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1 Groupe Régional d’Etudes sur le Cancer, Université de Caen Basse-Normandie, and 2 Registre Général des Tumeurs du Calvados, Centre François Baclesse, Caen, France
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Follicular lymphoma, one of the most common type of NHL accounting for 25–30% of cases, is characterized at 85% by the reciprocal translocation t(14;18)(q32;q21), which juxtaposes the BCL2 gene on chromosome 18q21 near the immunoglobulin heavy chain (IGH) locus at chromosome 14q32 (15) . Therefore, the BCL2 gene is subjected to the control of the IGH-Eµ enhancer leading to the overexpression of the antiapoptotic BCL2 protein and increasing cell survival (16) . Because of the involvement of the IGH locus as well as the presence of de novo nucleotides in BCL2-IGH junctions, the translocation was usually assumed to result, at least in part, from illegitimate V(D)J recombination in early B cells. But evidence to date has been mostly circumstantial, and recent breakpoint analysis tends to show that additional mechanisms could be involved (17) .
Since 1994, many studies reported the presence at low frequencies of BCL2-IGH rearrangements in peripheral blood lymphocytes of healthy individuals (18 , 19) . Depending on PCR sensitivity, it was observed that from 50 to 80% of healthy individuals beard BCL2-IGH rearrangements with frequencies varying over a range of 100-fold (usually between 1 x 10-5 and 1 x 10-7). In view of the initiating role of the BCL2-IGH translocation, it was hypothesized that individuals with an increased risk of lymphoma may have increased frequencies of BCL2-IGH translocation (20 , 21) . We then suggested that environmental factors (among which is occupational exposure to pesticides) could influence the prevalence, the frequency, or the molecular characteristics of BCL2-IGH bearing cells. In a study published previously, we found that neither the frequency of BCL2-IGH-bearing cells nor molecular characteristics of breakpoint junctions varied significantly in a healthy reference group over a 4-month period (between winter and spring), corresponding for farmers to the low or high pesticide use period, respectively (22) . We used the same approach to characterize BCL2-IGH rearrangements on a population of farmers occupationally exposed to pesticides, mostly involved in open field farming, and coming from a representative cohort of agricultural population from the Calvados area and having a well-defined pesticide exposure assessment.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For the present investigation, a subsample was selected among this cohort with the following criteria, males, without smoking history, and usual pesticide applicators on open field crops (Fig. 1)
. These criteria provided us with better conditions for data interpretation controlling for potential confounding factors (tobacco use) on a population of farmers with well-defined exposure assessment and who might be the most exposed to pesticides in the studied area (females only used insecticides on cattle or herbicides on farmyards). Among the 144 individuals matching these criteria, 56 were analyzed in the present study. Pesticide use varies considerably according to season with periods of high pesticide application (approximately from April to June in our area) and periods of little or no use. These two periods were designated as high pesticide and low pesticide use periods, respectively. All of the farmers (n = 24) from whom blood samples had been obtained during the high pesticide use period were examined, whereas 32 farmers were selected among the remaining 120 (Fig. 1)
. No significant differences were observed between the two groups for age (P = 0.8), farm area (P = 0.5), or farm area devoted to crops (P = 0.9) (Table 1)
. To control for potential season-related confounding factors, we collected individual daily sunlight data for the 1997–2000 period. The cumulative sunlight value (Joule/cm2) for the 3-week period before sampling was calculated and ranged from 5,663 to 48,573 Joule/cm2.
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Nested-PCR and DNA Sequencing for BCL2-IGH Translocation Status.
