCl. The comparison showed that the biological effects in each study were clearly dependent on the cumulative doses to critical organs.

Pulmonary carcinogenicity of relatively low doses of beta-particle radiation from inhaled 144CeO2 in rats.

This study was conducted to examine the carcinogenic effects of inhaled beta-particle-emitting radionuclides, particularly in lower dose regions in which there were substantial uncertainties associated with available information. A total of 2751 F344/N rats (1358 males and 1393 females) approximately 12 weeks of age at exposure were used. Of these, 1059 rats were exposed to aerosols of 144CeO2 to achieve mean desired initial lung burdens (ILBs) of 18 kBq (low level), 247 rats to achieve mean ILBs of 60 kBq (medium level) and 381 rats to achieve mean ILBs of 180 kBq (high level). Control rats (total of 1064) were exposed to aerosols of stable CeO2. Based on the 95% confidence intervals of the median survival times and the cumulative survival curves, there were no significant differences in the survival of groups of female and male exposed rats relative to controls. The mean lifetime beta-particle doses to the lungs of the rats in the four groups were: low level, 3.6 +/- 1.3 (+/-SD) Gy; medium level, 12 +/- 4.5 Gy; and high level, 37 +/- 5.9 Gy. The crude incidence of lung neoplasms increased linearly with increasing doses to the lungs (controls, 0.57%; low level, 2.0%; medium level, 6.1%; and high level, 19%). The estimated linear risk coefficients for lung neoplasms per unit of dose to the lung were not significantly different for the three dose levels studied. The risk coefficient at the lower level was 39 +/- 14 (+/-SE) excess lung neoplasms per 10(4) rat Gy; at the medium level the risk was 47 +/- 12; and at the higher level the risk was 50 +/- 9.0. The relationship of beta-particle dose to the lung and the crude incidence of lung neoplasms was described adequately by a linear function. We concluded that the risk of lung neoplasms in rats per unit of radiation dose did not increase with decreasing mean beta-particle dose to the lung over the range of 3.6 to 37 Gy. The weighted average of these three values was 47 +/- 6.4 (+/-SE) excess lung neoplasms per 10(4) rat Gy. To extend the risk coefficients for lung neoplasms to lower doses by experimentation will require much larger numbers of rats than used in this study.

Estimation of the dose of radon progeny to the peripheral lung and the effect of exposure to radon progeny on the alveolar macrophage.

Groups of rats were exposed to radon progeny attached to a vector aerosol of cigarette smoke particles in a closed-loop exposure system. The total exposures ranged from 70 to 1020 WLM over a 3-5-h period. Alveolar macrophages were lavaged from exposed and control rats (exposed to the vector aerosol alone). There were no differences between phagocytic capabilities of the macrophages lavaged from the controls and any group of exposed rats. The increases in the numbers of binucleated/multinucleated and micronucleated macrophages were enumerated using cytological preparations of lavaged alveolar macrophages after exposure compared to the numbers in the control rats. The peak response in numbers of binucleated/multinucleated and micronucleated macrophages occurred at 21 days after exposure, beyond which the numbers decreased. This peak response showed a significant exposure-dependent relationship. The number of micronuclei provided a sensitive indicator of exposure. The lowest exposure level used (70 WLM) resulted in a significant difference in the number of micronucleated macrophages between control and exposed rats. The alpha-particle dose to the peripheral lung was estimated by comparing formation of micronuclei in cultured macrophages exposed to alpha particles from 238Pu (where the dose was known) to micronuclei in lavaged macrophages from animals exposed to radon progeny (where the exposure concentration was known, but the dose was not known). The dose estimated in this way for the peripheral lung was 9.8 +/- 1.2 mGy/WLM. This value is three to five times higher than predicted from mathematical models currently available.

A modified Lucas cell for leakage measurement from encapsulated radium sources.

