Detection of beryllium sensitivity using a flow cytometric lymphocyte proliferation test: the Immuno-Be-LPT.

Measurement of lymphocyte proliferation to detect hypersensitivity to beryllium (Be-LPT) in vitro is done presently using a method based on tritiated thymidine incorporation. Although this method is sensitive it gives no information on cell viability or responding lymphocyte subsets. We have developed reliable and simple flow cytometric assays for lymphocyte proliferation testing (Immuno-Be-LPT) by combining immunophenotyping with bromodeoxyuridine (BrdU) incorporation or DNA content using propidium iodide (PI) or 4'6'-diimidazolin-2-phenylindole (DAPI). Evaluation of beryllium-induced lymphocyte proliferation in blood cells from seven patients with chronic beryllium disease (CBD) and 120 beryllium workers by both the Bc-LPT and the Immuno-Be-LPT showed agreement between the tests. The Immuno-Bc-LPT provided additional information about the specific type of lymphocytes responding. CD4+ lymphocytes proliferated in response to beryllium in blood samples from all seven CBD individuals and CD8+ lymphocytes proliferated in six of the seven. Four beryllium workers without CBD had positive responses to beryllium primarily in the CD8+ cells. The use of the individual's own plasma supported a greater beryllium or tetanus-induced proliferation of CD4+ lymphocytes when compared to commercial human serum. The response of CD4+ lymphocytes measured in the Immuno-Be-LPT may provide a new marker for the diagnosis of CBD.

A mortality study of employees of the nuclear industry in Oak Ridge, Tennessee.

An analysis was conducted of 27,982 deaths among 106,020 persons employed at four Federal nuclear plants in Oak Ridge, Tennessee, between 1943 and 1985. The main objectives were to extend the evaluation of the health effects of employment in the nuclear industry in Oak Ridge to include most workers who were omitted from earlier studies, to compare the mortality experience of workers among the facilities, to address methodological problems that occur when individuals employed at more than one facility are included in the analysis, and to conduct dose-response analyses for those individuals with potential exposure to external radiation. All-cause mortality and all-cancer mortality were in close agreement with national rates. The only notable excesses occurred for white males for lung cancer [standardized mortality ratio (SMR) = 1.18, 1,849 deaths] and non-malignant respiratory disease (SMR = 1.12, 1,568 deaths). A more detailed analysis revealed substantial differences in death rates among workers at the Oak Ridge plants. Evaluation of internally adjusted log SMRs using Poisson regression showed that workers employed only at Tennessee Eastman Corporation or K-25 and at multiple facilities had higher death rates than similar workers employed only at X-10 or Y-12, and that the differences were primarily due to non-cancer causes. Analysis of selected cancer causes for white males indicated large differences among the workers at the different facilities for lung cancer, leukemia and other lymphatic cancer. Dose-response analyses for external penetrating radiation were limited to a subcohort of 28,347 white males employed at X-10 or Y-12. Their collective recorded dose equivalent was 376 Sv. There was a strong "healthy worker effect" in this subcohort-all-cause SMR = 0.80 (4,786 deaths) and all-cancer SMR = 0.87 (1,134 deaths). Variables included in the analyses were age, birth cohort, a measure of socioeconomic status, length of employment, internal radiation exposure potential and facility. For external radiation dose with a 10-year lag, the excess relative risk was 0.31 per Sv (95% CI = -0.16, 1.01) for all causes and 1.45 per Sv (95% CI = 0.15, 3.48) for all cancer. The estimated excess relative risk for leukemia was negative but imprecisely determined. A preliminary dose adjustment procedure was developed to compensate for missing dose but not other dosimetry errors. Results of the analyses using the adjusted doses suggest that the effect of missing dose is an upward bias in dose-response coefficients and test statistics.

A method for estimating occupational radiation dose to individuals, using weekly dosimetry data.

Statistical analyses of data from epidemiological studies of workers exposed to radiation have been based on recorded annual radiation doses. It is usually assumed that the annual doses are known exactly, although it is generally recognized that the data contain uncertainty due to measurement error and bias. We propose the use of a probability distribution to describe an individual's dose during a specific period and develop statistical methods for estimating this distribution. The methods take into account the "measurement error" that is produced by the dosimetry system and the bias that was introduced by policies of recording doses below a threshold as zero. The method is applied to a sample of dose histories over the period 1945 to 1955 obtained from hard-copy dosimetry records at Oak Ridge National Laboratory (ORNL). The result of this evaluation raises serious questions about the validity of the historical personnel dosimetry data that are currently being used in studies of the effects of low doses in nuclear industry workers. In particular, it appears that there was a systematic underestimation of doses for ORNL workers. This may result in biased estimates of dose-response coefficients and their standard errors.

