Biomarkers of genetic damage and inflammation in blood and bronchoalveolar lavage fluid among former German uranium miners: a pilot study

Former East German uranium miners who are known to have been exposed to radon are estimated to be at high risk for lung carcinogenesis. Among these miners over 200 occupationally caused lung cancer cases are expected to occur each year, resulting in a total of 7,000–24,000 excess lung cancer cases in the coming years. It is still unknown whether there is a correlation between biomarkers and the exposure of the uranium miners to ionizing radiation that might enable us to trace those miners with high lung cancer risk. The primary aim of this pilot study was to test the possibility of performing a biomarker study in this unique cohort of former uranium miners in spite of several limitations that had to be taken into consideration when comparing them with healthy controls, such as old age, age-dependent diseases and potential confounding artefacts from dissimilar smoking patterns. The second aim was to test a range of biomarkers for DNA damage and inflammation in leukocytes and bronchoalveolar fluid for their ability to detect biological effects. In this cohort of miners we found an increased frequency of chromosomal aberrations in blood lymphocytes and an increased prevalence of both fibronectin and tumour necrosis factor α in the bronchoalveolar fluid. Received: 16 March 2000 / Accepted: 15 September 2000     

Differences in baseline lung cancer mortality between the German uranium miners cohort and the population of the former German Democratic Republic (1960–2003)

A previous analysis of the radon-related lung cancer mortality risk, in the German uranium miners cohort, using Poisson modeling techniques, noted internal (spontaneous) rates that were higher on average than the external rates by 16.5% (95% CI: 9%; 24%). The main purpose of the present paper is to investigate the nature of, and possible reasons for, this difference by comparing patterns in spontaneous lung cancer mortality rates in a cohort of male miners involved in uranium extraction at the former Wismut mining company in East Germany with national male rates from the former German Democratic Republic. The analysis is based on miner data for 3,001 lung cancer deaths, 1.76 million person-years for the period 1960–2003, and national rates covering the same calendar-year range. Simple “age–period–cohort” graphical analyses were applied to assess the main qualitative differences between the national and cohort baseline lung cancer rates. Some differences were found to occur mainly at higher attained ages above 70 years. Although many occupational risk factors may have contributed to these observed age differences, only the effects of smoking have been assessed here by applying the Peto–Lopez indirect method for calculating smoking attributability. It is inferred that the observed age differences could be due to the greater prevalence of smoking and more mature smoking epidemic in the Wismut cohort compared to the general population of the former German Democratic Republic. In view of these observed differences between external population-based rates and internal (spontaneous) cohort baseline lung cancer rates, it is strongly recommended to apply only the internal rates in future analyses of uranium miner cohorts.     

Three-dimensional simulations of airways within human lungs

Information regarding the deposition patterns of inhaled particles has important implications to the fields of medicine and risk assesment. The former concerns the targeted delivery of inhaled pharmacological drugs (aerosol therapy); the latter concerns the risk assessment of inhaled air pollutants (inhalation toxicology). It is well documented in the literature that the behavior and fate of inhaled particles may be formulated using three families of variables: respiratory system morphologies, aerosol characteristics, and ventilatory parameters. It is straightforward to propose that the seminal role is played by morphology per se because the structures of individual airways and their spatial orientations within lungs affect the motion of air and the trajectories of transported particles. In previous efforts, we have developed original algorithms to describe airway networks within lungs and employed them as templates to interpret the results of single photon emission computed tomography (SPECTs) studies. In this work, we have advanced the process of mathematical modeling and computer simulations to produce three-dimensional (3D) images. We have tested the new in silico model by studying two different branching concepts: an inclusive (all airways present) system and a single “typical” pathway system. When viewed with the glasses supplied with this volume, the 3D nature of airway branching networks within lungs as displayed via our original computer graphics software is clear. We submit that the new technology will have numerous and seminal functions in future medical and toxicological regimens, the most fundamental being the creation of a platform to view natural 3D structures in vivo with related biological processes (e.g., disposition of inhaled pharmaceuticals).     

