Document bB68o2y83vXQnnOgO3XJv0RRo

Cancer Causes and Control 11: 373380, 2000. 2000 Kluwer Academic Publishers. Printed in the Netherlands. 373 Increased lymphocyte replicative index following 2,4-dichlorophenoxyacetic acid herbicide exposure Larry W. Figgs1,*, Nina Titenko Holland2, Nathanial Rothman3, Shelia Hoar Zahm3, Robert E. Tarone3, Robert Hill4, Robert F. Vogt4, Martyn T. Smith2, Cathy D. Boysen5, Frederick F. Holmes5, Karen VanDyck6 & Aaron Blair3 1School of Public Health, St Louis University, 3663 Lindell Blvd. St Louis, MO 63108-3342. E-mail: ggslw@slu.edu; 2University of California at Berkeley, Berkeley, CA 94720; 3National Cancer Institute, Bethesda, MD 20892; 4Center for Disease Control and Prevention, Atlanta, GA 30341; 5Kansas University Medical Center, Kansas City, KS 66160; 6Battelle/Survey Research Associates, Baltimore, MD 21209 (*Author for correspondence) Received 15 June 1999; accepted in revised form 3 January 2000 Key words: herbicide, lymphocytes, micronucleus, occupation, 2,4-D. Abstract Objective: Evaluate peripheral blood lymphocyte proliferation (replicative index:RI) and micronuclei frequency (MF) among 2,4-D herbicide applicators. Methods: Twelve applicators spraying only 2,4-D provided a blood and urine specimen upon enrollment, several urine samples during the spraying season, and a blood specimen at the study's end. Nine controls provided blood and urine specimens upon enrollment and at the study's end. Gas chromatography/tandem mass spectroscopy determined urinary 2,4-D levels and standard in-vitro assays determined RI and MF scores. Applicator RI and MF were compared before and after spraying and with controls. Results: Applicators contributed 45 urine specimens with concentrations ranging from 1.0 to 1700 (lg 2,4-D/g creatinine/L urine) that logarithmically (ln) increased as spraying time increased. Applicator RI increased after spraying (p = 0.016), independent of tobacco and alcohol use, and demonstrated a weak doseresponse with increasing urinary 2,4-D levels (p = 0.15). Among 2,4-D applicators, pre-exposure complete blood counts and lymphocyte immunophenotypes were not signicantly dierent from post-exposure measurements. Conclusion: Urinary 2,4-D concentration, an exposure biomarker, may be associated with lymphocyte replicative index, a cell proliferation biomarker. Introduction 2,4-dichlorophenoxyacetic acid (2,4-D) is one of the most widely used postemergence pesticides in the United States [1] that acts by disrupting hormone balance and protein synthesis to cause a variety of plant growth abnormalities. Although, 2,4-D does not appear to cause cancer in rodent bioassays [2], associations with non-Hodgkin's lymphoma have been observed in some [26], but not all [7, 8] epidemiologic studies. Animal studies have shown that 2,4-D has a number of biological eects [918], but a possible carcinogenic mechanism(s) is not obvious in mammals [1922]. Humans primarily excrete unmodied herbicide in their urine, suggesting few metabolic intermediates [23]. To provide information on possible inconsistencies between epidemiologic data and biologic eects in humans, we conducted a pilot study of 12 herbicide applicators who exclusively sprayed 2,4-D from April to July 1994 to investigate the relationship between urinary 2,4-D levels and lymphocyte proliferation rates [24] and micronuclei frequencies [2527]. Materials and methods Study subjects Study participants were 13 herbicide applicators from county noxious weed oces in eastern Kansas, aged 17 56, who had no cancer history and no occupational 374 L.W. Figgs et al. pesticide exposure 6 months prior to 1 March 1994, and 12 non-applicators. County noxious weed oces are charged with controlling troublesome agricultural weeds (e.g. bindweed, wormwood, snakeweed, thistle, knapweed, larkspur, leafy spurge, locoweed, lupine, ironweed, skeleton weed) on public and private land. These pesticide applicators use only herbicides and often spray on a daily basis. Participants completed a questionnaire, maintained a daily activity log, and provided blood and urine