A new method to determine the leakage rate from encapsulated radium sources is proposed. The method is based on the principle of enclosing the source to be tested in a hermetically sealed container and measuring the radon radioactivity growth in the container as a function of time. A cylindrical modified Lucas cell, designed and built in our laboratory, is used for measuring the radon which are done by ZnS(Ag) alpha scintillation counting and can be easily compared with existing standard for leak tests. Results obtained show the applicability of the method for leak testing for radon leakage from sealed radium sources.

Predicted and observed early effects of combined alpha and beta lung irradiation.

The nonstochastic radiobiological effects of combined alpha and beta irradiation of the lungs of rats from inhaled radionuclides were studied. Both respiratory functional morbidity at 18 mo and mortality from radiation pneumonitis within 18 mo after exposure were examined for rats exposed to the beta-emitter 147Pm, the alpha-emitter 238Pu, or both combined. The results were used to validate hazard-function models that were developed (1) for respiratory functional morbidity at 18 mo and (2) for lethality from radiation pneumonitis within 18 mo. Both models were found to adequately predict the experimental observations for chronic alpha plus beta irradiation of the lung. Based on this 18-mo study, a relative biological effectiveness of approximately seven was obtained for 238Pu alpha radiation compared to 147Pm beta radiation for both respiratory functional morbidity and lethality from radiation pneumonitis. However, the relative biological effectiveness for the alpha radiation is likely to increase with longer follow-up.

The combined effects of alpha-particles and X-rays on cell killing and micronuclei induction in lung epithelial cells.

Understanding how cellular damage produced by high-linear energy transfer (LET) radiation interacts with that produced by low-LET is important both in radiation therapy and in evaluating risk. To study such interactions, rat lung epithelial cells (LEC) were grown on Mylar films and exposed to both X-rays and alpha-particles, separately or simultaneously. Cell killing, and the numbers of binucleated cells and micronuclei, were measured as indicators of damage. X-rays and alpha-particles given separately caused dose-related increases in cell cycle time, with alpha-particles producing greater mitotic delay than X-rays. Damage from alpha-particles and X-rays given simultaneously did not interact to alter further the cell cycle. Cell survival data following exposure to X-rays and alpha-particles, combined or individually, were fitted by linear-quadratic models. Survival curves following exposure to alpha-particles only, or to 1.0 Gy alpha-particles plus graded X-ray doses, were adequately described using only the linear (alpha) term of a linear-quadratic model with alpha coefficients of 0.9 +/- 0.04 and 1.03 +/- 0.18 Gy-1, respectively. Survival following exposure to X-rays only or to 0.06 Gy alpha-particles combined with X-rays was best fitted using both alpha and beta terms of the linear-quadratic model (0.12 +/- 0.03)D + (0.007 +/- 0.002)D2 and (0.57 +/- 0.08)D + (0.3 +/- 0.02)D2, respectively. The numbers of micronuclei produced by exposure to alpha-particles or X-rays alone increased linearly with dose, with slopes of 0.48 +/- 0.07 and 0.19 +/- 0.05 micronuclei/binucleated cell per Gy for alpha and X-rays, respectively. Simultaneous exposure to graded levels of X-rays and a constant alpha dose of either 1.0 or 0.06 Gy increased micronuclei frequency, with a slope of 0.74 +/- 0.05 or 0.58 +/- 0.04 micronuclei/binucleated cell per Gy, respectively. These slopes are similar to that produced by alpha-particles alone. These studies demonstrated that both cell killing and the induction of micronuclei were increased by combined exposures compared with that predicted for separate exposures.

Experimental measurements of the diffusion coefficient of 212Pb.