Radon dosimetry applications ofin vitro tracheobronchial-deposition measurements

Deposition efficiencies of monodisperse ammonium fluorescein aerosols have been measured in simulated human lungs made of replica laryngeal casts combined with trachebronchial systems. Other tests, with radiolabelled submicron-sized particles, combined the larynges with replica tracheobronchial casts. The laryngeal casts had internal flow rate-specific geometries. Data indicate thatin vitro bifurcations have ?hot spots? or highly localized deposits, particularly at carinal ridges, suggesting that epithelial cells at airway branching sitesin vitro receive increased exposures to inhaled particulate matter. For dosimetry purposes, therefore, the lung should be likened to a series of Y-shaped airway junctures. The data have risk assessment applications for ambient radon progeny and radioactive airborne particles found in uranium mining and milling operations.     

Vanadate as an inhibitor of plant and mammalian peroxidases

Vanadate ions are shown to inhibit horseradish, squash, and rat intestinal peroxidases by following the reaction spectrophotometrically in a wide range of vanadate concentrations. I₅₀ in phosphate buffer were 43, 9.4, and 535 μM, respectively. No inhibitory effect was found on cow milk lactoperoxidase and beef liver catalase. Gel filtration of peroxidases in the presence of vanadate, as carried out by radioactive⁴⁸V for horseradish peroxidases (either in aerobic or anoxic conditions) and neutron activation analysis (NAA) for squash peroxidase, demonstrated a binding of vanadium to these enzymes in stoichiometric amounts. Electron paramagnetic resonance spectra of the eluted peaks for the former peroxidase indicated that vanadium is in the +5 oxidation state, but an equilibrium between V (V) and V (IV) in the assay conditions cannot be discarded. Although the inhibitory mechanism remains obscure, some hypotheses are considered. The potential implications that the inhibitory effect of vanadium might have on plant and animal metabolism are also discussed.     

Pulmonary defense mechanisms in Boa constrictor

We studied aerosol deposition and the response to inhaled particles and irritants in lungs of Boa constrictor. Snakes which breathed submicrometric particles radiolabeled with 99mTc retained 41.4 +/- 9.9% of the aerosol in the trachea, 42.5 +/- 8.8% in the anterior faveolar regions, and 8.7 +/- 4.1% in the posterior saccular regions of the lungs. Low activity recovered in the gastrointestinal tract over a 5-h period following aerosol exposure indicated slow clearance of inhaled particles. In contrast to mammalian lungs, there are no macrophages resident on the surface of boa lungs, and uningested particles persist for up to 4 days without being phagocytized. Particles and irritant stimuli (Fe2O3, endotoxin, and N-formylmethionylphenylalanine) elicited only eosinophilic granulocytes that were not phagocytic. The numbers of these cells peaked at 24 h following exposure and declined gradually over the next 7 days. Lavage fluid from stimulated snake lungs contained many large lamellar figures continuous with tubular myelin, a form of surfactant. Very little of this material was recovered from control lungs. Response to inhaled particles and lung injury in boas increased surfactant release, elicited eosinophilic granulocytes, but did not recruit phagocytic mononuclear cells.     

大气中粉尘颗粒物对支气管肺泡上皮影响的实验研究

目的探讨大气污染物对呼吸道的病理变化,为预防大气污染对人体健康的损害提供理论依据。方法采用模拟大气污染方法,用大鼠制备大气污染物模型,对大鼠气管、肺进行透射电镜及气管扫描电镜检查。结果大气污染大鼠动物模型的气管、肺与对照组相比,实验组气管及肺泡上皮有渐进性损伤,且随量的蓄积损伤逐渐加重,最终发生肺实变。结论吸入的污染粉尘可造成气管及肺泡上皮的渐进性损伤,形态学变化与吸入粉尘的量呈一致性,并随吸人量的累积而逐渐变得严重,最终变为肺实变,残留肺组织为代偿性气肿。     

The relative biological effectiveness of alpha-radiation during human lung exposure to irradiation

