Hematopoietic neoplasia in C57BL/6 mice exposed to split-dose ionizing radiation and circularly polarized 60 Hz magnetic fields
Jane T. Babbitt1,9,
Alexander I. Kharazi1,3,
Jeremy M.G. Taylor4,
Carole B. Bonds5,
Stuart G. Mirell2,6,
Emanuil Frumkin7,
Dongliang Zhuang8 and
Theodore J. Hahn1,7
1 Department of Medicine and
2 Department of Radiology, University of California at Los Angeles, Los Angeles, CA 90095,
3 Immunotherapy Laboratory, St Vincent's Medical Center, Los Angeles, CA 90057,
4 Department of Biostatistics, University of Michigan, Ann Arbor, MI 48109,
5 Merle Norman Cosmetics, El Segundo, CA,
6 Nuclear Medicine and
7 Geriatric Research, Education and Clinical Center, V.A. Greater Los Angeles Healthcare System at West Los Angeles, Los Angeles, CA 90073 and
8 Scirex Corporation, Horsham, PA 19044, USA
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Abstract
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This study assessed the effect of chronic exposure to a 60 Hz circularly polarized magnetic field on the occurrence of ionizing radiation-induced lymphoma and other hematopoietic neoplasia in mice. Female C57BL/6 mice received lifetime exposure to either a magnetic field flux density of 1.42 mT for 18 h/day, or an ambient magnetic field of 0.13 µT. Beginning on the first day of magnetic field exposure, 1710 mice were treated with one of three levels of split-dose Cobalt-60
-radiation (cumulative 3.0, 4.0 or 5.1 Gy). The remaining 570 mice received sham irradiation treatment. Sections from 10 lymphoid tissues were evaluated histopathologically for hematopoietic neoplasia. The primary statistical analysis used the Poly3 method to compare lymphoma incidences in magnetic field (MF)-exposed and control mice. Secondary analyses used the Cox proportional hazards model to analyze incidence rates for mortality and development of specific types of neoplasia. The mortality incidence rate was increased by ionizing radiation treatment, and all neoplasms were observed sooner in irradiated mice. However, the lifetime incidence of hematopoietic neoplasia was similar in all experimental groups, including those that were not exposed to ionizing radiation. Chronic exposure to MFs did not affect the mortality incidence rates and did not change the relative incidences of hematopoietic neoplasia in mice that received the same ionizing radiation treatment, with the exception of a marginally significant reduced relative risk of 0.97 (P = 0.05) for lymphoblastic lymphoma in mice exposed to a magnetic field and treated with 5.1 Gy. Lymphomas and histiocytic sarcomas were first observed ~50 days sooner in mice that were exposed to magnetic fields but not ionizing radiation, although this comparison was not statistically significant and the incidence of hematopoietic neoplasia in these mice was not different from that of mice in the 0 T/0 Gy group.
Abbreviations: CI, confidence interval; FCC, follicular center cell; IB, immunoblastic; LB, lymphoblastic; LL, lymphocytic; MF, magnetic field; NOS, not otherwise specified; PC, plasma cell.
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Introduction
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During the period from 1920 to 1970 overall leukemia incidence rose considerably in the USA (1), and childhood leukemia in the USA, UK and Japan exhibited a 10-fold incidence rate increase (2). Today the highest worldwide incidence rates for leukemia, lymphoma and myeloid leukemia are found in economically developed countries, with non-Hodgkin's lymphoma increasing 34% annually (3). Using immunohistologic evaluations (4) to compare pediatric acute lymphocytic (LL) leukemia in developed and undeveloped countries, recent studies have associated increased risk for lymphoid cancers with Westernization (5). A variety of potential risk factors for lymphoid cancers that can be associated with Westernization have been identified (1,2). These include societal and occupational conditions such as viral infection, diet, smoking, low-dose ionizing radiation, magnetic field (MF) exposure from the distribution of electricity, pesticides, organic solvents and treatment-related immunosuppression associated with organ transplants.