samples. Subjects were monitored for 12 weeks or until 2,4-D use was discontinued, whichever came rst. Pesticide applicators received $250 and non-applicators received $50 remuneration at the study's end. Shortly after enrolling, one applicator relocated to a nonparticipating county, changed occupations, and withdrew from the study. Non-applicators were solicited by word-of-mouth and newspaper advertisements. We excluded persons with previous or current cancer medical histories, persons taking prescribed medications, or persons with current disease histories that might interfere with laboratory assays. Of the 150 subjects screened, nine controls were selected and matched to applicators by gender, 5-year age group, alcohol and tobacco use, and geography (preference was given to non-applicator county employees working in or adjacent to an applicator's county of employment). Each control subject received $50 remuneration. All participants signed informed-consent documents that were approved by the Human Subjects Review Committees of the National Cancer Institute and the University of Kansas Medical School. Questionnaires and daily diary All applicators and matched controls completed a 40minute, in-person, enrollment questionnaire to collect health and employment history data. Applicators also responded to a 15-minute post-study questionnaire to determine changes in health status, habits, or occupational history. Applicators also kept daily work diaries to record task, task duration, pesticide use, and personal protective equipment use during the 2,4-D spraying season. Biological specimen collection, processing, and assay methods Baseline blood and single-void urine specimens were collected at enrollment from applicators and controls. In addition, overnight urine samples were obtained from applicators every other week following a typical day of 2,4-D application. To insure that the urine was properly collected and not contaminated, participants were trained to ll containers without touching the urine stream to clothing or hands. Applicators were instructed to urinate at 6:00 p.m. and collect all urine thereafter, including the nal urine before reporting for work the next day. To retard bacterial growth, subjects stored overnight urine collections in a single plastic container in a refrigerator at home and transported the urine to the worksite in an ordinary Thermos7 cooler led with ice packs. Study technicians provided participants with new supplies as needed and prepared the urine specimens for transport to the laboratory. Upon receiving urine specimens, study technicians immediately pipetted 20 ml into each of two 25 ml glass serum vials (Wheaton), capped the vials with Teon stoppers, and sealed each with aluminum retainers. Each vial was immediately placed on dry ice and transported frozen to the University of Kansas Medical Center laboratory in Kansas City, Kansas. After determining the total volume, the unfrozen urine was discarded. All frozen samples were stored at )80 C and were shipped on dry ice to the Centers for Disease Control and Prevention for laboratory analyses at the end of the spraying season. To maintain stability, samples were thawed as needed. Baseline urinary 2,4-D estimates were determined from pooled urine that contained two enrollment, single-void urine samples selected randomly from applicators and controls, respectively. Urinary 2,4-D analyses followed procedures described by Hill et al. [28]. Frozen urines were thawed, 10 ml urine aliquots were prepared and C13 labeled 2,4-D was added as an internal standard. Enzyme hydrolysis, derivatization, clean-up and concentration to 100 ll (microliters) followed. Urine 2,4-D measurements were made using capillary gas chromatography combined with tandem mass spectrometry (GC/MS/MS) employing collision-associated decomposition of parent to daughter ions. Quality assurance methods included retention time evaluation, ion ratios, and controls with known 2,4-D concentrations. Long-term sample storage was similar to conditions described elsewhere [28] for quality control samples