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Organophosphate-based pesticides and genetic damage implicated in bladder cancer
Cancer Genetics and Cytogenetics 133 (2002) 112–117
Organophosphate-based pesticides and genetic damage implicated in bladder cancer Lucy R. Webster, Geoff H. McKenzie, Helen T. Moriarty* School of Biomedical Sciences, Faculty of Health Studies, Charles Sturt University, Wagga Wagga, 2678 Australia Received 19 June 2001; received in revised form 10 August 2001; accepted 14 August 2001
Abstract
Organophosphate-based pesticides have been associated with pathology and chromosomal damage in humans. There are also epidemiologic links with cancer. The few screening tests for low-level occupational exposure are of doubtful sensitivity; this investigation evaluated four methods. Blood samples were studied from 10 farmers before and after occupational exposure to organophosphate-based pesticides and five unexposed controls. The standard cholinesterase test was insensitive to the exposure (P0.815). However, a significant increase in Howell-Jolly bodies within erythrocytes was observed (P0.001). Cytogenetic studies on routine and aphidicolin-induced blood cultures revealed that following organophosphate exposure the total number of gaps and breaks on human chromosomes was significantly increased (P0.004 and P0.0006, respectively). We concluded that Howell-Jolly body and fragile site analysis were sensitive indicators of nuclear damage resulting from low-level occupational exposure to organophosphate. Such nuclear damage could be implicated in carcinogenesis. The development of bladder cancer is one such example. © 2002 Elsevier Science Inc. All rights reserved.
1. Introduction Organophosphate-based chemicals acting as anticholinesterase substances are widely used throughout the world [1]. A variety of short-term and chronic effects may be experienced following repeated or prolonged exposure to organophosphate-based pesticides, which include nausea, headache, and confusion [1,2]. Little information has been gathered with respect to the long-term effects of sublethal doses over a period of time, but reports of depression, memory loss, and chronic fatigue syndrome have been presented [1–3]; a carcinogenic effect of organophosphate-based pesticides has also been postulated [4,5]. Considerable attention has been given to the use of indicators of genetic damage such as chromosomal aberrations, sister-chromatid exchanges, and micronuclei to observe human populations occupationally exposed to organophosphate-based pesticides [4,6–8]. The precise effects of these chromosomal aberrations induced by occupational exposure to organophosphate-based pesticides are not fully known, but the increase in the number of aberrations following ex* Corresponding author. Tel.:61-2-6933-2195; fax: 61-2-69332587. E-mail address: [email protected] (H.T. Moriarty).
posure suggests an increase in the likelihood of cancer development induced by chromosomal damage [5,7,9]. In 1995, Sbrana and Musio [10] reported an increase in the expression of aphidicolin-induced chromosomal fragile sites following occupational exposure to pesticides. Aphidicolin induces 79 (67%) of the recognized fragile sites in the human genome and is the usual inducing agent used for environmental studies [10–14]. The study by Sbrana and Musio [10] reinforced the existence of a link between exposure to clastogenic agents (e.g., organophosphate-based pesticides) and fragile site induction. Furthermore, the analysis of the breakage effects of different carcinogens on fragile site regions appeared to be significant in the carcinogenic process of many cancers. Enhanced expression of particular fragile sites was implicated in cancers such as the leukemias and non-Hodgkin lymphoma and those sites were also the locations of numerous tumor suppressor genes and oncogenes [10,14]. These authors also reported that fragile site expression was a reproducible cell response to human exposure to pesticides [10]. Farmers are one occupational group with a high usage of organophosphate-based pesticides, and thus form a group of potential high risk to the types of genetic damage described here. In this connection, a number of studies have shown an increase in cancer incidence of numerous tumor types among the farm-
0165-4608/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S0165-4608(01)00 5 7 6 - 3
L.R. Webster et al. / Cancer Genetics and Cytogenetics 133 (2002) 112–117
ing population. These include bladder cancer [15,16], prostate cancer [15,17–19], non-Hodgkin lymphoma [17,18,20], multiple myeloma [17,18], skin cancer [18,21], kidney cancer [15], and lung cancer [21]. Of these tumor types, the bladder is potentially at a greater risk than any other organ with respect to carcinogenesis resulting from occupational pesticide exposure and, therefore, is of particular relevance to this study. The aim of this study was to test the hypothesis that occupational exposure of farmers to organophosphate-based pesticides enhances genetic damage, which may be implicated in bladder cancer.