BCL2-IGH fusions were amplified by nested PCR with a multitube approach using specific primers for the major breakpoint region in combination with a JH consensus primer, as described previously (20
, 22)
. The reaction conditions were an initial step at 94°C for 10 min (Applied Biosystems, Foster City, CA), followed by 40 cycles of 1 min at 94°C, 1 min at 60°C, 1 min at 72°C, with a final extension step at 72°C for 5 min. One µl of the first PCR product was reamplified under the same conditions described above for 20 additional cycles. PCR products were analyzed on 1% agarose gel electrophoresis and visualized under UV light. For all of the samples, DNA integrity was checked by amplification of the albumin gene. Negative and positive controls were added in each PCR reaction, with, respectively, no added template and a diluted sample of the RL cell line characterized by a t(14;18) translocation. Among the 56 farmers selected for analysis, 3 of them were unavailable due to an insufficient amount of peripheral blood lymphocyte DNA. Finally, 53 samples were evaluated for the presence of BCL2-IGH translocation. Fresh PCR products were directly purified on a GFX PCR column (Amersham Biosciences, Piscataway, NJ) and sequenced using the Big Dye Terminator cycling sequencing Kit v3.0 (Applied Biosystems, Foster City, CA) on an ABIPRISM 377. As a unique and specific junction segment characterized each rearrangement, we excluded cross-contamination between samples.
Quantification of Translocation Frequencies.
Translocation frequencies were estimated by a multitube approach and Poisson assumptions as described previously (20)
. For each sample, 30 replicates of 250 ng (equivalent to 40,000 cells) were amplified corresponding to a theoretical detection limit of 8.48 x 10-7. In our multitube approach, an individual was considered to be positive when at least one replicate gave a positive signal on agarose gel electrophoresis that was confirmed to be specific by DNA sequencing. To allow comparisons between farmers and the reference group published previously (22)
, we had to be at an identical detection limit for the two groups (e.g., 8.48 x 10-7). The calculation of prevalence was made with the detection limit used in the present study, conducting to a new prevalence of 50% among referents.
Statistical Analysis.
Median was used for comparison of the characteristics of farmers included or not included in the present study. Mean frequency value was calculated for all of the individuals. In this case, half of the detection limit (4.23 x 10-7) was assigned for individuals with no detectable BCL2-IGH translocation. To take into account modifications of prevalence among compared groups of individuals, a second value of frequency was calculated for BCL2-IGH positive individuals. Because the distribution of BCL2-IGH frequency was skewed, values were log transformed for our analysis. For categorical variables, 
2 or Fisher exact (when appropriate) tests were used. For continuous variables, Student t test or Wilcoxon nonparametric test and linear regression analyzes were performed using STATA software (STATA corporation release 7.0).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows the characteristics of the cohort (n = 758) and of the sub-sample selected for biological investigations analyzed (n = 56) or not (n = 88) in the present study (Fig. 1)
. The 56 analyzed farmers were slightly older (median age, 46 versus 41 years old; P = 0.02) and had a lower time since last pesticide use (P = 0.09). Except for the spraying equipment with the under-representation of the trailer sprayer among analyzed farmers, agricultural practices and exposure parameters used in this study (farm area, farm area devoted to crops, number of commercial products, and crop diversity) did not differ between the two groups. (Table 1)
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Characterization of BCL2-IGH Translocation in Peripheral Blood Lymphocytes from Farmers.
Prevalence of BCL2-IGH translocation in farmers occupationally exposed to pesticides was 71% (38 of 53). On the basis of the multitube approach, a frequency of BCL2-IGH-bearing cells was assigned to each farmer. The mean frequency of BCL2-IGH positive cells was 4.5 x 10-6 [< 8.4 x 10-6 - 57.6 x 10-6] for all of the farmers and 6.1 x 10-6 when only positive farmers were considered.
Seasonal Variation of BCL2-IGH Translocation.
According to the day of blood collection, farmers were divided into two groups corresponding to the high or low pesticide use period. Consistence of this classification was confirmed by the median time since last pesticide use, which was significantly lower for individuals belonging to the high pesticide use period (3 days versus 55 days; P = 0.0005). As seen in Table 2
, the prevalence of BCL2-IGH translocation was higher during the high pesticide use period, 81% (17 of 21) versus 65% (21 of 32; P = 0.10). We also noted a nonsignificant (P = 0.12) increase of the mean frequency of BCL2-IGH translocation from 2.9 x 10-6 [< 0.8 x 10-6 - 15.7 x 10-6] in the low pesticide use period to 6.9 x 10-6 [< 0.8 x 10-6- 58 x 10-6] in the high pesticide use period. When only positive individuals were included, the mean translocation frequency shared the same nonsignificant trend with period (P = 0.10). Despite a similar age distribution between the two groups [median age: 46 (range, 29–59) versus 47 (range: 27–62), for the low and the high pesticide use periods, respectively], a significant positive correlation was found with age only for the low pesticide use period (P = 0.01; r2 = 0.18). Considering the whole group of farmers, a nonsignificant positive correlation was found with age (P = 0.11; r2 = 0.04).