Statistical methods for the blood beryllium lymphocyte proliferation test.

The blood beryllium lymphocyte proliferation test (BeLPT) is a modification of the standard lymphocyte proliferation test that is used to identify persons who may have chronic beryllium disease. A major problem in the interpretation of BeLPT test results is outlying data values among the replicate well counts (approximately 7%). A long-linear regression model is used to describe the expected well counts for each set of Be exposure conditions, and the variance of the well counts is proportional to the square of the expected count. Two outlier-resistant regression methods are used to estimate stimulation indices (SIs) and the coefficient of variation. The first approach uses least absolute values (LAV) on the log of the well counts as a method for estimation; the second approach uses a resistant regression version of maximum quasi-likelihood estimation. A major advantage of these resistant methods is that they make it unnecessary to identify and delete outliers. These two new methods for the statistical analysis of the BeLPT data and the current outlier rejection method are applied to 173 BeLPT assays. We strongly recommend the LAV method for routine analysis of the BeLPT. Outliers are important when trying to identify individuals with beryllium hypersensitivity, since these individuals typically have large positive SI values. A new method for identifying large Sls using combined data from the nonexposed group and the beryllium workers is proposed. The log(SI)s are described with a Gaussian distribution with location and scale parameters estimated using resistant methods. This approach is applied to the test data and results are compared with those obtained from the current method.

Job factors, radiation and cancer mortality at Oak Ridge National Laboratory: follow-up through 1984.

A previous study of mortality among white men hired at Oak Ridge National Laboratory between 1943 and 1972 (n = 8,318) revealed an association between low-dose external penetrating ionizing radiation and cancer mortality in follow-up through 1984. The association was not observed in follow-up through 1977. This report considers the role of possible selection and confounding factors not previously studied. Control for hire during the World War II era and employment duration of less than 1 year had little effect on the radiation risk estimates. Risks associated with length of time spent in 15 job categories were considered as proxies for the effects of other occupational carcinogens. Adjustment for employment duration in each job category one at a time produced only small changes in the radiation risk estimate. Adjustment for potential exposures to beryllium, lead, and mercury also had little effect on the radiation risk estimates. These analyses suggest that selection factors and potential for chemical exposure do not account for the previously noted association of external radiation dose with cancer mortality. However, power to detect effects of chemical exposures is limited by a lack of individual exposure measures.

Concentration-dependent protection against X-ray-induced chromosome aberrations in human lymphocytes by the aminothiol WR-1065.

WR-1065, the free-thiol form of WR-2721, has radioprotective effects in various biological systems. We measured the efficiency of WR-1065 in modifying the induction of chromosome aberrations by X rays in human lymphocytes. G0 lymphocytes were incubated for 30 min in medium containing 1-12 mM WR-1065, exposed to 0 or 3.1 Gy 220-kV X rays, washed, and cultured for evaluations of chromosome aberrations and micronuclei (MN). Neither proliferation kinetics nor baseline frequencies of aberrations or MN were affected in nonirradiated cultures incubated in WR-1065 for up to 45 min. Radiation-induced chromosome aberrations and MN varied inversely as a logarithmic function of thiol concentration. At extracellular concentrations of 8-12 mM, WR-1065 protected against > 85% of X-ray-induced chromosome damage as measured by either cytogenetic end point. WR-1065 is more efficient in modulating X-ray-induced chromosome aberrations than dimethyl sulfoxide, which provides protection by scavenging OH radicals. Our data suggest that mechanisms in addition to OH radical scavenging are involved in radioprotection by WR-1065.

Comparison of two stocks of mice in spermatogonial response to different conditions of radiation exposure.

In a previous report (Generoso et al., 1985) it was shown that the two hybrid stocks of mice, (C3H/R1 x 101/R1)F1 and (SEC/R1 x C57BL/6)F1, differed in their responses to induction of chromosomal aberrations following exposure of the stem-cell spermatogonia to 500 R x 4 (4-week intervals) acute X-rays. The levels of response in the two stocks were paralleled by the effects on the length of the sterile period, which presumably results from stem-cell killing and repopulation. The present study was conducted in order to determine whether the differences between the two stocks in these parameters hold true also for other conditions of radiation exposure. Thus, comparative experiments were conducted using the following acute exposure regimens: 500 R single dose, 500 R + 500 R (24-h interval), 100 R + 900 R (24-h interval), and 500 R x 4 (8-week intervals). The endpoints measured were chromosome rearrangements in diakinesis/metaphase-I meiocytes, embryonic lethality in conceptuses, length of sterile period and testis weight. Trend analysis indicated that higher frequencies of chromosome rearrangements and embryonic lethality were recovered from (C3H/R1 x 101/R1)F1 than from (SEC/R1 x C57BL/6)F1 males, that there were no significant differences between stocks in testis weight reductions, and that there was no consistency in the direction of the significant differences that occurred in the length of the sterile period. A definitive conclusion regarding the possible association between induction of chromosomal aberrations and induction of cell killing awaits direct histological analysis of the stem-cell population.