2. Methods and materials Ten male farmers were recruited from the Riverina region, Australia. These farmers ranged from small-scale hobby farmers to larger scale broad-acre and sheep farmers. A small number of fruit growers were also sampled. The subjects were all healthy and occupationally exposed to organophosphate-based pesticides. Blood samples were collected from each of the 10 farmers on two separate occasions. The first sample was collected when the subjects had not been exposed to pesticides for at least 1 month. It was expected that the pesticide-induced changes would be transient and any nuclear damage should have recovered or at least decreased during that time [22]. The second blood samples were collected within 1 week of occupational exposure to organophosphate-based pesticides. The samples were collected over a 6-month period, subject to the various agricultural requirements for pesticide use by the group. Five normal control male samples were also collected to allow a comparison between occupational exposure to pesticides and nonexposure. The mean age was 45 years for both test and control groups. These control subjects had no exposure to organophosphate-based pesticides during the study and lived in townships within the farming area studied. Samples were collected from these subjects on a single occasion over the same 6-month period as the farmers. Both the farmers and the control group completed a questionnaire indicating pesticide exposure, type of pesticide used, and safety precautions taken (if appropriate). The procedures followed were in accordance with the ethical standards of the Charles Sturt University Ethics in Human Research Committee. 2.1. Howell-Jolly body analysis An EDTA blood sample was used to prepare blood films using the standard wedge smear technique. These films were then fixed in 100% methanol, and stained with May-Grünwald-Giemsa (Fronine, Riverstone, NSW, Australia). The number of Howell-Jolly bodies observed per 1500 erythrocytes per sample was recorded. 2.2. Plasma cholinesterase test Venous blood was collected into a lithium heparin Vacutainer tube (Beckton Dickinson, Lane Cove, NSW, Austra-
113
lia), which was centrifuged within 3–8 hours of collection for 10 minutes at 1460 g. Plasma cholinesterase levels were determined by the method of Dietz, Rubinstein, and Lubrano [23], using the Cobas Mira analyzer (Roche, Frenchs Forest, NSW, Australia). 2.3. Stimulated peripheral blood cultures Routine blood culture was performed by incubating whole blood in complete RPMI 1640 blood culture media with phytohemagglutinin at 37C for 96 hours. Standard cytogenetic techniques were used for the harvesting and dropping of all blood cultures [24]. Slides were stained with 10% Giemsa solution. 2.4. Aphidicolin-induced blood cultures Identical techniques as used for the stimulated peripheral blood culture methods were utilized for this procedure except that aphidicolin was added to the culture media to a final concentration of 0.4 M 24 hours prior to harvest. 2.5. Analysis of routine and aphidicolin-induced blood cultures Two hundred metaphases from each of the farmers preand postexposure samples were examined for breakage— 100 aphidicolin-induced and 100 noninduced. Similarly 200 metaphases from each of the control samples were examined. The degree of breakage was evaluated by scoring each metaphase for the number of gaps and breaks in chromatids and chromosomes. A chromatid gap was scored as “1” where as a chromosome gap was scored “2” (as it involved both chromatid arms); chromosome gaps and breaks were scored similarly. The gaps and breaks per 100 cells were then added. 2.6. Statistical techniques Farmer pre- and postexposure Howell-Jolly body examinations and cholinesterase data were analyzed by paired two sample t tests. A square root transformation was performed on all cytogenetics data as it followed an approximate Poisson distribution. To compare the number of gaps and/or breaks per 100 cells between pre- and postexposure farmer samples paired, two sample t tests were used. To compare results between control samples and farmer samples (where the sample size was different), two sample t tests, assuming unequal variances, were used. All statistical analyses were performed using Microsoft Excel software. 3. Results 3.1. Plasma cholinesterase Exposure to organophosphate-based pesticides that causes a depression in plasma cholinesterase levels by 60% is regarded as a marker to cease exposure [25]. The data obtained in this study indicated that no statistically significant depression in cholinesterase levels had occurred postexposure and, therefore, the chemical exposure experienced by
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these farmers was not acute or significant (P0.815). The largest depression in baseline cholinesterase levels (Table 1) was 15.0% for farmer 9, while farmer 4 had a 27.6% increase in cholinesterase levels following pesticide exposure. No statistically significant difference was observed between the cholinesterase levels in the control group and the postexposure farmers (P0.765). 3.2. Howell-Jolly bodies Blood films were examined from each farmer and control subject to detect the presence of Howell-Jolly bodies within erythrocytes. A statistically significant increase in the number of Howell-Jolly bodies present was observed in the farmers following pesticide exposure (P0.001) (Fig. 1). The number of Howell-Jolly bodies observed in the control samples was one or nil. 3.3. Noninduced cultures Statistically significant increases in the total number of noninduced gaps and breaks per 100 lymphocytes were seen when unexposed populations (control and preexposure samples) were compared with the exposed population (postexposure sample) (P0.004 and P0.008, respectively). Figs. 2a, b, and c demonstrate the different total numbers of aberrations per 100 cells for each group tested. It can be seen from Fig. 2c that the majority of the postexposure samples had more than 3 aberrations per 100 cells while the majority of control and preexposure populations (Figs. 2a and b, respectively) had two or less aberrations per 100 cells.