Knowledge of the diffusion coefficient of Rn progeny is necessary for assessing the radiation exposure resulting from exposure to Rn and its progeny. The diffusion coefficient for 220Rn progeny was determined in ambient air by two independent methods, measuring deposition using a cylindrical tube or screens. A sampling train consisting of a diffusion tube and a screen-type diffusion battery was used for the experimental study. A range of flow rates and relative humidities was investigated. For 35% less than or equal to RH less than or equal to 85%, results from the two systems agree with each other. The diffusion coefficient of 212Pb was 0.036 +/- 0.002 cm2 s-1 and 0.037 +/- 0.004 cm2 s-1 for the tube and screen penetration methods, respectively. In low humidity air (RH less than 30%), a linear relationship between the diffusion coefficient of 212Pb and relative humidity was observed. The observed diffusion coefficient is strongly affected by the amount of material agglomerated onto the 212Pb atom. Further studies on the effects of trace gases and organics are required to fully understand the results.

Continuous monitoring for airborne alpha emitters in a dusty environment.

Disposal of radioactive wastes in underground facilities requires continuous monitoring for airborne radioactive materials, both on the surface and underground. In addition to a natural background of nonradioactive and radioactive aerosols, there may be a sizeable dust contribution from ongoing work such as mining and vehicular traffic. In the monitoring of alpha-emitting radionuclides, these aerosols may lead to self-absorption in the source and a deterioration of the energy spectrum of the detected alpha particles. In this paper, the influence of a realistic background aerosol on the performance of an alpha monitoring system is evaluated theoretically. It is shown that depositing alpha emitters and background aerosol on a surface for counting leads rapidly to a considerable loss of counts, a deterioration of the alpha spectra, an eventual saturation of the count rates, and interference from the natural background of Rn daughters.

Natural radioactivity of some Spanish building materials.

The penetrating radiation from 226Ra, 232Th and 40K in conventional building materials has been measured by gamma spectrometry. A simple model is applied to the results to calculate the external radiation dose for people living in a typical Spanish house constructed with these materials. The radioactivity concentrations in the building materials were found to be similar to those reported for other countries. The average specific concentrations of 226Ra, 232Th and 40K for the total of 125 samples analyzed ranged from 5 to 421, 3 to 266 and 11 to 599 Bq kg-1, respectively. The results are examined in the light of standards for indoor exposure adopted by the Federal Republic of Germany and the criteria set up in the OECD/NEA Report, 1979.

Collection and characterization of aerosols from metal cutting techniques typically used in decommissioning nuclear facilities.

This study was designed to collect and characterize aerosols released during metal cutting activities typically used in decommissioning radioactively contaminated facilities. Such information can guide in the selection of appropriate control technologies for these airborne materials. Mechanical cutting tools evaluated included a multi-wheel pipe cutter, reciprocating saw, band saw, chop saw, and large and small grinding wheels. Melting-vaporization cutting techniques included an oxy-acetylene torch, electric arc cut rod and plasma torch. With the exception of the multi-wheel pipe cutter, all devices created aerosols in the respirable size range (less than 10 micron aerodynamic diameter). Time required to cut 2-in. (5-cm) Schedule 40, Type 304L, stainless steel ranged from about 0.6 min for the plasma torch to about 3.0 min for the reciprocating saw. Aerosol production rate ranged from less than 10 mg/min for the reciprocating saw to more than 3000 mg/min for the electric arc cut rod. Particles from mechanical tools were irregular in shape, whereas particles from vaporization tools were spheres and ultrafine branched-chain aggregates.

Studies of the temporal and spatial distribution of aerosols in multi-tiered inhalation exposure chambers.

Two multi-tiered whole body inhalation exposure chambers with nominal volumes of 1 m3 (H-1000) and 2 m3 (H-2000) were evaluated for their performance in terms of the temporal and spatial distribution of test aerosols within the chamber. Parameters investigated included chamber type, single-chamber-single-aerosol generator versus two-chamber-single-aerosol generator systems, chamber air supply and exhaust systems, particle size, and aerosol diluter type. Results indicated that: 1) particle size has an effect on chamber aerosol concentration distribution, with the larger particle resulting in a higher variation; 2) the single-chamber-single-generator system is more stable than the two-chamber-single-generator system; 3) the H-2000 chamber has a lower aerosol spatial variability than the H-1000 chamber; and 4) the aerosol distribution within the chamber could be improved with the use of a newly designed diluter.