The assessment of the relative biological effectiveness (RBE) for alpha-radiation was held in the cases of inhalation of radon progeny and incorporation of plutonium in lungs. It is based on simulation of lung cancer radiation risk for different types of radiation. Specific radiation risk models developed according to the results of direct epidemiological studies are used for the simulation. These include two published risk models for uranium miners and nuclear workers of the Mayak facilities in the former Soviet Union. Additionally two lung cancer risk models are developed and described for the following cases: population indoor radon exposure and low-linear-energy-transfer reference radiation exposure. By the results of lifetime lung cancer risk simulation the RBE values range from 11 to 12 and from 1.7 to 4.9 for the cases of plutonium incorporation and of radon progeny exposure accordingly. The significant uncertainty of radiation risk models results in significant variation of RBE assessments. Rough estimations of RBE values 90% confidence interval are from unit fraction to 25 and from 2 to 50 for the cases of radon progeny exposure and plutonium incorporation accordingly.     

Risk of lung cancer mortality in relation to lung doses among French uranium miners: follow-up 1956-1999

The aim of this study was to assess the risk of lung cancer death associated with cumulative lung doses from exposure to α-particle emitters, including radon gas, radon short-lived progeny, and long-lived radionuclides, and to external γ rays among French uranium miners. The French "post-55" sub-cohort included 3,377 uranium miners hired from 1956, followed up through the end of 1999, and contributing to 89,405 person-years. Lung doses were calculated with the ICRP Human Respiratory Tract Model (Publication 66) for 3,271 exposed miners. The mean "absorbed lung dose" due to α-particle radiation was 78 mGy, and that due to the contribution from other types of radiation (γ and β-particle radiation) was 56 mGy. Radon short-lived progeny accounted for 97% of the α-particle absorbed dose. Out of the 627 deaths, the cause of death was identified for 97.4%, and 66 cases were due to lung cancer. A significant excess relative risk (ERR) of lung cancer death was associated with the total absorbed lung dose (ERR/Gy = 2.94, 95% CI 0.80, 7.53) and the α-particle absorbed dose (4.48, 95% CI 1.27, 10.89). Assuming a value of 20 for the relative biological effectiveness (RBE) of α particles for lung cancer induction, the ERR/Gy-Eq for the total weighted lung dose was 0.22 (95% CI: 0.06, 0.53).     

Evaluation of trace elements in lung samples from coal miners using neutron activation analysis

In this study, instrumental neutron activation analysis was applied to the determination of Sc, La, Ce, Nd, Sm, Eu, Tb, Yb, Lu, Hf, Th, and U in lung samples from miners working in coal mines located in the state of Santa Catarina, Brazil. These results were compared to those from a control group constituted of healthy individuals. The results showed that the elements determined exhibit considerable intersubject variability within a single group of individuals and the mean values of concentrations in miners’ lungs were higher than those of normal individuals. Lung samples presented U concentrations varying from 11 to 890 Μg/kg. Therefore, for some samples, the contribution of the uranium fission products in the analysis of La, Ce, Nd, and Sm was considered by determining the interference correction factors. The accuracy of the results was evaluated by analyzing certified reference materials.     

The influence of multiple types of occupational exposure to radon, gamma rays and long-lived radionuclides on mortality risk in the French "post-55" sub-cohort of uranium miners: 1956-1999

The adverse health effects of radon on uranium miners, especially on their lungs, are well documented, but few studies have considered the effects of other radiation exposures. This study examined the mortality risks associated with exposure to radon, external γ rays and long-lived radionuclides (LLR) in the French "post-55" sub-cohort, which includes uranium miners first employed between 1956 and 1990 for whom all three types of exposure were assessed individually. Exposure-risk relationships were estimated with linear excess relative risk models and a 5-year lag time. The post-55 sub-cohort includes 3377 miners, contributing 89,405 person-years, followed up through the end of 1999 with a mean follow-up of 26.5 years. Mean cumulative exposure was 17.8 WLM for radon, 54.7 mSv for γ rays, and 1,632 Bq.m(-3).h for LLR. Among the 611 deaths observed, 66 were due to lung cancer. Annual individual exposures were significantly correlated. Increased mortality was observed for lung cancer (SMR = 1.30; 95% CI: 1.01, 1.65) and for brain and central nervous system (CNS) cancer (SMR = 2.00; 95% CI: 1.09, 3.35). Cumulative exposure to radon, γ rays and LLR was associated only with a significant risk of lung cancer. These new results could suggest an association between lung cancer and exposure to γ rays and LLR. They must nonetheless be interpreted with caution because of the correlation between the types of exposure. The calculation of organ doses received by each of these exposures would reduce the collinearity.     