Mortality among workers at Oak Ridge National Laboratory. Evidence of radiation effects in follow-up through 1984.

White men hired at the Oak Ridge (Tenn) National Laboratory between 1943 and 1972 were followed up for vital status through 1984 (N = 8318, 1524 deaths). Relatively low mortality compared with that in US white men was observed for most causes of death, but leukemia mortality was elevated in the total cohort (63% higher, 28 deaths) and in workers who had at some time been monitored for internal radionuclide contamination (123% higher, 16 deaths). Median cumulative dose of external penetrating radiation was 1.4 mSv; 638 workers had cumulative doses above 50 mSv (5 rem). After accounting for age, birth cohort, a measure of socioeconomic status, and active worker status, external radiation with a 20-year exposure lag was related to all causes of death (2.68% increase per 10 mSv) primarily due to an association with cancer mortality (4.94% per 10 mSv). Studies of this population through 1977 did not find radiation-cancer mortality associations, and identical analyses using the shorter follow-up showed that associations with radiation did not appear until after 1977. The radiation-cancer dose response is 10 times higher than estimates from the follow-up of survivors of the bombings of Hiroshima and Nagasaki, Japan, but similar to one previous occupational study. Dose-response estimates are subject to uncertainties due to potential problems, including measurement of radiation doses and cancer outcomes. Longer-term follow-up of this and other populations with good measurement of protracted low-level exposures will be critical to evaluating the generalizability of the results reported herein.

Poisson regression analysis of the mortality among a cohort of World War II nuclear industry workers.

A historical cohort mortality study was conducted among 28,008 white male employees who had worked for at least 1 month in Oak Ridge, Tennessee, during World War II. The workers were employed at two plants that were producing enriched uranium and a research and development laboratory. Vital status was ascertained through 1980 for 98.1% of the cohort members and death certificates were obtained for 96.8% of the 11,671 decedents. A modified version of the traditional standardized mortality ratio (SMR) analysis was used to compare the cause-specific mortality experience of the World War II workers with the U.S. white male population. An SMR and a trend statistic were computed for each cause-of-death category for the 30-year interval from 1950 to 1980. The SMR for all causes was 1.11, and there was a significant upward trend of 0.74% per year. The excess mortality was primarily due to lung cancer and diseases of the respiratory system. Poisson regression methods were used to evaluate the influence of duration of employment, facility of employment, socioeconomic status, birth year, period of follow-up, and radiation exposure on cause-specific mortality. Maximum likelihood estimates of the parameters in a main-effects model were obtained to describe the joint effects of these six factors on cause-specific mortality of the World War II workers. We show that these multivariate regression techniques provide a useful extension of conventional SMR analysis and illustrate their effective use in a large occupational cohort study.

Comparisons of dose-response parameters for radiation-induced acentric fragments and micronuclei observed in cytokinesis-arrested lymphocytes.

We employed cytochalasin B to block cytokinesis in human lymphocytes exposed to 220 kV X-radiation. When 3 micrograms/ml of cytochalasin B was used, most cells escaped the block whereas at 6 micrograms/ml greater than 90% of the mitogen-responsive cells became binucleate. Using the higher concentration of cytochalasin B, we observed a linear-quadratic (i.e. Y = gamma + alpha D + beta D2) dose dependency for X-ray-induced micronuclei (MN) in preparations from three donors. When dose-response parameters were compared with those for total acentrics scored in first division metaphases, we observed no significant differences in estimates of the background (gamma) or linear (alpha) coefficients, but a 2-fold reduction in the beta coefficient for MN. We interpret our data as providing evidence that radiation-induced micronuclei are derived from acentric fragments (AF); that virtually all AF are recovered as MN in binucleate interphase daughter cells at low radiation doses; and that for our data, the relative proportion of AF that will be observed as independent MN decreases by a constant factor of approximately one-half as radiation dose increases.

Modulation of radiation-induced chromosome aberrations by DMSO, an OH radical scavenger. 1: Dose-response studies in human lymphocytes exposed to 220 kV X-rays.