Use of a jet mill for dispersing dry powder for inhalation studies.

Compressed air-powered jet mills are used in the chemical and food industries for grinding and classifying powders. We adapted one type of these fluid energy mills as a powder generator for inhalation experiments. The generating system included a jet mill and a screw feeder, the jet mill consisting of an elongated channel, a feeding jet to deliver the material into the channel, and two high-speed air jets. High speed air circulating in the channel created turbulence and centrifugal forces to disperse powder. The jet mill used can be operated from 25 to 100 psig at flow rates of 300 to 900 L/min. Two test materials--a solvent yellow dye and a dye mixture of solvent green and solvent yellow--were used. Both dyes were soft and sticky and could not be dispersed with several other powder generators tested at the concentrations required for toxicity studies. Aerosol concentrations ranging from 10 to 1500 mg/m3 at a flow rate of 400 L/min were obtained by adjusting the feed rate to the jet mill. Stability of aerosol concentration during six-hour continuous generation was 15 to 20%. Comparisons of several generators for producing sticky organic powders are also discussed.

The use of sister-chromatid exchange in Chinese hamster primary lung cell cultures to measure genotoxicity.

Primary cell cultures derived from Chinese hamster lung (CHL) were established, and their response for the induction of sister-chromatid exchange (SCE) by direct- and indirect-acting mutagens was characterized. An increase in SCE frequency was induced in CHL cells by 3-methylcholanthrene (MCA), benzo[a]pyrene (BaP), and 2-aminoanthracene (2AA). The SCE frequency increased slightly after exposure to cyclophosphamide, but did not respond to the hepatocarcinogen dimethylnitrosamine (DMN). A slight increase in SCE frequency by DMN was observed in the CHL system with use of Aroclor-1254-induced rat liver homogenate fraction (S9). This response to DMN in CHL cells was lower than that seen when CHO cells were the target in the presence of S9. At low (1) and high (20) passages, the CHL cells responded with a similar dose-related increase in SCE frequency to direct- (ethyl methanesulfonate, EMS) and indirect-(MCA) acting mutagens. This response indicates that even after prolonged culturing in vitro, the cells retained the ability to metabolically activate xenobiotic promutagens. The induction of SCE by MCA occurred at concentrations that also induced macromolecular binding. SCE induction was also examined in primary lung cell cultures from animals exposed by nose-only inhalation to MCA aerosol. A significant increase in SCE frequency above controls was observed in cells from animals after a single exposure to MCA. No detectable increase in SCE frequency was observed after repeated inhalation exposures. Because CHL cells are of lung origin and showed metabolic activity, the CHL system appears to be appropriate for study of the genotoxic potential of inhaled compounds.

Characterization of an aerosol sample from the auxiliary building of the Three Mile Island reactor.

Analyses for radioisotopic composition and dissolution characteristics were performed on an aerosol filter sample collected for a week by an air sampler located in the auxiliary building of the Three Mile Island nuclear reactor. The major radioisotopes found on the filter were 89Sr, 90Sr, 134Cs and 137Cs. Greater than 90% of both 89-90Sr and 134-137Cs dissolved within 48 hr in an in vitro test system. Scanning electron microscopic analyses showed the presence of respirable size particles as well as larger particles ranging up to 10 micron in diameter. The major matrix components were Fe, Ca, S, Mg, Al and Si. Although the radionuclides were present in a heterogeneous matrix, they were in a soluble form. This information enables a better evaluation of bioassay data and predictions of dose distribution resulting from an inhalation exposure to this aerosol. Further, the combination of techniques used in this study may be applicable to the characterization of other aerosols of unknown composition.