Fig. 1. Comparison of the number of Howell-Jolly bodies per 1500 erythrocytes in the farmer pre- and postexposure blood films.
of gaps and breaks present in this farmer was consistently high suggesting some residual chemical damage or that other forms of exposure may have been present in the preexposure sample. For the nine other farmers significant increases in the total number of gaps and breaks in 100 lymphocytes were seen following pesticide exposure. A significant difference was also found between the control samples and the postexposure samples (P1.96 105). No significant difference was observed between the control and preexposure samples (P0.382 noninduced and P0.128 aphidicolin-induced).
3.4. Aphidicolin-induced blood cultures
4. Discussion
A statistically significant increase in the total number of aphidicolin-induced gaps and breaks was seen in the farmers’ pre- and postexposure blood samples (P0.0006; Fig. 3). Fig. 3 also shows that only farmer 2 had a preexposure breakage level close to the postexposure level. The number
The results presented in this article show that even at low levels of exposure to organophosphate-based pesticides there was a statistically significant increase in the number of chromosomal fragile sites and of Howell-Jolly bodies. This suggests that these tests may be better indicators of pathology at low levels of exposure than traditional standard tests for exposure to organophosphate-based pesticides. In this connection, the cholinesterase test was insensitive to the exposure events experienced by the farmers in this study. Numerous previous studies on the effect of organophosphate exposure on plasma cholinesterase have yielded conflicting results, with some studies showing little significant depression, and others showing considerable depression. In addition, the use of a baseline preexposure plasma cholinesterase measurement had little influence on whether depression was observed or not. Our results reinforce the notion that at present the plasma cholinesterase levels are not a reliable indicator of low-level occupational exposure to organophosphate-based pesticides. This is only the second worldwide study where the impact of pesticide exposure on induced fragile site expression was observed. This method has been proposed as a more sensitive test for chemical mutagenesis than standard noninduced cultures [26]. Clastogenic exposure too weak to produce aberrations in standard culture is amplified in aphid-
Table 1 Plasma cholinesterase results for farmer and control subjects Case no.
Preexposure (U/L)
Postexposure (U/L)
% Depression in cholinesterase
1 2 3 4 5 6 7 8 9 10
10590 11361 11620 8276 13390 13420 12300 9380 11850 11620
9688 10510 11070 10560 11950 13740 11550 10072 10072 13370
8.52% 7.49% 4.73% 27.60% 10.75% 2.38% 6.10% 7.38% 15.00% 15.06%
Case no.
Cholinesterase level (U/L)
1 2 3 4 5
10310 13070 9431 11350 10960
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Fig. 3. Comparison of the total number of gaps and breaks per 100 lymphocytes in the farmer pre- and postexposure aphidicolin-induced blood cultures.