The widespread distribution of electricity to residences in developed countries makes MF exposure a likely candidate for investigation. However, it was not until 1979 that an epidemiological study by Wertheimer and Leeper (6) associated powerline wirecodes with elevated risk for childhood cancers in Denver, CO. The results of subsequent studies by other investigators have varied (713). Weakly positive associations between cancer occurrence and presumed MF exposure have generally been correlated with wirecodes rather than field measurements (14,15). Chronic occupational exposures to 60 Hz MF have also been evaluated relative to adult lymphoma/leukemia risk (16). Positive associations have been established for leukemia deaths correlated to specific job titles that imply increased MF exposures (17,18) and, for leukemia, incidence linked to actual MF measurements (19,20). Frequently, however, reanalysis of leukemia mortality data, after replacing the original surrogate estimates with actual contemporary MF measurements, has failed to confirm the previously observed increased risk (21). Critics have questioned whether the association of elevated cancer risk with job titles is due entirely, or in part, to workplace exposures other than MF (11). All retrospective studies have been criticized for their inability to either document actual MF levels during critical time periods or identify relevant types of exposure (15,16,2124), thus emphasizing the need for animal studies that can examine the carcinogenic potential of chronic MF exposure under controlled conditions and exposures.
The biophysical mechanisms by which weak residential-frequency MFs interact with biological systems are unknown (16). The energy levels involved are thought to be insufficient to damage genetic material directly (16,24), although there is some evidence suggesting that magnetic fields may enhance mutagenesis (2527). Other MF effects on cancer development are likely to influence promotion or progression. In general, animal exposure studies have not found evidence of increased tumor incidences as a result of chronic MF exposure (2835), but increases in tumor growth rate (2830), tumor size (28,29), malignant conversion (28) and metastatic infiltration (35) have been reported, supporting a possible co-promotional effect of MF exposure.
To investigate the effect of chronic exposure to residential-frequency MF on leukemogenesis, we applied to the C57BL/6 mouse substrain the model of ionizing radiation-induced lymphoma in C57 Black mice that was published by Kaplan and Brown in 1952 (36). Both strains spontaneously develop lymphoma during middle age. After 2 years of age, the cumulative lymphoma incidence in C57BL/6 mice reaches 2931% (37). Whole body exposure to split dose
- or X-irradiation, beginning at 4 weeks of age, induces a dose-related incidence of thymic lymphoma that is observed within 412 months following treatment. Total cumulative doses of 7.0 Gy can produce thymic lymphoma in >90% of treated mice after several months (38). Over the past 40 years this model has been elucidated as being an example of retroviral tumorigenesis (3941), characterized by a lengthy premalignant phase that involves multiple molecular and cellular events and results in the sequential accumulation of altered regulatory pathways. The exact mechanisms underlying lymphogenesis in this model remain unclear. Nevertheless it offers a useful system in which to evaluate possible promotional and co-promotional effects of chronic exposure to residential-frequency MF.
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Materials and methods
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Animals
Female C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were received at 2124 days of age and quarantined for 68 days in a limited access AAALAC accredited animal research facility. Throughout the experiment, mice were housed in standard polycarbonate cages with stainless steel tops and micro-isolator lids. Certified Purina Rodent Chow #5002 and acidified water (pH 2.5 ± 0.2) were provided ad libitum. Virgin pulp cellulose fiber bedding was changed every 34 days. Environmental parameters were maintained at a room temperature of 21 ± 3°C, with 3070% relative humidity and a 12/12 h light/dark cycle. To minimize the effects of environmental differences related to the positioning of cages within exposure modules and housing racks, cages positions were rotated monthly according to a computerized procedure. Inspections for morbidity and moribundity were conducted daily on all animals, and killing for humane reasons or excessive weight loss was instituted according to strict guidelines. Monthly clinical observations and body weights were recorded, except that during the first 6 months weighings were conducted bi-weekly. A monthly screening procedure for parasites and a serological viral antibody profile were performed on sentinel animals from each experimental room. All experimental procedures and recording of data were carried out in a blinded fashion. The study adhered to Good Laboratory Practices regulations of the US Environmental Protection Agency 40 CFR 792. In accordance with these regulations, an independent quality assurance review was conducted.
MF exposure system
The MF exposure system (Electric Research and Management, Inc., State College, PA) consisted of three identical stationary large exposure modules, having the same directional orientation, located in separate rooms within the animal facility. For each module, resilient vibration-damping brackets were used to mount an array of 19 large coils on a fiberglass support structure. Six fiberglass shelves capable of holding up to 156 standard rodent cages (780 mice) within the uniform field area of each module were supported by a separate free-standing fiberglass rack. Two of the MF exposure modules were operated to produce simultaneous vertical and horizontal (eastwest) 1.0 mT 60 Hz sinusoidal MF having a temporal phase separation of
cycle (90°), which resulted in a total root mean square field vector of constant magnitude (1.42 mT) that rotated at a rate of 60 Hz in the plane normal to the northsouth axis of the building. The third module, which housed the sham-exposed mice, was not energized. Ambient static MF, principally of geomagnetic origin, was not modified in the modules.