measured several times over a 33-month analysis period to determine if 2,4-D concentrations remained within the experimental error of the method (6.2 lg/L (ppb) 8.7% (CV)). The detection limit for 10 ml urine samples was 1 lg/L (parts per billion). All 2,4-D concentrations were creatinine adjusted (lg/g creatinine/L urine). Blood specimen collection and processing Typically, all blood specimens were collected between 6:00 a.m. and 10:00 a.m. in 10 ml, green top vacutainer Lymphocyte alterations following 2,4-D exposure 375 tubes and appropriately packaged for overnight shipment and delivery. For the replicative index/micronucleus assay, lymphocytes were isolated using FicollPaque (Pharmacia, Piscataway, NJ) density gradients and cultured [26]. Briey, blood was diluted 1:1 with phosphate buered saline (PBS), layered onto FicollPaque with a ratio of cells + PBS:Ficoll-Paque maintained at 4:3, and centrifuged at 170g for 35 minutes at room temperature. The lymphocyte layer was removed, washed twice in PBS at 150170g for 10 minutes each, and then washed with RPMI 1640 media. A hemocytometer was used to achieve an initial culture density of 1 106 cells in 2.0 ml of culture medium. Culture medium consisted of RPMI 1640 supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 2 mM L-glutamine, 100 units/ml penicillin, 100 lg/ml streptomycin (Gibco, Grand Island, NY) and 1.5% phytohemagglutinin (PHA, HA15, Burroughs-Wellcome, Greenville, NC). The lymphocyte cultures were grown in a humidied incubator with 5% CO2 at 37 C in 15.0 ml conical polystyrene centrifuge tubes. Cytochalasin B (Sigma, St Louis, MO) (5.0 lg/ml) was added to lymphocyte cultures at 44 hours post-initiation as described by Fenech and Morley [27]. Cytochalasin B prevents complete cytokinesis, which results in multinucleated cells. At 72 hours, lymphocytes were spun directly (48 g, 10 minutes) onto glass slides using a cytocentrifuge (Shandon, Sewickley, PA). Slides were air-dried before xing with methanol at room temperature for 15 minutes. Slides were stored at )20 C in a sealed box, desiccated, under a N2 atmosphere. Cell division kinetics were calculated by scoring at least 400 cells per sample (200 cells per duplicate), by counting the percent of cells containing one, two, three or more nuclei per individual. A replicative index (RI) was calculated as follows: RI f1% mononuclear cells 2% binuclear cells 3% trinuclear cells 4% tetranuclear cells ga100X Antikinetochore antibody staining procedures followed those described by Eastmond and Tucker [29]. Methanol-xed slides were incubated for 5 minutes in PBS containing 0.1% Tween 20. Excess uid was drained from slides and 4050 ll of the antikinetochore antibody (Chemicon, Temecula, CA) diluted 1:1 with PBS containing 0.2% Tween 20 was applied. The slide's working surface was covered with a glass coverslip and placed in a humidied chamber at 37 C for 1 hour. Following two washes in PBS containing 0.1% Tween 20 for 5 minutes each, excess uid was again drained, slides were covered with a 1:50 dilution of uorescent goat anti-human IgG (Chemicon, Temecula, CA), and incubated again for 1 hour. Because uorescent-labeled antibodies fade upon exposure to light, this and all subsequent steps were conducted in yellow light. The slides were rinsed twice in buer plus 0.1% Tween 20 and counterstained with DNA-dye 4,6-diamidino-2phenylindole (DAPI) (2 lg/ml) in an antifade solution [30]. Slides were refrigerated for up to a week prior to microscopic examination. Randomized and coded slides were scored using a Nikon microscope equipped with epiuorescent illumination and lters for uorescein (excitation at 470 nm, dichroic at 510 nm, barrier at 520560 nm) and quinacrine (excitation at 400440 nm, dichroic at 450 nm, barrier at 470 nm). At least 1000 binucleate lymphocytes (those that have undergone one mitotic division) were scored for the number of micronuclei. When a micronucleus was located using the quinacrine lter, the presence or absence of kinetochore staining was determined by switching to the uorescent lter. Scoring criteria were