Chromium content of normal and pathologically altered lung tissue

Flameless atomic absorption spectrophotometry was used to determine the chromium (Cr) content of samples taken at autopsy from the lungs, bronchi, and regional hilar lymph nodes of 50 randomly selected patients from urban and rural areas; these patients were not known to have been excessively exposed to Cr. On the average, the Cr concentration in the lungs of patients younger than 40 yr of age was approximately 2 μg/g; for patients over 40, the average Cr values were between 5 and 15 μg/g dry wt. The highest values were found in samples from the apex of the lung. The Cr concentration in lung and lymph nodes increased in proportion to age and the degree of anthracosis. Chromium content in the bronchial wall was very low in all cases. Chromium values up to ten times greater as compared to age-matched average values were found in scarred lung tissue, probably caused by a postinflammatory lymph vessel blockade. Slightly elevated Cr values were found in smokers' lungs. Chromium values in tissue from primary lung carcinomas (n=9) were lower than those in neighboring lung tissue. Based on the results of this study the amount of Cr of lung and bronchial tissue does not appear to be associated with the induction of bronchial carcinoma.     

Sodium vanadate toxicity in adult and developing rats

The toxic effect of vanadium (sodium metavanadate) during pregnancy and lactation was studied by feeding vanadium to pregnant, Sprague-Dawley rats at levels of 1 (control) or 75 μg V/g diet through d 21 postpartum, at which time they were killed. Vanadium-fed dams had lower food intakes and weight gains than controls during pregnancy. Survival until d 21 postpartum was significantly lower in the vanadium pups compared to controls. In addition, the surviving pups gained less weight than control pups, despite similar birth weights. On a relative body weight basis, vanadium pups had larger livers, brains, and testes than controls, suggesting that these animals were developmentally delayed. Vanadium dams and pups had higher concentrations of hepatic vanadium than controls. Vanadium pups also had higher concentrations of hepatic zinc than control pups. Maternal hepatic zinc concentrations were not affected by diet. Also, no significant differences in hepatic iron, copper, or manganese concentrations were observed for either dams or pups. Hepatic thiobarbituric acid reactivity was higher in whole cell and isolated mitochondria for vanadium dams and pups than for control dams and pups, indicating that these animals may have had higher levels of lipid peroxidation. This idea was supported by the observation of lower concentrations of reduced glutathione in the livers of vanadium pups compared to controls. In contrast, kidney and brain glutathione levels were not affected by diet. In conclusion, animals during periods of rapid growth are susceptible to vanadium toxicity, and increased lipid peroxidation may be one factor underlying this toxicity.     

Vanadium levels in urine and cystine levels in fingernails and hair of exposed and normal persons

Vanadium was determined by radiochemical neutron activation analysis (RNAA) with proven accuracy in urine of workers occupationally exposed to vanadium-rich dust in a vanadium pentoxide production plant, and values in the range of 3.02–762 ng/mL (median 33.0 ng/mL) were found. In a control group consisting of administrative workers of the plant, urinary vanadium levels were found in the range of 1.05–53.4 ng/mL (median 2.53 ng/mL), whereas in an another control group of occupationally nonexposed persons, these values amounted to 0.066–0.489 ng/mL (median 0.212 ng/mL). Accuracy of the results was tested by analysis of reference material IAEA A-13 Animal Blood and NIST SRM-1515 Apple Leaves, and very good agreement was found with literature and the NIST certified values, respectively. Unlike urine, no significant differences were found for cystine levels in fingernails and hair of exposed and control persons.     