Actual measured conditions, including the directional parameters of the geomagnetic and artificial fields are summarized in Table I
. Field intensity and polarization was adjusted by power supply equipment controls located in a separate engineering room. Low total harmonic distortion was maintained by resonant circuits formed by connecting capacitors in series between coils. Together with special damping circuits in the power supplies, this circuitry essentially eliminated transient fields whenever the system was turned on or off. A computer-based monitoring system (Electric Research and Management, Inc.) recorded environmental parameters. Measured values for the MF in each module, the current and voltage to each module, and the air temperature, relative humidity and lighting status in each exposure room were sampled every 10 s and saved to permanent files as hourly averages and hourly spot samples. Mean recorded values for these parameters are given in Table I
. Comprehensive measurements to characterize ambient and exposure MFs were performed before, during, and after the experiment by project staff, the manufacturer, and independently by a representative of the National Institute of Standards and Technology (42).
Treatment of animals and MF exposure
Each animal was uniquely identified by ear punch and tail tattoo. Animals were stratified by initial quarantine body weight into five weight classes and were assigned to experimental cages within treatment groups by a computer-generated randomization procedure which allotted one animal from each weight class to each cage. The experimental design and mean treatment values are summarized in Table II
. Animals treated with split-dose ionizing radiation received one quarter of their total cumulative dose during each of four weekly
-radiation exposures to a Cobalt-60 source at a dose rate of 19.8620.13 cGy/min. The total doses of ionizing radiation received were 0, 3.0, 4.0 and 5.1 Gy. Exposures were determined by averaging dosimetric readings. Beginning at 2832 days of age on the first day of ionizing radiation treatment, animals were exposed to either a 1.4 mT 60 Hz circularly polarized MF, having 1.0 mT horizontal and vertical components, for ~18 h daily (Table I
), or a nominal 0 T ambient MF (average 0.13 µT). MF exposure continued for the duration of the study, including the 7 week terminal sacrifice period.
The approximate maximum practical exposure level achievable under current engineering designs for exposure systems is 1 mT. Circularly polarized MFs were chosen because they produce more uniform and average higher induced currents in experimental animals. Rodents require a 1020-fold greater MF exposure than that required by humans to induce similar current density within the body (43,44). Assuming that induced electric fields are important for MF bioeffects (45), it can be extrapolated that biological effects documented in mice exposed to a 1.4 mT MF approximate biological effects anticipated in humans exposed to a 70140 µT MF. The three separate exposure modules in the animal experimental facility allowed for a maximum of three different exposure levels. Because energized modules generate appreciable stray fields, particularly if circularly polarized fields are used, it was necessary to locate the sham module >30 m from the two energized modules. Due to this limitation of the facility layout, the experimental design was constrained to using twice as many animals in MF-exposed groups as in unexposed groups.
Negative control (NC) animals did not receive sham ionizing radiation treatments and were exposed only to ambient MF. Spot measurements suggested that this MF exposure may have been higher than that of the experimental sham exposed (0 T/0 Gy) group (Table II
). The NC mice were not included in the statistical analysis. However, data relevant to their general health such as growth rate, mortality, and clinical observations were compared with those of the sham control mice in order to identify possible effects due to environmental variables not associated with experimental treatments. Histopathological data from one half of the NC cohort (190 randomly selected mice) was unblinded to establish baseline pathology for untreated female mice of the C57BL/6 strain housed in this facility. All animals were kept on study until natural death, euthanasia or the terminal sacrifice, which was initiated when the mean lifespan of the NC cohort (852 days) had been reached.
Histopathology evaluation
All animals were necropsied and tissues and carcasses were preserved in neutral buffered formalin. All preserved materials were shipped to Pathology Associates International, Inc. (Frederick, MD) for histological preparation and histopathological evaluation of hematoxylin and eosin stained slides prepared from the following tissues: thymus, spleen, lymph nodes (cervical and mesenteric), lungs and mainstem bronchi, sternum (including bone marrow), kidneys, liver (two sections including left lateral lobe and median lobe), brain (three sections including frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons) and gross lesions in the preceding tissues (42). Histopathological evaluation was performed by Robert Kovatch, (Diplomate of the American College of Veterinary Pathology) of Pathology Associates International. The classification for mouse hematopoietic neoplasia proposed by Pattengale and Frith (46,47) was used. The scheme recognizes the following morphological categories of lymphoid neoplasia: lymphoblastic (LB), lymphocytic (LL), immunoblastic (IB), plasma cell (PC) and follicular center cell (FCC) lymphomas. Categories of non-lymphoid neoplasia include myelogenous leukemias, histiocytic sarcoma, and mast cell tumors. In this experiment the immunocytochemical parameters of the classification were not assessed. Neoplasms that could be identified as lymphoma, but could not be assigned to a specific morphological category due to inferior quality of the specimen, were classified as lymphoma not otherwise specified (NOS).