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for each analyzed farmer. As expected, the mean daily sunlight intensity exhibited a similar pattern than pesticide use and was higher during the high pesticide use period [1701 ± 663 J/m2 (255–3105)] compared with the low pesticide use period [894 ± 668 J/m2 (23–2882)]. However, no significant correlation was observed between BCL2-IGH frequency and sunlight radiation estimated with individual cumulative sunlight record the 1, 2, or 3 weeks before sampling (P = 0.21; r2 = 0.03).
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). Farmers with detectable BCL2-IGH translocation had a higher total farm area (97 versus 64 ha; P = 0.01), a higher farm area devoted to crops (65 versus 43 ha; P = 0.03), and a shorter mean time since last pesticide use (54 versus 89 days; P = 0.08). We then correlated exposure data with BCL2-IGH frequency. Significant positive correlations between frequency of BCL2-IGH-bearing cells and farm area (P = 0.03), and farm area devoted to crops (P = 0.03) were observed (Fig. 3)
. When analysis was performed separately for each pesticide use period, only farmers from the low pesticide use period demonstrated a significant positive correlation with farm area (P = 0.006; r2 = 0.23) or farm area devoted to crops (P = 0.04; r2 = 0.13). No correlation was observed between farm area and age (P = 0.48) excluding an influence of age for the observed effect. No significant relationship was found between BCL2-IGH translocation frequency and the number of commercial products used during the previous year, the type of spraying equipment, or the crop diversity.
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Molecular Characteristics of BCL2-IGH Translocation and Seasonal Variation.
Sequence analysis performed in most positive individuals (n = 36 of 38) confirmed that amplified DNA fragments consisted of the juxtaposition of the BCL2 oncogene near the IGH locus with an intervening sequence of nontemplated nucleotides. Of the farmers, 47.5% (17 of 36) presented with several BCL2-IGH rearrangements, the diversity of which was closely related to the frequency value. Consequently, oligoclonality was slightly higher during the high pesticide use period (Table 2)
. BCL2-major breakpoint region tended to fall into three clusters with an unbalanced use of the J6 segment. The frequency of breakpoints inside the three hotspot regions of the BCL2 gene was similar for the low and the high pesticide use periods (82.4% versus 88.9%). Although there were slight discrepancies, such as an excess of breakpoint clustered in the region 3115 for farmers outside the pesticide use period (44% versus 25.9%) and a new microcluster at bp 3135 for the high pesticide use period, no major differences of breakpoint pattern were observed in the two populations (Fig. 4)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using a common sensitivity level of 
1 x 10-6 of translocation-bearing lymphocytes, studies conducted on healthy Caucasians without known exposure to genotoxins consistently reported that 50% of people present BCL2-IGH-bearing cells (19)
. When applying this level of sensitivity in our reference population (22)
, we observed a similar prevalence. In these conditions, farmers exhibited a 71% prevalence of BCL2-IGH translocation.
