Thyroid Hormones and Cytogenetic Outcomes in Backpack Sprayers Using Ethylenebis(dithiocarbamate) (EBDC) Fungicides in Mexico Kyle Steenland,1 Leonor Cedillo,2 James Tucker,3 Cynthia Hines,' Karen Sorensen,3 James Deddens,1 and Veronica Cruz2 'National Institute for Occupational Safety and Health, Cincinnati, OH 45226 USA; 2lnstituto Nacional de Salud Publica, Cuernavaca, Mexico; 3Biology and Biotechnology Research Program, Lawrence Livermore Laboratory, Livermore, CA 94551 USA Ethylenebis(dithiocarbamate) (EBDC) f are hea`il in the Unid St ED (e.g, mancozeb, maneb) are metabolized to ethylene ir(ETU). The EPAc ifies EMU as a carcinogen, based on thyroid and othercancers ird n a e use o EBDC, while requiring to use p ie carcinogenicty of EBDCs in huma'ns, and re is on ne hu n ETU is known to cause de s of thyroxine,C) a ie in (TSH) in rodents. We ve stied among 49 heavily exposed workers witout ro u ipme: spraying D o tma in Mexico. We also studied 14 lighdy e nd 1 s ETU was used to compare exposure bT among applicators compard to controls, but;no de i (T). W-e found increases in sister chromatid exchang (P 0.03) and in c omo t ati (romo- some aberrations that penrist tough ce On) 0.05). However, t-he subset of rec trpl s ). O data suggest that EBDCs affect the tyroid nd an h ce a n h exposed workers. However, our data are limtedto.sbciicll tical1; significanice, and should be int ed e s ethylenebis(dithiocarbamates), EBDC, ethylen thiourca, ETU, eticde i& Envimrn Health Perspect 105:1126-1130 (197). ph n; Ethylenebis(dithiocarbamate) (EBDC) fungicides (maneb, mancozeb, and metiram) are among the most common in the United States, with approximately 8-12 million pounds of active ingredients used annually prior to 1990 (1). Different EBDCs share a common molecular structure, but differ by metal cation (Fig. 1). EBDCs are absorbed primarily dermally and are metabolized to ethylene thiourea (ETU). Dermal absorption of EBDCs range from 1 to 10%, and approximately 7.5% of the absorbed dose is converted to ETU (1). EBDCs and ETU are mildly genotoxic in bacterial and animal sys- tems (1). The EPA classifies ETU as a car- cinogen, based on animal data showing that it causes thyroid and other cancers in rodents. Because of concern about carcino- genicity, in 1992 the EPA canceled the use of EBDCs on 11 crops, but EBDCs are still used in the United States on a wide variety of fruit, nut, and vegetable crops such as apples, almonds, and tomatoes (1). There are no data on the potential carcinogenicity of EBDCs in humans. In the one study on human genotoxicity, 44 workers producing a fungicide containing mancozeb showed sig- nificantly increased chromosomal aberrations (CAs; primarily breaks) in exposed non- smokers compared to controls and non- significant elevations among exposed smok- ers compared to controls (4. CAs are visible at metaphase as chromosomal breaks or rearrangements and are often lethal to the cell. These same data showed nonsignificant elevations of sister chromatid exchanges (SCEs) of exposed versus nonexposed work- ers after stratification by smoking. ETU has also been shown to cause a sig- nificant decrease in thyroxine (T4) in pro- duction workers (mixers) who were highly exposed versus controls, although the num- ber of mixers was small and calculated vari- ances of T4 levels do not appear to have taken into account that there were repeated measures on the same person (3). ETU has been shown to have the same effects in rodents, causing a decrease in T4 and an increase in thyroid-stimulating hormone (TSH). It has been hypothesized that this disruption of thyroid homeostasis, which correlated with morphological changes in the thyroid gland, may be related to the thy- roid tumors observed in rodents (4). We sought to investigate further the pos- sible cytogenetic and thyrotoxic effects of EBDC fungicides. Our study population was composed of Mexican backpack sprayers applying EBDCs to tomatoes, a group of lesser exposed landowners, and a nonex- posed comparison population. Urinary ETU was measured to evaluate current exposure. Cytogenetic outcomes included sister chromatid exchange and chromosome aberrations. The latter outcome was mea- sured by chromosome painting, which enables detection of stable aberrations such as translocations, as well as unstable events such as dicentrics and acentric fragments. Materials and Methods Selection of subjects. Data were collected on a population of 49 backpack applicators, 14 small landowners, and 31 nonexposed con- trols