Independent internal and external peer reviews of the diagnoses were conducted. Consensus diagnoses for several unresolved cases were determined by a pathology working group committee (PWG) selected for expertise in rodent pathology and murine lymphoma. The PWG concluded that LB and LL lymphoma, histiocytic sarcoma and granulocytic leukemia were distinct morphological diagnoses and could be analyzed statistically as separate categories. The PWG also found that there was considerable overlap in the morphology of the FCC, IB and PC lymphomas, and recommended that these categories be combined for statistical analysis.
Statistical analysis
Since the primary objective was to test for an effect of MF rather than to assess a possible doseresponse relationship, power calculations indicated that a design with one MF exposure level in which twice as many animals were exposed (2:1 design) had greater statistical power to detect an effect of MF than either a design with both intermediate and high exposure levels (1:1:1 design) or a design without an intermediate exposure level which used fewer animals (1:1 design). In planning the study, the statistical powers for a number of realistic lymphoma incidence rates were compared to determine the required sample size. The hypothesized incidence rates for the MF unexposed arms were based on published literature and a preliminary experiment using different doses of ionizing radiation. A range of hypothetical effects of MF exposure was considered. These comparisons showed that the design and sample size used in the experiment had, in most cases, at least 80% power to detect an odds ratio of 1.5 or higher for the effect of MF exposure on the incidence of lymphoma.
For the primary analysis, the Poly3 method of Bailer and Portier (48) was used to test the null hypothesis of equality of lifetime lymphoma incidence between the MF-exposed and control groups. The secondary analyses employed the Cox proportional hazard model (49) to test the null hypothesis of equality of rates of death (mortality), or rates of death with specific neoplasms present, as a measure of tumor onset. For the secondary analyses, animals were considered uncensored if they died or were killed prior to the terminal sacrifice, and were considered censored if they were killed at the terminal sacrifice. Comparisons between MF-exposed and unexposed animals were made at each level of ionizing radiation treatment. No comparison of the effects of treatment between different levels of ionizing radiation was undertaken. In all analyses, a
2 test statistic was calculated for each radiation dose level. These statistics were combined to give a
2 test with 4 d.f. to test the overall null hypothesis that MFs do not influence the likelihood of lymphoma at the 5% significance level. With this approach no assumptions were made regarding the possible effect of MF on the various ionizing radiation treatment groups, and the latter were compared separately for MF exposure effects before being analyzed for an overall effect. KaplanMeier estimated proportions (50) were used to compare treatment groups for mortality from all causes and for the occurrence of specific neoplasms that were present at death.
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Results
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Ionizing radiation treatment effects
Mortality
Few deaths occurred before 18 months of age in untreated mice. Ionizing radiation treatment resulted in more and earlier deaths from all causes (Figure 1
). Cause of death was not determined, and for most of the study, ~50% of the mice that died did not have a hematopoietic neoplasm present. However, almost all mice that died during the first year were diagnosed with radiation-induced thymic lymphoblastic lymphoma, which can reasonably be considered to be the cause of death (Figures 1 and 2
). Deaths that might be attributable to acute radiation disease were rare. Of the 1710 irradiated mice, only 11 died within the first 100 days.

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Fig. 1. KaplanMeier estimates of mortality from all causes, comparing MF-exposed to unexposed animals at each level of ionizing radiation treatment. Cox regression analysis of the incidence rate data resulted in the following RRs for MF-exposed animals. At 0 Gy, RR = 1.01 (P = 0.59); at 3.0 Gy, RR = 1.01 (P = 0.34); at 4.0 Gy, RR = 0.98 (P = 0.09); at 5.1 Gy, RR = 1.01 (P = 0.46).