Fig. 2. The number of gaps and breaks present in control, pre- and postexposure subjects per 100 lymphocytes.
icolin treated cultures, as a result of aphidicolin’s ability to inhibit effective DNA synthesis, and consequently even mild exposure to clastogenic agents can be detected [10]. The data obtained strongly supports the findings by Sbrana and Musio [10] where statistically significantly increased numbers of aphidicolin-induced gaps and breaks were found. It should be noted that this statistical significance was achieved between the farmers’ pre- and postexposure populations as well as the nonexposed control and postexposure farmer populations. The use of a preexposure farmer population is perhaps a more accurate measure of damage relating to an environmental mutagen than a nonexposed control group as any baseline damage and individual variation is accounted for
(providing that a period of at least 1 month of nonexposure can be achieved). The observation of blood films for Howell-Jolly bodies (DNA fragments) was performed in this study as an additional indicator of chromosomal damage resulting from pesticide exposure. The increase of these micronuclei within red blood cells of the farmers complements the findings from the lymphocytes as it confirms that the chromosomes were being broken and pieces subsequently detached following organophosphate pesticide exposure. One farmer did not have an increase in Howell-Jolly bodies and this coincided with virtually no increase in the number of breaks and gaps in the aphidicolin-induced postexposure sample (332 preexposure compared to 337 postexposure). However, the persistent high numbers of breaks and gaps recorded for this farmer suggest that he was in fact experiencing exposure of which he was unaware (according to his questionnaire response). Apart from their use as a general indicator of environmental damage, cytogenetic breakpoint studies were performed in this project to determine whether there might be an association between fragile sites and bladder cancer. Indeed, increasing importance is being placed on such a link. The three fragile sites studied in detail so far, FRA3B, FRA7G, and FRA16D, have demonstrated strong correlations with carcinogenesis, suggesting that perhaps this is a universal feature of all common fragile sites [27]. Other positive discoveries include FRA11B being located within the CBL2 oncogene and FRA3B being within the FHIT tumor suppressor gene [28]. Other studies that support the theory of fragile site involvement in carcinogenic processes include those of Ribas et al. [12] and Fundia et al. [11]. The latter authors reported that 73% of induced common fragile sites coincided with cancer breakpoints; 81% of those breakpoints were especially associated with bands involved in structural rearrangements in chronic lymphocytic leukemia. Further suggestions that increased fragile site expression may be a marker for genetic predisposition to particular cancer types have been proposed [13,29].
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Focusing specifically on bladder cancer, the link between fragile sites and chromosome aberrations seen in that cancer has yet to be established. However, preliminary studies by our group (Webster, McKenzie, Moriarty; unpublished observations) indicate a likely link. Of the total 118 common and rare fragile sites, 77 (65%) were implicated in one or more cases of bladder cancer. More than half of those implicated fragile sites were found to be the location of one or more genes that are involved in cell regulation and have been associated with human cancer. Aphidicolin-induced fragile sites were implicated in 60% of sites involved in bladder cancer and 52% of these were associated with cancer genes. This preliminary evidence, together with the study by Sbrana and Musio [10] favor fragile sites being involved in at least a proportion of bladder cancer cases. Sustained chromosomal breakages and ineffective DNA repair mechanisms resulting from continued exposure to the damaging agent could result in carcinogenesis through gene disruption and loss of heterozygosity. Schantz and Hsu [30] explored chromosome instability with an in vitro bleomycin-induced lymphocyte assay and have postulated a similar theory. Hsu, Spitz, and Schantz [31] further suggested that variable host susceptibility might be explained by the range of chromosomal lesions between individuals. The cytogenetics of bladder cancer is highly heterogeneous with no specific aberrations reported. It is postulated here that there are a variety of combinations of genetic changes that separately result in a bladder cancer phenotype. Further analysis is being undertaken in our laboratory to identify the breakpoints from the subjects in this study to reveal consistent sites that have been damaged (i.e., particular gene involvement). Those studies may also reveal a particular template of chromosomal breakage sites consistent with each individual’s Mendelian pattern of inheritance of fragile sites and thereby expose potential hidden fingerprints of carcinogenic mechanisms. Acknowledgments From CSU: Kerry Cullis, School of Information Studies; John Kent, Farrer Centre; Todd Walker, School of Biomedical Sciences. John Kemp, Cytogenetics Unit, The New Children’s Hospital, Westmead, Sydney. Sources of support: The School of Biomedical Sciences, Faculty of Health Studies, Charles Sturt University; CSU Small Grant Scheme. References [1] Gordon CJ, Rowsey PJ. Poisons and fever. Clin Exp Pharmacol Physiol 1998;25:145–9. [2] O’Malley M. Clinical evaluation of pesticide exposure and poisonings. Lancet 1997;349:1161–5. [3] Marrs TC. Organophosphate poisoning. Pharmacol Ther 1993;58:51–66. [4] Rupa DS, Reddy PP, Reddi OS. Chromosomal aberrations in peripheral lymphocytes of cotton field workers exposed to pesticides. Environ Res 1989;49:1–6.