who were employed by city govern- ment in construction jobs. Blood samples for hormone analysis and questionnaires were also collected from an additional group of 14 controls to replace two ship- ments of blood, which were delayed in shipment and were damaged because they arrived without ice. All subjects were males and lived near Cuernavaca in central Mexico. Subjects were selected via contact with small landowners who grow tomatoes in the area. All workers employed by a recruited landowner were also recruited, and recruit- ment continued until the target sample size of 50 exposed subjects was assured. Demographic data were collected by questionnaire, including data on smoking and alcohol use. All subjects were compensated $10 for their time and inconvenience, and all subjects signed consent forms explaining the study. All blood was drawn in the morning. The backpack applicators applied pesti- cides seasonally (une to November), pri- marily to tomatoes. Many applicators were migrants who returned to their home province for part of the year. Owners were full-time tomato farmers and were generally present during application; owners were likely to have some exposure, although less than the applicators. Exposure. EBDCs were applied to toma- toes in a liquid mixture of water, plant nutri- ents, and other pesticides as needed. These other pesticides typically included an organophosphate, e.g., chlorpyrifos or methamidophos, as well as foliar nutrients and growth regulators. On occasion another Address correspondence to K. Steenland, National Institute for Occupational Safety and Health, Mailstop R13, 4676 Columbia Parkway, Cincinnati, OH 45226 USA. This work was performed in part at the Lawrence Livermore National Laboratory under the auspices of the U.S. Department of.Energy contract W- 7405-Eng-48. Received 3 February 1997; accepted 9 June 1997. Volume 105, Number 10, October 1997 * Environmental Health Perspectives 1126 Articles * Thyroid hormone and cvtogenetic effects of EBDC fungicides Figure 1. A) Structure of maneb and mancozeb (chelate and metal). B) Structure of ethylene thiourea (ETU). Maneb, R = Mn2+; mancozeb, R= Mn2+ + Zn2+. fungicide, captan, was used along with the EBDCS, but this was not done commonly. A typical mixture induded 0.75 kg mancozeb (powder), 0.33 liter liquid methamidophos (48% by weight), 1 kg plant regulator (pow- der), 0.25 liter foliar nutrients (liquid), and 200 liters water in a large barrel. Mixing was done without protective equipment; the pes- ticides were poured into the mix by hand and then stirred with a stick. A bucket was then dipped into the mixture, filled, and poured into backpack sprayers containing approxi- mately 20 liters. Spills onto the skin were common in this process. Crews typically had three to six members. Depending on the size of the field, workers might apply 5-10 barrels a day over a period of perhaps 4-6 hr. Use of fungicides varied with the amount of rain; wetter conditions required more fungicides. Applicators wore only light clothing and san- dals, and a fine spray of pesticides generally drifted onto the clothes and exposed body surfaces of the applicators. Each crew, typical- ly employed by the owner over the season, applied pesticides once or twice a week. Collection of biological samples. Different numbers of subjects were available for analysis of different endpoints. Blood was drawn in the morning for both exposed and nonexposed subjects. All blood sam- ples were shipped on ice within 24 hr to the laboratory. For the exposed subjects, an attempt was made to collect blood in the morning on a day exposure took place and to collect the first-void urine the morning of the next day from the same worker. The urinary elimination half-life of ETU in humans has been estimated to be approxi- mately 32-100 hr, with highest levels excreted in the urine in the first 24 hr after exposure (4,5). Most subjects contributed both urine and blood; however, some subjects con- tributed only one or the other (no subject contributed more than one sample for either blood or urine). The urine was used to confirm exposure and to serve as a basis of comparison with other studies (ETU is generally not present in the urine of indi- viduals without EBDC exposure). Urinary ETS was not used for exposure-response analyses, so having blood and urine sam- ples on the same people was not essential. Serum thyroid hormone data were orig- inally available on all 31 nonexposed sub- jects and 51 of 63 exposed subjects. Because two shipments of