as follows: (1) cells appeared round or oval with an intact cytoplasm, (2) nuclei appeared round or oval with an intact nuclear membrane, (3) cells having undergone one nuclear division were scored for the presence of micronuclei, (4) micronuclei had to be one-third or less the size of the main nuclei, (5) micronuclei were stained similar to the main nuclei, and (6) micronuclei were clearly separated from the main nuclei. Two scorers performed scoring with 10% of slides being rescored. A third scorer additionally assessed all questionable micronuclei. Other laboratory analyses included lymphocyte phenotypes and complete blood counts (CBC). Lymphocyte phenotypes were determined by ow cytometry using two-color immunouorescence and a whole-blood stainand-lyse method [31] under guidelines approved for clinical laboratory analyses [32]. The CBC was performed to determine leukocyte type and number to assess immune function and to characterize erythrocyte size, shape, and number for signs of anemia. Total white blood cells, lymphocytes, monocytes, granulocytes (eosinophils and basophils), and erythrocytes were counted in each blood sample. Hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, and platelet count were estimated. All determinations were made using standard methods with a Coulter counter (Beckman Coulter, Inc., Fullerton, CA 92834, USA). Statistical Analyses Our design compared pre-exposure (baseline) measurements with post-exposure measurements of the applica- 376 L.W. Figgs et al. tors and post-exposure measurements of applicators with non-applicators controls. Means were presented with standard deviations (mean SD) unless otherwise indicated. Urinary 2,4-D concentrations were time adjusted (mean 2,4-D concentration/mean hours spraying 2,4-D) where indicated. Paired t-test analyses, stratied by tobacco use history or alcohol use, compared micronuclei and replicative index scores before and after 2,4-D application. An independent sample (unpaired) t-test was used to compare applicator micronuclei and replicative index scores with non-applicator controls. A non-parametric test [33] was used to test for a trend of increasing replicative index scores and replicative index dierence (RIpost-exposure ) RIpre-exposure) across three time-adjusted urinary 2,4-D concentration groups. All statistical data analyses and tests were performed using Stata 5.0 (Stata Corporation, 702 University Drive East, College Station, TX 77840) and SPSS 7.5.1 for Windows (SPSS Inc., 444 N Michigan Avenue, Chicago, IL 60611-3962). Results Twelve white males ranging in ages from 17 to 56 years (mean: 27.5 12.5 years) were enrolled as applicators (Table 1). Two were past users and four were current users of tobacco. All subjects drank beer and consumption ranged from 1 to 30 cans per week. Three applicators consumed hard liquor. Nine controls, eight white and one white-Hispanic, ranged in age from 19 to 32 with a mean of 24.7 4.3 years. Four non-applicators used tobacco and four consumed beer. Forty-ve urine specimens were collected from applicators following 204 spraying hours of 2,4-D and a mean of 4.5 1.9 hours per specimen prior to collection. The herbicide was typically sprayed from a long, exible wand attached to a truck bed reservoir. Urinary 2,4-D concentration increased as hours spent spraying herbicide increased (Figure 1). The 2,4-D concentration grand mean among all applicators after spraying 2,4-D was 240 100 (SE) ppb. The means among serial urine samples from applicators taken after spraying ranged from 12 5.2 pbb (n = 4) to 1285 336 ppb (n = 4). Mean micronuclei scores and replicative index scores among applicators before and after spraying 2,4-D and scores between applicators and non-applicator controls are shown in Table 2. No signicant dierences in micronuclei scores were observed, but post-exposure scores among applicators were lower (21.5%) than preexposure scores and lower among applicators (17.2%) Table 1. Comparison of pesticide applicators and controls by age, race, tobacco use, and alcohol use Participantsa Age Tobacco use Alcohol