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Fig. 2. KaplanMeier estimates of mortality with LB lymphoma present, comparing MF-exposed to unexposed animals at each level of ionizing radiation treatment. Cox regression analysis of the incidence rate data resulted in the following RRs for MF-exposed animals. At 0 Gy, RR = 0.94 (P = 0.67); at 3.0 Gy, RR = 0.94 (P = 0.21); at 4.0 Gy, RR = 1.03 (P = 0.44); at 5.1 Gy, RR = 0.97 (P = 0.05).
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Histopathology
The spontaneous lymphomas that were observed beginning at 18 months of age in untreated (0 T/0 Gy) mice were found to be primarily of the FCC, IB and PC morphologic subtypes (Table III
). A substantial number of late occurring histiocytic sarcomas were also observed in these animals. Lymphoblastic lymphomas were rare among untreated mice, but treatment with ionizing radiation, particularly at the 5.1 Gy level, considerably increased the incidence of this lymphoma in young mice (Figure 2
; Table III
). Together, the late developing FCC, IB and PC lymphomas, histiocytic sarcomas and early occurring LB lymphomas accounted for the great majority of hematopoietic neoplasia observed during the experiment. Smaller numbers of LL lymphoma and a few granulocytic leukemias were represented in most treatment groups. Other subtypes of myelogenous leukemias and mast cell tumors which complete the Pattengale/Frith classification are rarely found in mice (51). Over the course of the study, a concomitant decrease in the number of late-occurring hematopoietic neoplasms offset the early increased incidence of LB lymphoma in irradiated mice. As a result, the final frequencies for total combined hematopoietic neoplasms were remarkably similar across all treatment groups (Table III
).
Incidence rate of neoplasia
Although no data were collected which could establish the actual onset of neoplasia for an individual animal, necropsy records were used to identify the date when neoplasia was first observed. The choice of a parameter by which to measure the onset of neoplasia is an arbitrary decision. For purposes of this statistical analysis, tumor onset is defined as death with neoplasm present. Hematopoietic neoplasias of every morphologic type were first observed at an earlier age in mice that had been treated with ionizing radiation (Figures 1 and 36



).

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Fig. 3. KaplanMeier estimates of mortality with FCC, IB, or PC lymphoma present, comparing MF-exposed to unexposed animals at each level of ionizing radiation treatment. Cox regression analysis of the incidence rate data resulted in the following RRs for MF-exposed animals. At 0 Gy, RR = 1.02 (P = 0.31); at 3.0 Gy, RR = 1.00 (P = 0.95); at 4.0 Gy, RR = 0.99 (P = 0.44); at 5.1 Gy, RR = 1.00 (P = 0.85).
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Fig. 4. KaplanMeier estimates of mortality with histiocytic sarcoma present, comparing MF-exposed to unexposed animals at each level of ionizing radiation treatment. Cox regression analysis of the incidence rate data resulted in the following RRs for MF-exposed animals. At 0 Gy, RR = 1.03 (P = 0.22); at 3.0 Gy, RR = 1.02 (P = 0.45); at 4.0 Gy, RR = 0.97 (P = 0.17); at 5.1 Gy, RR = 1.01 (P = 0.64).
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Fig. 5. KaplanMeier estimates of mortality with any lymphoma present, comparing MF-exposed to unexposed animals at each level of ionizing radiation treatment. Cox regression analysis of the incidence rate data resulted in the following RRs for MF-exposed animals. At 0 Gy, RR = 1.02 (P = 0.25); at 3.0 Gy, RR = 0.99 (P = 0.68); at 4.0 Gy, RR = 0.99 (P = 0.75); at 5.1 Gy, RR = 0.99 (P = 0.31).
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Fig. 6. KaplanMeier estimates of mortality with any hematopoietic neoplasm present, comparing MF-exposed to unexposed animals at each level of ionizing radiation treatment. Cox regression analysis of the incidence rate data resulted in the following RRs for MF-exposed animals. At 0 Gy, RR = 1.02 (P = 0.10); at 3.0 Gy, RR = 0.99 (P = 0.85); at 4.0 Gy, RR = 0.99 (P = 0.44); at 5.1 Gy, RR = 0.99 (P = 0.43).
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MF exposure effects
Histopathology
No new morphological categories of hematopoietic neoplasia were observed among mice exposed to MFs. The relative frequencies and general occurrence of hematopoietic neoplasia were similar for both MF-exposed and unexposed mice that had received the same ionizing radiation treatment (Table III
; Figures 26



), with the exception of reduced incidence rates for LB lymphoma in the 3.0 Gy (P = 0.21) and 5.1 Gy (P = 0.05) MF-exposed animals (Table IV
).