blood (21 nonexposed, 8 exposed subjects) were delayed and arrived after ice melted in hot summer tempera- tures (altering hormone levels), these sam- ples could not be used for the hormone analysis, although most were able to be used for cytogenetic analysis. Fourteen additional controls were then recruited, and blood samples were obtained from them for hor- mone analysis only. Cytogenetic data were available for 30 of 31 nonexposed subjects and 44 of 63 exposed subjects. Urine data were available for all 31 nonexposed sub- jects and for 50 of 63 exposed subjects. Data were missing for a variety of reasons. Some subjects refused to supply the sample (typically the blood), others failed to collect and/or deliver the sample (typically the urine), and some samples were inadvertent- ly lost in transport (blood). Urine samples were stored on dry ice and shipped to the lab. Double blind sam- ples spiked with known amounts of ETU were also sent to the lab for quality control and confirmation of the limit of detection. Analysis of urine for ETU was done via high performance liquid chromatography using an electrochemical detector (6), with a limit of detection of 10 ppb. Blood samples for thyroid hormone analysis were collected in 5-ml serum sepa- ration tubes and spun down to separate the erythrocytes prior to shipping. Both T4 and TSH were analyzed via enzyme immunoassays (MEI or microparticle enzyme assay for TSH via IMX, CEDIA enzyme immunoassay for T4). Blood sam- ples for cytogenetic analysis were collected in 7-ml heparinized tubes. Cytogenetic procedures. Cell culture and cytogenetic analyses were conducted in Livermore, California, without knowledge of the exposure category of samples. Whole blood (0.8 ml) was added to 10 ml of RPMI 1640 medium (Gibco/BRL, Gaithersburg, MD) supplemented with 15% fetal bovine serum, 100 IU/ml penicillin, 100 pg/ml streptomycin, 1% sodium heparin, 2% phy- tohemagglutinin, and 2 mM L-glutamine. Four cultures were prepared for each subject, two of which were supplemented with 100 mM bromodeoxyuridine (BrdU) to obtain SCEs. Cultures were incubated at 37?C in a 5% CO2 environment. The culture duration was 52 and 72 hr for aberrations and SCEs, respectively; in each case, 0.1 pg/ml Colcemid was added for the last 4 hr of cul- ture. Cells were harvested by swelling in 0.075 M KCl and then fixed a minimum of three times in 3:1 (v/v) methanol:glacial acetic acid. Well-spread metaphase cells, low in cytoplasm, were prepared as described elsewhere (7). All slides were stored at -20?C in sealed plastic bags in the presence of a des- iccant and N2 until needed for hybridization. For SCEs, slides were stained with the fluorescence-plus-Giemsa method (8,9). Fifty second-division cells were scored, 25 from each of two replicate cultures. Two readers each scored one culture per donor. Most cells had 46 chromosomes, although some had as few as 45 or as many as 48 chromosomes. The SCEs were normalized to 46 chromosomes per cell prior to analysis. For chromosome painting, slides were hybridized with SpectrumOrange chromo- some-specific DNA probe (Vysis, Downers Grove, IL) for chromosomes 1, 2, and 4 simultaneously according to the manufactur- er's instructions, with minor modifications as described elsewhere (7,10). The slides were mounted in a DAPI antifade solution (Sigma, St. Louis, MO) (11) and coded. All cells were visualized with a dual band pass fil- ter (Vysis), which allows simultaneous view- ing of SpectrumOrange labeled and DAPI counter-stained chromosomes. Metaphase cells were considered scoreable if they met the following criteria: 1) the cells appeared intact with well-spread chromosomes, 2) the centromeres of all chromosomes were readily visible, 3) the centromeres from all six paint- ed chromosomes were present, and 4) the SpectrumOrange label was sufficiently bright to detect exchanges between painted and unpainted chromosomes. Every abnormal metaphase cell was photographed with Kodak Ektachrome 400 film (Eastman Kodak, Rochester, NY). These photographs provide a permanent record of each aberrant cell and were used to resolve ambiguous aberrations. All aberrant cells were catego- rized according to the Protocol for Aberration Identification and Nomenclature Terminology (PAINT) (12). Following the initial evaluation of aberrations, transloca- tions were redassified as apparently recipro- cal if two color junctions, in one each of two Environmental Health Perspectives * Volume 105, Number 10, October 1997 1127 Articles * Steenland et