use Cigarettes Other Beer (cans) 1. Applicator 2. Applicator 3. Applicator 4. Applicator 5. Applicator 6. Applicator 7. Applicator 8. Applicator 9. Applicator 10. Applicator 11. Applicator 12. Applicator 1. Control 2. Control 3. Control 4. Control 5. Control 6. Control 7. Control 8. Control 9. Control 56 25 30 24 20 21 24 26 50 17 18 20 31 25 32 26 22 21 25 23 19 40/day Never Never Never Never Never 7/dayb Never Never Never 5/day Never Never Never Never Never 20/day Never 20/dayb Never Never Never Never Never Never 1.0 oz/day (chew) Never 1.0 oz/week (snu)b 1.2 oz/day (chew) 1.5 oz/day (chew)b Never 1.0 oz/day (snu)b Never 1.5 oz/day (chew) Never Never Never 1.0 oz/day (snu) Never 1.0 oz/day (snu) 1.0 oz/day (chew)b Never 24/week 6/week <1/week 6/week 12/week 8/week 12/week 12/week 30/week 4/week 6/week 6/week 12/week No 10/week No 2/week 4/week No No No a All subjects were male. b Former user. Liquor (shots) No No No No No No No 3/week No No 3/week 3/week No No No No No No No No No Lymphocyte alterations following 2,4-D exposure 377 Fig. 1. 2,4-D Levels among urine samples grouped by spraying duration category. Table 2. Comparison of the micronuclei and replicative index mean scores for 2,4-D applicators and controls Assay n Mean (SD)a pd Micronuclei Applicators Before 2,4-D applied After 2,4-D applied Applicator vs. controls Applicators Controls Replicative index Applicators Before 2,4-D applied After 2,4-D applied Applicators vs. controls Applicators Controls 12 12 12 9 12 12 12 9 11.6 (2.8) 9.1 (6.2) 9.1 (6.2) 11.0 (4.8) 1.33 (0.2) 1.47 (0.2) 1.47 (0.2) 1.29 (0.2) 0.289 0.454 0.016b 0.046c a Replicative index score = 1 %M1 + 2 %M2 + 3 M3 + 4 M4, where Mx is the number of a cell division cycle per 1000 cells. b Paired t-test. c Independent sample t-test. d p = Probability of observing applicator score dierences before and after spraying 2,4-D. than among non-applicators. In contrast, replicative index scores among applicators were signicantly higher following 2,4-D exposure (p = 0.016) and higher than non-applicators' scores (p = 0.046). A comparison of pre- and post-exposure lymphocyte immunophenotypes among applicators and between applicators and controls did not show signicant dierences or trends (not shown). Categorical analyses of mean urinary 2,4-D concentration and RI scores (Table 3) showed an increase in RI scores as time-adjusted urinary 2,4-D levels increased, but no statistically signicant trend (p = 0.15) was observed. The replicative index increased among applicators after spraying regardless of tobacco and alcohol use (Table 4). Both before and after spraying, replicative index was higher among applicators who did not use tobacco and among those who drank six or fewer beers than among tobacco users or more frequent beer drinkers. Alcohol and/or tobacco use signicantly decreased replicative index scores, but because of the considerable overlap in usage, independent eects of their use could not be determined. Among applicators, lymphocyte immunologic phenotypes and complete blood counts (CBC) before spraying 2,4-D were not statistically dierent after spraying 2,4D, nor were there signicant dierences between 2,4-D applicators and controls after applicators had sprayed the herbicide (not shown). Discussion This study was conducted to evaluate several biologic outcomes in relation to urinary levels of 2,4-D in humans. Measurable levels of 2,4-D occurred in applicators' urine but not among non-applicators. Applicators' urinary 2,4-D levels increased with the number of spraying hours, but overall were intermediate among other occupational 2,4-D users similarly exposed [14, 15, 18, 34] and generally above population-based references [35]. Urinary 2,4-D concentration was associated with Table 3. Doseresponse relationship between urinary 2,4-D concentration categories, mean replicative index scores, and replicative index dierencea among herbicide applicators Concentration category (mean ppb/mean hours spraying 2,4-D) n Mean RI after spraying (SD) Trend test across categories Mean RI dierence (SD) Trend test across categories <20 2040 >40 4 1.32 (0.21) Z = 1.44 0.05 (0.15) Z = 0.34 3 1.53 (0.17) pb = 0.15 0.20 (0.14) p = 0.40 5 1.57 (0.22) 0.16 (0.18) a Replicative index dierence = RIbaseline ) RIend of study. Trend test across categories is a nonparametric test for trend across ordered groups developed by Cuzick [33] is an extension of the Wilcoxon rank-sum test; bp is equal to the probability of observing a value greater than |Z|. 