Recent advances in pulmonary drug delivery using large, porous inhaled particles

The abilityto deliver proteins and peptides to the systemic circulation byinhalation has contributed to a rise in the number of inhalationtherapies under investigation. For most of these therapies, aerosolsare designed to comprise small spherical droplets or particles of massdensity near 1 g/cm³ and meangeometric diameter between ~1 and 3 µm, suitable for particlepenetration into the airways or lung periphery. Studies performedprimarily with liquid aerosols have shown that these characteristics ofinhaled aerosols lead to optimal therapeutic effect, both for local andsystemic therapeutic delivery. Inefficient drug delivery can stillarise, owing to excessive particle aggregation in an inhaler,deposition in the mouth and throat, and overly rapid particle removalfrom the lungs by mucocilliary or phagocytic clearance mechanisms. Toaddress these problems, particle surface chemistry and surfaceroughness are traditionally manipulated. Recent data indicate thatmajor improvements in aerosol particle performance may also be achievedby lowering particle mass density and increasing particle size, sincelarge, porous particles display less tendency to agglomerate than(conventional) small and nonporous particles. Also, large, porousparticles inhaled into the lungs can potentially release therapeuticsubstances for long periods of time by escaping phagocytic clearancefrom the lung periphery, thus enabling therapeutic action for periodsranging from hours to many days.

     

8-Hydroxyguanosine formed in human lung tissues and the association with diesel exhaust particles

Diesel exhaust particles consist of various organic chemicals, heavy metals, and carbon particles. Knowledge of the fate of organic chemicals and carbon particles in the lungs is important to determine the mechanisms responsible for lung tumors. In the present study, diesel particle extracts were found to show mutagenicity for YG3003, a sensitive strain to some oxidative mutagens, as well as other mutant strains, and those of lung tissues obtained from lung cancer patients exhibited potent mutagenicity. Formation of 8-hydroxyguanosine (8-OHdG) as a biomarker of oxidative damage was analyzed with in vitro and in vivo assay systems. The 8-OHdG was detected in all 22 cases of lung tissues with carcinomas tested and their levels increased with the increasing age of the patients, suggesting a correlation between age and the presence of carbon particles in lung tissues. Therefore, the formation of 8-OHdG due to diesel exhaust particles was investigated via intratracheal injections into mice. 8-OHdG formation was elevated when carboneceous particles, after removal of organic chemicals with various solvents, were administered to mice, but it was not elevated when polyaromatic compounds such as benzo[a]pyrene, 1,8-dinitropyrene, and 1-nitropyrene were used in the same procedure in mice. The carboneceous particles were formed from a giant particle that was aggregated by micro-particles with diameters of 1.47 +/- 1.34 to 1.05 +/- 0.83 microm. These results suggest that carboneceous particles, but not mutagens and carcinogens, promote the formation of 8-OHdG, and that as a mechanism, alveolar macrophages may be involved in oxidative damage. The oxidative damage may be due to the fact that the mutation is involved with the generation of a hydroxyl radical during phagocytosis, and the hydroxyl radical leads to hydroxylation at the C-8 position of the deoxyguanosine residue in the DNA.     

Evidence for active control of perfusion within lung microvessels

Vasoconstrictors cause contraction of pulmonary microvascular endothelial cells in culture. We wondered if this meant that contraction of these cells in situ caused active control of microvascular perfusion. If true, it would mean that pulmonary microvessels were not simply passive tubes and that control of pulmonary microvascular perfusion was not mainly due to the contraction and dilation of arterioles. To test this idea, we vasoconstricted isolated perfused rat lungs with angiotensin II, bradykinin, serotonin, or U46619 (a thromboxane analog) at concentrations that produced equal flows. We also perfused matched-flow controls. We then infused a bolus of 3 μm diameter particles into each lung and measured the rate of appearance of the particles in the venous effluent. We also measured microscopic trapping patterns of particles retained within each lung. Thirty seconds after particle infusion, venous particle concentrations were significantly lower (P ≤ 0.05) for lungs perfused with angiotensin II or bradykinin than for those perfused with U46619, but not significantly different from serotonin perfused lungs or matched flow controls. Microscopic clustering of particles retained within the lungs was significantly greater (P ≤ 0.05) for lungs perfused with angiotensin II, bradykinin, or serotonin, than for lungs perfused with U46619 or for matched flow controls. Our results suggest that these agents did not produce vasoconstriction by a common mechanism and support the idea that pulmonary microvessels possess a level of active control and are not simply passive exchange vessels.     