al. chromosomes, were visible (13). If only one color junction was evident, the translocation was considered to be nonreciprocal. Analyses were conducted for all translocations and for the subset of reciprocal translocations. The analysis of aberrations by chromo- some painting involves several assumptions (14). With painting, only a fraction of all events are seen, specifically, only those with exchanges occurring between painted and unpainted chromosomes. The fraction of all translocations detected by hybridization (Fh) is determined from the amount of the genome painted and is related to the fraction observable by G-banding (Fr,) by the equation Fh = 2fs(1-fs) Fb where J is the proportion of the genome painted (13) and Fb is assumed to be 1. The fraction of all dicentrics detected is determined by the same formula. In the work presented here, chromosomes 1, 2, and 4 were painted simultaneously. These chromosomes represent 22.05% of the genome (15) and detect 34.4% of all exchanges. In this study, approximately 1500 metaphase cells were scored per sub- ject. This is equivalent to 1500 x 0.344 = -500 metaphase cells if the full genome were scored (defined as cell equivalents). Statistical analysis. Statistical analysis for the hormone and SCE data was done by ordinary linear regression analyses, and least square means for exposure groups were cal- culated, adjusted for covariates. Residuals for these models were approximately normal. Analysis of the chromosome aberration data was done via Poisson regression, as positive outcomes were rare events in these data. The number of cell equivalents was taken into account by including it as an offset (constant term) in the Poisson regression; results for exposure effect are invariant to proportional changes in this offset and are therefore inde- pendent of our assumptions regarding cell equivalents. Age (categorized by 5-year inter- vals: <20, 20-24, 25-29, 30-34, 35-39, >40) was included as a covariate in all regres- sions for hormone outcomes, SCEs, chromo- some translocations, and reciprocal transloca- tions. Other aberrations were rare (making a model with fewer parameters preferable) and have been shown to increase approximately linearly with age (7). Therefore, models for dicentrics, insertions, fragments, and rings included exposure and a single (continuous) parameter for age. There were only seven subjects over 40 years of age in the study, and six of these were exposed. Given this imbalance in the highest age category and because of the known importance of age for several outcomes, par- ticularly chromosome translocations (16), we also conducted additional analyses for all out- comes after restricting the subjects to those aged 40 or less. Smoking was thought a priori to be a potential confounder for sister chromatid exchange and, to a lesser extent, for chromo- somal translocations. A dichotomous vari- able for current smoking was added to mod- els for all outcomes and tested. It was not a significant predictor in any model, possibly because smokers in this population were very light smokers (4-5 cigarettes/day); therefore, the smoking variable was dropped for all models but the SCE analysis. It was retained in the final model for SCEs because it had some predictive value (p = 0.16) and some slight confounding effect on the expo- sure effect. Alcohol consumption was also tested for both SCEs and CAs, but was not an important predictor for either outcome; it was not included in final models. Analyses were conducted using SAS (PROC GLM or PROC REG for linear regression, PROC GENMOD for Poisson regression; SAS Institute, Cary, NC) (17). Results Table 1 gives the results for age, smoking, and urinary ETU by three exposure cate- gories (nonexposed, owners, applicators). The exposed applicators and owners were somewhat older, on the average, than the nonexposed. The majority of all groups smoked, but smokers smoked few cigarettes (subjects who smoked one cigarette or less per week were considered nonsmokers). All of the nonexposed subjects had urinary ETU below the limit of detection (10 ppb). The same was true for 13 of 38 applicators (34%) and 6 of 12 owners (50%). After assigning half the limit of detection (i.e., 5 ppb) to exposed subjects with an ETU level below the limit of detec- tion, the mean was 58 ppb (geometric mean 19) for applicators and 12 ppb (geo- metric mean 9) for owners. The ETU exposure of the applicators was somewhat higher than prior urinary data for applica- tors in Europe (18) (maximum 13 ppb) or