Human G0 lymphocytes were exposed to 220 kV X-radiation in the presence or absence of DMSO, an efficient selective scavenger of OH radicals. Our studies demonstrate that DMSO affects a concentration-dependent modulation of induced asymmetrical aberrations in human lymphocytes exposed to approximately 3.0 Gy, with maximum protectible fractions of approximately 70 percent at DMSO concentrations of greater than or equal to 1 M. The dose dependency for dicentrics in lymphocytes acutely exposed to X-ray doses of 0.51 to 4.98 Gy in the absence of DMSO is adequately described by the linear-quadratic dose-response function Y = alpha D + beta D2. Data from duplicate cultures exposed in the presence of 1 M DMSO produce an excellent fit to the regression function modified as follows: Y(+ DMSO) = alpha(delta D) + beta(delta D)2 where the 'dose modifying' factor delta = 0.501. We interpret these findings as providing evidence that OH radical-mediated lesions in DNA account for approximately 50 percent of the dose dependency for dicentrics resulting from either one-track or two-track events, following exposures of non-cycling cells to moderate-to-high doses of low LET radiation. These data may be used in additional calculations to derive an estimate of approximately 6 x 10(8) s-1 for the rate of reaction of OH radicals with DNA targets involved in aberration formation.

Chemical exposures and central nervous system cancers: a case-control study among workers at two nuclear facilities.

In a nested case-control study of workers employed between 1943 and 1977 at two nuclear facilities, we evaluated the possible association of primary CNS cancers with occupational exposure to chemicals. Seventy-two white male and 17 white female workers who, according to the information on death certificates, died of primary CNS cancers were identified as cases. For each case, four controls were matched on race, sex, facility at which initially employed (cohort), year of birth, and year of hire. Each job title/department combination was subjectively evaluated for potential exposure to each of 26 chemicals or chemical groups. Statistically significant associations were not found between CNS cancer deaths and any of the 26 chemicals. An increased risk of CNS cancer occurrence was observed among subjects employed for more than 20 yr (OR = 7.0, 95% CI = 1.2,41.1, cases = 9).

Brain cancer and nonoccupational risk factors: a case-control study among workers at two nuclear facilities.

In a nested case-control study of nuclear workers, 82 brain cancer cases were compared with 328 matched controls to investigate the possible association with nonoccupational risk factors such as histories of epilepsy or head injury. We observed a moderately strong association between brain cancer occurrence and history of epilepsy (OR = 5.7, 95 per cent CI: 1.0, 32.1), but did not find a positive association with previous head injury (OR = 0.9, 95 per cent CI: 0.2, 4.2).

Skin tumorigenic potential of crude and refined coal liquids and analogous petroleum products.

The skin tumorigenic potential of seven complex hydrocarbon mixtures was determined: a coal-derived raw blend composed of light and heavy oils, a low- and high-severity hydrotreated product of that blend, and naphthas and fuel oils from the raw blend or from natural petroleum. Male and female C3H/Bdf mice were exposed three times per week to each test mixture by dermal application of 50 microliters of neat, 50, or 25% (w/v) preparations. Room, vehicle, and benzo[alpha]pyrene control groups were run concurrently. The raw blend produced an almost 100% incidence of skin tumors at all three doses while tumorigenicity was considerably decreased by hydrotreating the blend both in terms of incidence and onset. The tumorigenicities of the naphthas and fuel oils derived from the raw blend or from petroleums were low relative to that of the parent mixture. Although tumorigens in the raw blend were much reduced by hydrotreatment, tumorigenicity of the other agents did not parallel the content of polycyclic aromatic hydrocarbons known to be good tumor initiators.

CNS cancers and radiation exposure: a case-control study among workers at two nuclear facilities.

A nested case-control study was conducted among workers employed in two nuclear facilities to investigate the possible association of primary CNS cancers with occupational exposure to radiation from external and internal sources. External radiation monitoring data from film badges were available for 27 cases and 90 matched controls. The radiation dose to the lung from internally deposited uranium was estimated for 47 cases and 120 matched controls from area and personnel monitoring data and was used in analyses in lieu of the brain dose. No association was observed between deaths from CNS cancers and exposure to ionizing radiation from external or internal sources. However, due to the small number of monitored subjects and low doses, a modest association could not be ruled out.

Pulmonary toxicity of cytostatic drugs: cell kinetics.