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Table IV. Cox regression on the effect of MF exposure on the incidence rate of lymphoblastic lymphomas present at death
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Primary statistical analysis: incidence of combined lymphomas
For the primary statistical analysis, Poly3-adjusted lifetime lymphoma incidences of the MF-exposed groups were compared with unexposed groups. Data from all the lymphoma categories (LB, LL, FCC, PC, IB and NOS) were combined for this analysis and included the terminal sacrifice animals. Total tumor incidences for MF-exposed groups were not appreciably different from those of unexposed animals (Table V
), and the combined test statistic was not significant (P = 0.55). The large sample size and numbers of events (Table III
) are responsible for the narrow confidence intervals. This analysis has sufficient power to detect the magnitudes of risk reported in EMF epidemiological studies of lymphoma/leukemia.
Secondary statistical analyses
In order to evaluate possible effects of chronic MF exposure on incidence rates within the subcategories of lymphomas and other hematopoietic neoplasia included in the Pattengale/Frith morphological classification, we used Cox regression analysis to compare MF-exposed with unexposed groups within dose levels of ionizing radiation treatment. Terminal sacrifice data were excluded from these analyses.
Mortality
Mortality incidence rates were also not different for mice exposed to MF (Figure 1
). Secondary analysis of deaths from all causes using Cox regression showed no overall effect of chronic MF exposure on mortality rate (Table VI
), although a trend toward reduced mortality was noted in the MF-exposed 4.0 Gy group which had a relative risk (RR) of 0.98 (P = 0.09).
Hematopoietic neoplasms in unirradiated mice
Data collected from the 0 T/0 Gy and l.4 mT/0 Gy animals was unconfounded by ionizing radiation treatment effects. Table VII
summarizes the statistical analyses of incidence rate data comparing these 0 Gy groups and reflects the fact that the combined FCC, IB and PC lymphomas and histiocytic sarcomas, both of which develop spontaneously in unirradiated mice, were observed ~50 days earlier in 1.4 mT/0 Gy exposed animals (Figures 3 and 4
), although the P-values for these comparisons do not approach statistical significance. Comparable differences in mortality rates for these two groups were not found, indicating that the early observation of neoplasia in the MF-exposed animals was not the result of earlier deaths in that group.
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Table VII. Cox regression on the effect of MF exposure on the incidence rates for mortality and hematopoietic neoplasms present at death in unirradiated mice
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Incidence rate of ionizing-radiation-induced lymphoblastic lymphoma
The fast-developing LB lymphomas induced by ionizing radiation treatment were found exclusively in the thymuses of both MF-exposed and unexposed mice. Cox regression analyses of LB lymphoma incidence rate are shown in Table IV
. When compared by ionizing radiation dose, differences in LB incidence rates for MF-exposed animals were not significant except at the 5.1 Gy level, where a significantly reduced RR for LB lymphoma (0.97, P = 0.05) was observed among animals exposed to MF. A similar reduction in LB frequency (Table III
) and RR (Table IV
) at the 3.0 Gy level was not statistically significant (RR = 0.94, P = 0.21), but the power to detect a difference was low because of the small number of events. The combined test statistic (
2 = 6.036, P = 0.20) was weakened by the inconsistency of the effect at the other ionizing radiation treatment levels.
Incidence rates of other hematopoietic neoplasia
Combined FCC, IB and PC lymphomas.
A comparison by Cox regression analysis of the incidence rates for combined FCC, IB and PC lymphomas observed at necropsy demonstrated no significant effect of MF exposure on the presence of this morphological lymphoma subtype among mice that died, either when the ionizing radiation treatment groups were stratified, or when combined (
2 = 1.675, P = 0.80). Statistical data from the comparison of the incidence rate of this lymphoma subtype between unirradiated groups is shown in Table VII
(RR = 1.02, P = 0.31). RRs for MF-exposed animals treated with 3.0, 4.0 or 5.1 Gy were 1.00 (P = 0.95), 0.99 (P = 0.44) and 1.00 (P = 0.85), respectively. Corresponding confidence intervals (CIs) were within 0.951.05.
Histiocytic sarcoma.