Effect of Relative Humidity on Dynamic Aerosols of Adenovirus 12

Dynamic aerosols of adenovirus 12 were generated in the same Henderson apparatus under conditions of high, medium, and low relative humidity. High relative humidities resulted in more recovery of adenovirus 12 from aerosols and lungs of newborn Syrian hamsters. At 89, 51, and 32% relative humidity, the total infectious virus recovered from a 20-min aerosol was 10(6.7), 10(6.0), and 10(4.3) TCD(50), respectively. Hamsters exposed to these 20-min aerosols retained measured lung doses of 10(3.0), 10(2.4), and 10(1.0) TCD(50), respectively. The measured retained lung doses were compared to calculated inhaled lung doses based on both total virus aerosolized and total virus recovery from the aerosols.     

Association of mast cells with lung function in chronic obstructive pulmonary disease

Background

Surfactant protein D (SP-D), an innate immune molecule, plays an important protective role during airway inflammation. Deficiency of this molecule induces emphysematous changes in murine lungs, but its significance in human COPD remains unclear.

Methods

We collected bronchoalveolar lavage fluid from 20 subjects with varying degrees of COPD (8 former smokers and 12 current smokers) and 15 asymptomatic healthy control subjects (5 never smokers, 3 remote former smokers, and 7 current smokers). All subjects underwent a complete medical history and pulmonary function testing. SP-D was measured by Enzyme-Linked ImmunoSorbent Assay. Statistical analysis was performed using nonparametric methods and multivariable linear regression for control of confounding. The effect of corticosteroid treatment on SP-D synthesis was studied in vitro using an established model of isolated type II alveolar epithelial cell culture.

Results

Among former smokers, those with COPD had significantly lower SP-D levels than healthy subjects (median 502 and 1067 ng/mL, respectively, p = 0.01). In a multivariable linear regression model controlling for age, sex, race, and pack-years of tobacco, COPD was independently associated with lower SP-D levels (model coefficient -539, p = 0.04) and inhaled corticosteroid use was independently associated with higher SP-D levels (398, p = 0.046). To support the hypothesis that corticosteroids increase SP-D production we used type II alveolar epithelial cells isolated from adult rat lungs. These cells responded to dexamethasone treatment by a significant increase of SP-D mRNA (p = 0.041) and protein (p = 0.037) production after 4 days of culture.

Conclusion

Among former smokers, COPD is associated with lower levels of SP-D and inhaled corticosteroid use is associated with higher levels of SP-D in the lung. Dexamethasone induced SP-D mRNA and protein expression in isolated epithelial cells in vitro. Given the importance of this molecule as a modulator of innate immunity and inflammation in the lung, low levels may play a role in the pathogenesis and/or progression of COPD. Further, we speculate that inhaled steroids may induce SP-D expression and that this mechanism may contribute to their beneficial effects in COPD. Larger, prospective studies are warranted to further elucidate the role of surfactant protein D in modulating pulmonary inflammation and COPD pathogenesis.     

Lung Changes in Woodworkers

Pathological changes were observed in the lungs of two workers who had been exposed to wood dust for many years. The cause of death in each case was unrelated to the lung condition. The histopathological changes in the lung were: (1) centrilobular fibrosis and emphysema, (2) the presence of intra-alveolar basophilic particles which had excited a histiocytic and foreign body reaction. Special studies of these bodies tended to confirm the suspicion that they were particles of wood dust. Studies have shown that woodworkers are in an environment heavily saturated with wood dust. The present study suggests that the wood dust is inhaled into the alveoli and may lead to changes in the lungs.