Mice were treated with three cytostatic drugs: cyclophosphamide, busulfan, or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The alveolar labeling index was measured following drug administration with a pulse of 3H-labeled thymidine and autoradiography. In cyclophosphamide-treated animals, peak alveolar cell proliferation was seen 5 days after injection of the drug. In animals treated with busulfan or BCNU, proliferation was even more delayed (occurring 2-3 weeks after administration). In contrast, with oleic acid, the highest alveolar cell labeling was found 2 days after intravenous administration. In animals exposed to a cytostatic drug, proliferation of type II alveolar cells was never a prominent feature whereas in animals treated with oleic acid there was an initial burst of type II cell proliferation. It is concluded that the patterns of pulmonary repair vary between chemicals designed to interfere with DNA replication as compared to agents which produce acute lung damage such as oleic acid.

Intratracheal versus intravenous administration of bleomycin in mice: acute effects.

We examined whether intratracheal instillation (IT) of bleomycin would produce similar or dissimilar lesions when compared to lung damage following intravenous (iv) injection of the drug. BALB/c mice were treated with either 4 U/kg IT or 100 U iv bleomycin and killed at intervals up to 21 days after treatment. Cell proliferation, histopathology, lung lavage, and hydroxyproline content were examined. There was a biphasic response in the cell proliferation in the IT-treated mice, while the iv-treated mice showed a single delayed peak in proliferation. The histopathologic features of interstitial pneumonitis, elevation of lung lavage enzyme activities, and lung hydroxyproline content were qualitatively similar between the two routes of administration, although the IT mice response was always greater in magnitude. Differences exist between the lung reaction to these two routes of administration, but these differences reflect nonspecific inflammatory response and magnitude of initial injury. We conclude that the response to bleomycin administered IT is basically similar to the changes produced by intravenous injection of the drug.

Maximum likelihood estimation for cytogenetic dose-response curves.

In vitro dose-response curves are used to describe the relation between chromosome aberrations and radiation dose for human lymphocytes. The lymphocytes are exposed to low-LET radiation, and the resulting dicentric chromosome aberrations follow the Poisson distribution. The expected yield depends on both the magnitude and the temporal distribution of the dose. A general dose-response model that describes this relation has been presented by Kellerer and Rossi (1972, Current Topics on Radiation Research Quarterly 8, 85-158; 1978, Radiation Research 75, 471-488) using the theory of dual radiation action. Two special cases of practical interest are split-dose and continuous exposure experiments, and the resulting dose-time-response models are intrinsically nonlinear in the parameters. A general-purpose maximum likelihood estimation procedure is described, and estimation for the nonlinear models is illustrated with numerical examples from both experimental designs. Poisson regression analysis is used for estimation, hypothesis testing, and regression diagnostics. Results are discussed in the context of exposure assessment procedures for both acute and chronic human radiation exposure.

Epidemiologic programs for computers and calculators. Use of Poisson regression models in estimating incidence rates and ratios.

Summarizing relative risk estimates across strata of a covariate is commonly done in comparative epidemiologic studies of incidence or mortality. Conventional Mantel-Haenszel and rate standardization techniques used for this purpose are strictly suitable only when there is no interaction between relative risk and the covariate, and tests for interaction typically are limited to examination for departures from linearity. Poisson regression modeling offers an alternative technique which can be used for summarizing relative risk and for evaluating complex interactions with covariates. A more general application of Poisson regression is its utility in modeling disease rates according to postulated etiologic mechanisms of exposures or according to disease expression characteristics in the population. The applications of Poisson regression analysis to problems of summarizing relative risk and disease rate modeling are illustrated with examples of cancer incidence and mortality data, including an example of a nonlinear model predicted by the multistage theory of carcinogenesis.

The analysis of rates using Poisson regression models.

Models are considered in which the underlying rate at which events occur can be represented by a regression function that describes the relation between the predictor variables and the unknown parameters. Estimates of the parameters can be obtained by means of iteratively reweighted least squares (IRLS). When the events of interest follow the Poisson distribution, the IRLS algorithm is equivalent to using the method of scoring to obtain maximum likelihood (ML) estimates. The general Poisson regression models include log-linear, quasilinear and intrinsically nonlinear models. The approach considered enables one to concentrate on describing the relation between the dependent variable and the predictor variables through the regression model. Standard statistical packages that support IRLS can then be used to obtain ML estimates, their asymptotic covariance matrix, and diagnostic measures that can be used to aid the analyst in detecting outlying responses and extreme points in the model space. Applications of these methods to epidemiologic follow-up studies with the data organized into a life-table type of format are discussed. The method is illustrated by using a nonlinear model, derived from the multistage theory of carcinogenesis, to analyze lung cancer death rates among British physicians who were regular cigarette smokers.