for grape thinners exposed to residues in the United States (19) (maximum 25 ppb). Table 2 presents the unadjusted and age- adjusted results of the thyroid hormone analysis. All subjects were within normal lim- its for all hormone tests. There were no sig- nificant differences between groups for T4: the controls and the heavily exposed applica- tors had virtually identical results. The unad- justed data also showed no significant differ- ences for TSH (applicators compared to nonexposed, p = 0.20). Age was negatively correlated with TSH and acted as a negative confounder in these data. The data adjusted for age indicated an exposure effect. The applicators had a significantly higher amount of TSH (0.52 mIU/l, p = 0.05) than the nonexposed, while the landowners had a minimal and nonsignificant elevation of TSH (0.13 mIU/l, p = 0.74). Increased TSH may indicate hypothyroidism even in the absence Table 1. Demographic variables and urinary ethylene thiourea (ETU) for study subjects Applicators (n= 49) Owners (n= 14) Nonexposed (n= 31) Mean age ? SE 26.2 ? 1.6 31.6 ? 2.5 22.0 ? 1.2 Smokers (%) 70 50 67 Cigarettes/day smokers ? SE 3.5 ? 0.8 4.8 ? 1.3 5.1 ? 2.5 Mean urinary ETU ? SE (ppb)a 58 ? 26 12 + 3 NA Geometric mean urinary ETU (geometric SD) 19 (4) 10 (2) NA Abbreviations: SE, standard error; SD, standard deviation; NA, not available. allrine samples were available for 38 applicators, 12 owners, and 31 nonexposed subjects. All nonexposed subjects had levels below limit of detection (10 ppb) as did 34% of applicators and 50% of landowners. Samples for exposed subjects below the limit of detection were assigned half the limit of detection. Table 2. Thyroid hormone resultsa Applicators (n = 32) Owners (n = 11) Nonexposed (n = 24) Unadjusted for age MeanT4 ? (pg/dl)b 7.65 ? 0.18 8.11 ? 0.43 7.68 ? 0.41 Mean TSH ? SE (mlU/1) 2.09 ? 0.18 1.45 ? 0.23 1.79 ? 0.13 Adjusted for age (5 categories)c Mean T4 ? SE (pg/dl) 7.68 ? 0.22 8.24 ? 0.43 7.59 ? 0.29 Mean TSH ? SE (mlU/1) 2.13 ? 0.15* 1.73 ? 0.30 1.61 ? 0.19 Abbreviations: SE, standard error; mlU, milli-international unit; T4, thyroxine; TSH, thyroid-stimulating hor- mone. aLaboratory reference ranges for T4 and TSH were 4.5-11.5 and 0.32-5.00, respectively. Four subjects were excluded from hormone analyses because the lab reported hemolysis, which might have affected results. bSI unit conversion: pg/dl = 12.9 nmol/l. cLeast square means adjusted for age (categorical); adjustment for age using a continuous variable yield- ed similar results. *Significantly increased compared to the nonexposed; p = 0.05. Volume 105, Number 10, October 1997 * Environmental Health Perspectives 1128 Articles - Thyroid hormone and cytogenetic effects of EBDC fungicides of a decrease in T4 levels (20), although in this population, TSH levels are all within normal range. The linear regression model for TSH and T4, including exposure and age variables, explained 23% of the variance (r2) for TSH, but only 3% for T4. Adjustment for age using a continuous instead of a cate- gorical variable showed similar results for TSH (difference between applicators and nonexposed, p = 0.03). Restriction of the data to those under 30 years of age (87% of appli- cators and nonexposed controls) showed a similar pattern of higher TSH among the applicators in the data with no age adjust- ment (2.25 pg/dl for applicators; 1.81 pg/dl for controls, p = 0.07). Table 3 presents the cytogenetic out- comes by exposure group. Both unadjusted and adjusted (for age, smoking, via linear regression) data indicated that the applica- tors had significantly higher SCEs than nonexposed controls (0.62 increase in SCE per cell, p = 0.02, adjusted data). Owners were not significantly different from con- trols. The proportion of variance explained by the regression model was 22%. Table 3 also presents the unadjusted and age-adjusted results for chromosomal aberra- tions. The unadjusted data show significant differences between applicators and owners compared to nonexposed controls for translocations and for the subset of reciprocal translocations. However, translocations increase markedly with age, and age acts as a confounder in these data. The Poisson regression analyses controlling for age indi- cated that applicators had significantly more total translocations than did the nonexposed subjects (1.50 increase, p = 0.05), while own- ers had a nonsignificant increase (0.85 increase, p = 0.31). Additional analyses using terms for age and age squared to control for age, rather than age