378 L.W. Figgs et al. Table 4. Comparison of tobacco and alcohol use and replicative index scores among applicators and non-applicators User group n Pre-exposure Post-exposure Summary replicative indexa replicative index p value 2,4-D Applicator Ever use tobacco No Yes p-Valueb Alcohol use >6 cans/week No Yes p-Value Non-applicator Ever use tobacco No Yes p-Value Alcohol use >6 cans/week No Yes p-Value 6 6 6 6 4 5 6 3 1.44 0.24 1.24 0.14 0.11 1.49 0.08 1.18 0.02 0.005 1.28 0.15 1.29 0.19 0.89 1.31 0.12 1.24 0.26 0.56 1.61 0.17 1.34 0.18 0.03 1.64 0.06 1.32 0.06 0.004 n.a. n.a. n.a. n.a. 0.006 0.0001 a Replicative index score = 1 %M1 + 2 %M2 + 3 M3 + 4 M4, where Mx is the number of nuclei per cell of a cell division cycle per 1000 cells. b Probability of observed t (test that mean dierences are equal to zero using two sample t-test and assuming equal variances). Summary p-value is the probability of obtaining the observed distribution of means. increased peripheral blood lymphocyte replicative index scores, but not with micronucleus formation. The replicative index for lymphocytes was higher among applicators than non-applicators and it was higher among applicators after spraying than before spraying. Age, tobacco, or alcohol use could not explain these dierences. Other factors that might explain replicative index dierences among the exposed and unexposed are medical and pharmacological history, previous occupational or heterogeneous occupational pesticide exposures, or chance. However, we excluded unhealthy subjects initially and upon completion of the study (pre-existing or concurrent morbidity) and included among controls only individuals with no previous occupational pesticide exposure. Changes in sister chromatid exchange and micronucleus formation have been associated with other pesticides [1] and replicative index changes have been associated with aging [3638]. Our ndings, while preliminary, are the rst to show a relationship between urinary 2,4-D concentration and increased replicative index in humans. Increased replicative index scores may be important because they suggest stimulated cell growth that could contribute to carcinogenesis. Our nding of no relationship between the frequency of micronuclei and urinary 2,4-D level does not support a human chromosome-damage outcome at mean urinary 2,4-D levels ranging from 12 to 1285 ppb. The link between 2,4-D function and lymphocyte replicative index is unclear. However, as in animal cells, plant cell growth regulatory receptor proteins may act on plasma membrane ion channels, inositol phospholipid signaling pathways, or inside the nucleus to regulate gene expression [3944]. Whatever the mechanism, our results and other recent investigations [19, 20] do not support a genotoxic pathway. This pilot investigation was designed to appraise genetic and epigenetic mechanisms and orient our interdisciplinary research regarding epidemiologic associations between NHL and 2,4-D exposure [4] and potential relationships between urinary 2,4-D levels, lymphocyte proliferation rates [24], and micronucleus frequencies [2527]. The replicative index was used as a measure of cell proliferation and the micronucleus assay as a measure of 2,4-D genotoxicity. These assays were selected because they were simple, inexpensive, and had been eectively used in numerous occupational studies [39] including a malathion study by co-investigators Titenko-Holland and Smith [40]. The relationship between urinary 2,4-D levels and lymphocyte proliferation observed in this pilot suggests that a larger investigation is needed to further evaluate the association. 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