The mean frequency of BCL2-IGH-bearing cells was not significantly different among farmers compared with the reference population (4 BCL2-IGH-positive cells in 1,000,000 normal cells). However, when we looked at dose-effect relationships between BCL2-IGH translocation and several pesticide exposure parameters, a positive correlation between the frequency and farm area was observed. This correlation was essentially due to the influence of the group of farmers sampled outside the high pesticide use period. Taken together, our results would indicate that a recent exposure induced a transient increase in prevalence and frequency of BCL2-IGH translocation during the high pesticide use period and mask a cumulative effect suggested by the relationship observed between farm area and frequency. The present data are highly consistent with previous results regarding the frequency of hybrid T-cell receptor 
/ß antigen receptor-gene, mediated by the V(D)J recombinase complex, in peripheral blood lymphocytes from agricultural workers. A 10-fold increase of hybrid T-cell receptor /ß frequency was observed in agricultural workers (n = 12) compared with control population (n = 10). In addition, the relative frequency of hybrid T-cell receptor /ß was higher during their active pesticide exposure returning to baseline level after cessation of exposure (25)
. In the only study evaluating the relation between pesticide exposure and t(14;18) translocation in healthy farmers, no influence was found on t(14;18) prevalence that might be the result of a very low detected prevalence (8% for the whole population, 10% and 6% for the rural and the urban control group, respectively; 26
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The observed effect during the period of high pesticide use could be related to a direct induction of breaks on the BCL2 gene. Genotoxic effects of at least some pesticides have been demonstrated by cytogenetic investigations conducted on farmers exposed to phosphine fumigators (27) or to a mixture of pesticides in open field farming (12) with an excess in breaks and rearrangements involving specific band 14q32 and 18q21 observed in comparison with control subjects (12) . Epigenetic mechanisms might also be involved. Modification of chromatin structure by pesticide exposure could facilitate the accessibility to the BCL2 and IGH loci, which are preferentially in close spatial proximity in normal B cells favoring the genesis of translocations (28) . Apart from genotoxicity, some pesticides might also exhibit immunotoxic activities (29) . BCL2-IGH-positive cells with an enhanced resistance to apoptosis due to BCL2 overexpression could be less sensitive to an immunosuppressive effect. Bell et al. (23) postulated that smoking induced the proliferation of pre-existing BCL2-IGH clones. This might be the same for pesticide exposure, as judged from the increased frequency and the similarity of clonal diversity, whatever the sampling period. Breakpoint distribution in farmers is similar to those published previously in follicular lymphoma (30 , 31) and in other healthy individuals (22 , 31 , 32) , which is in favor of a nongenotoxic effect. However, our PCR strategies targeted only the MBR region, which concerns 70% of the BCL2 breakpoints in lymphoma. Other breakpoint clusters like mcr or icr, which seem to be more prevalent in follicular lymphoma than expected previously, were not considered (33) .
We were concerned that the increased prevalence and frequency for farmers during the high pesticide use period could be influenced by other season-related environmental factors. Particularly, sunlight has been shown previously to influence the level of DNA damage or BCL2-IGH translocation frequency during summer (24 , 34) . In the present study, mean daily sunlight clearly displayed a similar yearly pattern than the pesticide exposure with a peak level of sunshine during the period of high pesticide use. However, analyzed farmers were sampled throughout the year, and no dose-effect relationships were found between BCL2-IGH frequency and individual daily or cumulative sunlight data, arguing against a role of sunlight in the observed frequency variations.
In conclusion, our results suggest for the first time that occupational exposure to pesticides might increase prevalence of positive individuals and frequency of BCL2-IGH-bearing cells, both as a consequence of cumulative exposure and transient effect of recent exposure. These results could be brought together with those of a recent case-control study on NHL showing that farming and exposure to selected pesticides (cyclodienes, lindane, captan, atrazine, and so forth) were strongly related to t(14;18)-positive NHL but not to t(14;18)-negative NHL (35) . They support the interest of BCL2-IGH translocation in etiologic research. Studies focusing on chronic effect of pesticide exposure use different parameters to evaluate the intensity of pesticide exposure (farm area, duration of exposure, spraying equipment, and so forth), but none of these parameters have been firmly validated (36) . Moreover, only recently has some integrative assessment of pesticide exposure been proposed (37) . To confirm our results and to use a model that will be based on field exposure data (38) , we will extend the study to the remaining 88 pesticide-exposed, never-smoking males from the cohort.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Sandrine Roulland, Groupe Régional d’Etudes sur le Cancer, Centre François Baclesse, Avenue du Général Harris,14076 Caen CEDEX 05, France. Phone: 33-231-455-070; Fax: 33-231-455-172; E-mail: s.roulland{at}baclesse.fr
Received 11/17/03. Revised 1/ 6/04. Accepted 1/ 7/04.
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) or farmers belonging to the high pesticide use period (
). All remaining daily sunlight records for the period 1997–2000 are indicated by gray points.
) represents the high pesticide use period and (
) represents the low pesticide exposure period. B, distribution of BCL2 breakpoints from the previously published reference population (
) is plotted to allow comparisons of BCL2 breakpoints spectra