categories, yielded essen- tially the same results. Analyses restricted to those age 40 and below resulted in a slightly lower predicted increase for applicators (1.25 translocations) compared to the nonexposed (p = 0.14). Analyses restricted to reciprocal translocations also yielded a lower and non- significant exposure effect for applicators ver- sus the nonexposed (p = 0.24). Poisson regression analyses of other chro- mosome outcomes (dicentrics, insertions, fragments, and rings) did not show any sig- nificant differences between the exposed group and the nonexposed referents. Total translocations and reciprocal translocations were both significantly corre- lated with SCEs, with Spearman correla- tions of 0.28 (p = 0.02) and 0.27 (p = 0.02), respectively. Discussion We have studied two groups of subjects exposed to EBDCs during application to tomatoes in Mexico. Urinary ETU data confirmed that the applicators were more highly exposed than the landowners, while Table 3. Cytogenetic outcome resultsa Applicators (n = 31) Owners (n = 13) Nonexposed (n = 30) SCEs per cell Unadjusted 8.50 ? 0.22* 7.91 ? 0.21 7.80 ? 0.18 Adjusted for age, smokingb 8.52 ? 0.19* 7.61 ? 0.31 7.90 ? 0.20 Chromosome aberrations Unadjusted All translocations 4.61 ? 0.94 6.00 ? 1.00 2.76 ? 0.54 Reciprocal translocations 2.70 ? 0.53 3.38 ? 0.65 1.73 ? 0.32 Dicentrics 0.42 ? 0.18 0.30 ? 0.13 0.23 ? 0.10 Insertions 0.06 ? 0.04 0.15 ? 0.10 0.03 ? 0.03 Rings 0.10 ? 0.05 NA 0.13 ? 0.08 Fragments 0.87 ? 0.2 1.07 ? 0.31 0.76 ? 0.20 Adjusted for agec All translocations 6.07 + 0.97** 5.44 ? 1.00 4.59 ? 0.82 Reciprocal translocations 3.39 + 0.74 2.92 ? 0.72 2.71 ? 0.65 Dicentrics 0.40 + 0.30 0.26 ? 0.13 0.23 ? 0.09 Insertions 0.06 + 0.04 0.11 ? 0.08 0.03 ? 0.04 Rings 0.08 + 0.05 NA 0.13 ? 0.07 Fragments 0.81 0.15 0.81 ? 0.22 0.76 ? 0.16 Abbreviations: SCEs, sister chromatid exchanges; SE, standard error; NA, not available. aValues given are mean ? SE. bAdjusted SCE results are least-square means. CChromosome aberrations per 500 cell equivalents. The adjusted data chromosome translocations and the subset of reciprocal translocation are predicted from a Poisson regression model with exposure and age (five categories), while other aberration data uses a model with exposure and age as a continuous variable. Predicted results are for a subject 25-30 years of age. Variance of predictions derived from the standard error of X)i using the delta method. *Significantly increased compared to the nonexposed; p = 0.03 for adjusted data and p = 0.04 for nonad- justed data. **Significantly increased compared to the nonexposed, p = 0.05. all nonexposed subjects had urinary ETU levels below the limit of detection. The fact that one-third of the applicators and one- half of the landowners had urinary ETU levels below the limit of detection can be explained by 1) a lack of sensitivity of the assay to detect low ETU levels, even when there was exposure; 2) differences in metab- olism of EDBCs to ETU between subjects; and 3) different work practices and cleanup practices between subjects. Landowners generally had less contact with the pesti- cides, and it is perhaps not surprising that half had urinary ETU below the limit of detection. Among the applicators, division of the population by number of baths per week showed that those who bathed daily had a higher proportion of urinary ETU samples below the limit of detection, and had significantly lower mean urinary ETU, than those who bathed less often (data not shown). Another possible reason for urinary data below the limit of detection is misclas- sification; the urine collection was not observed directly and some samples could have been for the wrong person. Although all the thyroid hormone data were within normal range, after age-adjust- ment the TSH level was significantly increased in the applicators compared to the nonexposed subjects (and nonsignificantly increased among the landowners, suggesting a dose response). In our data, age was nega- tively correlated with TSH and acted as a negative confounder. Several reports in the literature indicate that TSH levels decrease with age in healthy populations (21,22), but other studies have not shown such an effect (23). The exposure effect for applica- tors versus nonexposed controls for TSH was also apparent without age adjustment (p = 0.07) when the data were restricted to younger subjects (<30 years of age, 87% of applicators and controls). Because T4 levels were not decreased among the exposed compared to the nonex- posed, TSH