Histiocytic sarcomas are neoplasms of mononuclear phagocytic lineage rather than lymphomas. However, they share common precursors and developmental pathways with lymphoid progenitors, and are considered hematopoietic neoplasms. The incidence rates of histiocytic sarcomas among animals exposed to MF compared with unexposed animals were not different when analyzed by ionizing radiation level, or as a combined statistic (
2 = 4.178, P = 0.38). Table IV
presents the regression analysis of the histiocytic sarcoma incidence rates for MF-exposed and unexposed groups of unirradiated animals (RR = 1.03, P = 0.22). RRs for MF-exposed animals treated with 3.0, 4.0 or 5.1 Gy were 1.02 (P = 0.45), 0.97 (P = 0.17) and 1.01 (P = 0.64), respectively. Corresponding CIs were within 0.931.07.
Combined lymphomas.
Cox regression of the incidence rates for all combined lymphomas comparing MF-exposed to unexposed mice that died prior to the terminal sacrifice showed no significant differences when stratified by ionizing radiation level or when combined (
2 = 2.655, P = 0.62). The statistical comparison of the incidence rates of combined lymphomas in unirradiated groups either exposed or unexposed to MF is shown in Table VII
(RR = 1.02, P = 0.25). RRs for MF-exposed animals treated with 3.0, 4.0 or 5.1 Gy were 0.99 (P = 0.68), 0.99 (P = 0.75) and 0.99 (P = 0.31), respectively. Corresponding CIs were within 0.961.02.
Combined hematopoietic neoplasia.
For this analysis, data from granulocytic leukemias, histiocytic sarcomas and all lymphoma categories were combined. Although the combined test statistic demonstrated no effect of MF exposure on incidence rate (
2 = 3.89, P = 0.42), the trend toward increased risk (RR = 1.02, P = 0.10) at the 0 Gy level reflects the earlier observation of lymphomas and histiocytic sarcomas in MF-exposed mice that were not irradiated (Table VII
). RRs for MF-exposed animals treated with 3.0, 4.0 or 5.1 Gy were 0.99 (P = 0.85), 0.99 (P = 0.44) and 0.99 (P = 0.43) respectively. Corresponding CIs were within 0.971.02.
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Discussion
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Mortality incidence rate was not increased by MF exposure. The unusually large size of the treatment groups allowed small differences in treatment effects to be detected, particularly when the number of observations was substantial. Consequently, the results of the statistical analyses of combined lymphomas, combined hematopoietic neoplasia, combined FCC, IB and PC lymphomas, and histiocytic sarcomas convincingly establish a lack of overall effect of treatment with a single high level of chronic MF exposure on the incidence of these neoplasms. For the same reason, the significantly reduced relative risk for LB lymphoma in the 1.4 mT/5.1 Gy mice should not be discounted, and together with the non-significant reduced incidence of LB lymphoma in the 1.4 mT/3.0 Gy group, should be considered for further investigation. However, although observed trends should not be ignored, the interpretation of borderline statistically significant findings for secondary endpoints should be performed with a degree of caution because the analysis did not include any formal adjustment for multiple comparisons. The statistical analysis of the MF effect on LB lymphoma incidence combining all radiation levels is weakened by the small numbers of observations in the groups treated with lower levels of ionizing radiation. One question that arises is whether the observed number of LB lymphomas would be substantially increased if the NOS lymphomas could be assigned to their appropriate morphological categories. Using the following criteria for speculative reassignment to the LB categorythat the NOS lymphoma must have occurred in the thymus within the first 18 months of the studythree mice of the l.4 mT/5.1 Gy NOS group, and one mouse from the 0 T/5.l Gy NOS group would be eligible for reassignment to the LB category. However, this change would have no substantial effect on the analysis and is not a credible explanation for the observed differences. Other investigators have occasionally observed significantly reduced incidences of specific tumors in MF-exposed animals relative to sham-exposed animals (34).
In determining the experimental design for this study, priority was given to providing sufficient statistical power to detect small differences in tumor occurrence between MF-exposed and unexposed treatment groups. This decision and its implications regarding commitment of animals, resources and staff precluded the incorporation of scheduled interim sacrifices and bioassays that could have contributed information on transient or permanent alterations in DNA repair, genomic stability, cellular stress responses, and endocrine function that would be required to elucidate the mechanisms underlying observed MF exposure effects on tumor incidence rate. The choice of the C57BL/6 strain, which is moderately susceptible to both spontaneous lymphomas and ionizing-radiation- induced thymic lymphoma, rather than a strain having little or no spontaneous lymphoma incidence, provided increased statistical power by insuring a high frequency endpoint. However, in the absence of data relating to biochemical and molecular effects, such a model is unable to evaluate a possible role for MF in altering the initiation process in cells subjected to challenge by repeated doses of ionizing radiation. A non-susceptible strain would probably prove to be less useful in addressing this issue. The same animals that were evaluated for hematopoeitic neoplasia in this study were also evaluated for primary brain tumors (unrelated to lymphoma), even though C57BL/6 mice are not known to be susceptible to brain tumors (52). Ionizing radiation treatment did alter both the tumor type and incidence, but the numbers were so small that an evaluation of the additional effect of MF exposure was not possible (53).