may be a more sensitive indica- tor of an effect on the thyroid than T4 levels (20). Most of these workers had been exposed to EBDCs and ETU for several weeks or months at the time of testing; homeostasis with normal T4 but elevated TSH might be expected after several weeks of exposure (24). The effect on TSH is con- sistent with animal data (1). In the one study of ETU-production workers with presumed high exposure, the five most highly exposed mixers had significantly decreased T4 and one had abnormally high TSH with dinical signs of premyxedema (3). We did not observe any dinical signs of hypothyroidism in our study population, but we performed no clinical exams. If hypothyroidism does occur in these workers, it is possible that Environmental Health Perspectives * Volume 105, Number 10, October 1997 1129 Articles l Steenland et al. affected subjects could not continue in this type of relatively strenuous work. Our data on genotoxic outcomes indi- cate that the applicators had significantly increased SCEs, with or without adjustment for age and smoking. Chromosome translo- cations were significantly elevated for both applicators and owners (p<0.001) before age adjustment, compared to nonexposed sub- jects. However, age acted as a positive con- founder in these data; after age adjustment, the exposure effect was diminished, although it was still present for applicators versus nonexposed controls (p = 0.05). When analyses were restricted to the subset of reciprocal translocations, the increase for applicators failed to reach conventional sta- tistical significance after age adjustment (p = 0.24). In the age-adjusted data, again the landowners showed less pronounced eleva- tions than applicators, suggesting a possible dose response. Reciprocal translocations are thought to be more stable than nonrecipro- cal translocations (13), and the somewhat more pronounced effect seen for all translo- cations (including nonreciprocals) may reflect the effects of more recent exposure. Chromosome outcomes other than translo- cations did not significantly differ between the exposed subjects and controls, but most of these outcomes were quite rare and results lack statistical precision. The increased SCEs indicate that expo- sure is affecting the genome, but it is not established that increased levels of SCEs are predictive of cancer or any other health out- come. The one epidemiologic study on the question has not, to date, observed any effect of elevated SCEs on cancer, based on 6 years of follow-up and 49 observed cancers in a Nordic 6-year prospective study of 3,182 people (25). The increased levels of all translocations are somewhat more worri- some, as translocations are common in tumors and often occur at sites of genes affecting cell cycle and tumor suppression (26). Increased levels of chromosome breaks have been shown to be predictive of cancer in humans in the same Nordic study cited above (66 cancers observed) (25). Those in the highest tercile of aberrations had a twofold significant risk of cancer compared to the general population, while the lowest two terciles had no increased risk. A similar study in Italy based on 1,455 subjects (42 cancers) found an 80% increased cancer risk for those with medium or high levels of aberrations (27) after control for smoking and occupational exposures. There is some potential for confounding of the genotoxic results by other genotoxic pesticides, specifically by another fungicide, captan. Captan has been found to be muta- genic in some animal and bacterial systems and has been found to cause cancer in some rodent studies (28). However, captan use has been uncommon in tomato appli- cation; owners relied primarily on EBDCs. The organophosphate pesticides commonly used with the EBDCs are not generally genotoxic. In conclusion, our data are consistent with animal and some human data in sug- gesting a toxic effect of EBDCs on the thy- roid and on the genome (SCEs, chromoso- mal translocations). However, the exposure effects observed in our study were subclini- cal and of uncertain future clinical signifi- cance. Furthermore, exposure effects were modest, generally on the borderline of con- ventional statistical significance, and were based on a single field study with limited sample size. The observed effects should therefore be interpreted with caution. REFERENCES 1. U.S. EPA. 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San Diego, CA:Academic Press, 1991. 1130 Volume 105, Number 10, October 1997 * Environmental Health Perspectives