A unique feature of the Pattengale classification is the assessment of immunocytochemical markers in neoplastic tissues following morphological evaluation. These markers allow the immunological identity of neoplasms to be correctly determined, particularly within lymphoma categories whose morphological characteristics are not unique to a specific lineage. The lack of definitive morphological characteristics within the FCC, IB and PC morphologic subtypes underscores the need for the immunologic phenotyping recommended by the Pattengale classification, especially since it is anticipated that in rodents, as in humans, immunoblastic lymphomas may occur either as T-cell or B-cell clones (51). In addition, even though they are distinguishable morphologically, LB and LL lymphomas are widely recognized to be immunologically heterogeneous. Immunocytochemical evaluations have identified T-cell, B-cell and non-T/non-B clones among both LB and LL lymphomas (51). It is often thought that all LB lymphomas found in the thymus are exclusively of T-cell origin, as is the case with certain strains of mice treated with high levels (4x1.75 Gy) of split-dose ionizing radiation (38). However, immunohistological analyses using monoclonal antibodies now in progress in our laboratory suggest that in C57BL/6 mice treated with a lower level of split-dose ionizing radiation (4x0.75 Gy), examples of Thy1.2+/B220+ and Thy1.2/B220 thymic LB lymphomas can be found among both MF-exposed and unexposed animals (unpublished data). Similar evaluation of the LL lymphomas among the 0 T/3.0 Gy and 1.4 mT/3.0 Gy groups has also identified markers for B-cell, T-cell and non-T/non-B clones. Because the total numbers of LB (184) and LL (56) lymphomas observed during the study were substantial, further assessment of the LB and LL lymphomas in other treatment groups using immunohistological techniques, as recommended by Pattengale and Frith, would allow these lymphomas to be recombined according to immunologic lineage, and their comparative phenotypic frequencies analyzed for possible MF related alterations.
The present study design cannot adequately assess the influence of chronic MF exposure on the initiation of neoplastic lesions. However, another proposed effect of MF exposure has been the acceleration of neoplastic growth and progression (2830,35). The present study data provide some supporting experimental evidence for this postulate, including the observation that histiocytic sarcomas and FCC, IB and PC lymphomas were found earlier in unirradiated mice exposed to MF, although this comparison was not statistically significant. The lack of a well-defined mechanism for MF effects on biological systems makes it difficult to assess the suitability of models used to clarify such effects. Interpretation of the present study's findings would also benefit from a more exact knowledge of the etiology of ionizing-radiation-induced lymphomas. The observation that the final frequency of combined hematopoietic neoplasms is similar for all experimental groups of both irradiated and unirradiated mice, suggests that treatment within the range of split-dose ionizing radiation used for this experiment promotes the differentiation and growth of specific preneoplastic hematopoietic precursors, rather than initiating lymphomagenesis. The data from this study suggest that tumor development is promoted in those animals which received only ionizing radiation treatment or only MF exposure. The data also suggest an interaction of ionizing radiation treatment effects and MF exposure effects in the development of radiation-induced thymic LB lymphomas. The mechanisms by which these two different levels of radiant energy affect tumor development remains to be determined, as does the mechanism of their interaction. Future studies to elucidate these relationships are warranted.
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Notes
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9 To whom correspondence should be addressed Email: jbabbitt{at}ucla.edu 
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Acknowledgments
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The success of this experiment was largely due to the expertise and dedication of the research technical staff: Nataliya Bufius, Domingo Ruiz, Mohamad Saatara, Arlo Kurian, Federico Maledda, Don Babbitt, Doug Campbell, Maria Maligaya, Maziar Izadi, Sean Corbett, Blake Parsons and Stacy Crevello. We wish to thank Felicia Hesler and Darcy Richardes for project administration, and Judy Sy for statistical assistance. We are grateful to Janet Halter for conducting the quality assurance review. This study was supported by research contracts W02965-03, W02965-11, W02965-31, W02965-33 and W02965-34 with the Electric Power Research Institute, Palo Alto, CA, with co-funding from BC Hydro.
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Received November 23, 1999;
revised March 27, 2000;
accepted March 30, 2000.