[5] Figa-Talamanca I, Mearelli I, Valente P, Bascherini S. Cancer mortality in a cohort of rural licensed pesticide users in the Province of Rome. Int J Epidemiol 1993;22:579–83. [6] De Ferrari M, Artuso M, Bonassi S, Bonatti S, Cavalieri, Z., Pescatore D, Marchini E, Pisano V, Abbondandolo A. Cytogenetic monitoring of an Italian population exposed to pesticides: chromosome aberration and sister-chromatid exchange analysis in peripheral blood lymphocytes. Mutat Res 1991;260:105–13. [7] Kourakis A, Mouratidou M, Barbouti A, Dimikiotou M. Cytogenetic effects of occupational exposure in the peripheral blood lymphocytes of pesticide sprayers. Carcinogenesis 1996;17:99–101. [8] Joksic G, Vidakovic A, Spasojevic-Tisma V. Cytogenetic monitoring of pesticide sprayers. Environ Res 1997;75:113–18. [9] Sorsa M, Ojajarvi A, Salomaa S. Cytogenetic surveillance of workers exposed to genotoxic chemicals. Teratog Carcinog Mutagen 1990;10: 215–21. [10] Sbrana I, Musio A. Enhanced expression of common fragile sites with occupational exposure to pesticides. Cancer Genet Cytogenet 1995;82:123–7. [11] Fundia A, Giere I, Larripa I, Slavutsky I. Spontaneous breakage and fragile site expression in chronic lymphocytic leukaemia. Cancer Genet Cytogenet 1998;103:144–8. [12] Ribas M, Miro R, Gelabert A, Egozcue J. Chromosome instability in lymphocytes from two patients affected by three sequential primary cancers. Cancer Genet Cytogenet 1999;110:133–5. [13] Tunca B, Egeli U, Zorluoglu A, Yilmazlar T, Yerci O, Kizil A. The expression of fragile sites in lymphocytes of patients with rectum cancer and their first-degree relatives. Cancer Lett 2000;152:201–9. [14] Sbrana I, Musio A. Aphidicolin-sensitive specific common fragile sites: A biomarker of exposure to pesticides. Environ Mol Mutagen 1997;29:250–5. [15] Forastiere F, Quercia A, Miceli M, Settimi L, Terenzoni B, Rapiti E, Faustini A, Borgia P, Calvariani F, Perucci CA. Cancer among farmers in central Italy. Scand J Work Environ Health 1993;19:382–9. [16] Viel JF, Challier B. Bladder cancer among French farmers: does exposure to pesticides in vineyards play a part? Occup Environ Med 1995;52:587–92. [17] Wiklund K, Dich J. Cancer risks among male farmers in Sweden. Eur J Cancer Prev 1995;4:81–90. [18] Zahm SH. Mortality study of pesticide applicators and other employees of a lawn care service company. J Occup Environ Med 1997;39: 1055–67. [19] Sharma-Wagner S, Chokkalingam AP, Malker HS, Stone BJ, McLaughlin JK, Hsing AW. Occupation and prostate cancer risk in Sweden. J Occup Environ Med 2000;42:517–25. [20] Zahm SH, Babbitt PA, Weisenburger DD, Blair A, Saal RC, Vaught JB. The role of agricultural pesticide use in the development of nonHodgkin’s lymphoma in women. Arch Environ Health 1993;48:353–7. [21] Wesseling C, Antich D, Hogstedt C, Rodriguez AC, Ahlbom A. Geographical difference of cancer incidence in Costa Rica in relation to environmental and occupational pesticide exposure. Int J Epidemiol 1999;28:365–74. [22] Ford J. The use of the micronucleus test for monitoring workplace exposure to chemical ‘cocktails’. Aust J Med Sci 2000;21:59–60. [23] Dietz AA, Rubinstein HH, Lubrano T. Colorimetric determination of serum cholinesterase and its genetic variants by the propionylthiocholinedithiobis (nitrobenzoic acid) procedure. Clin Chem 1973;19:1309–13. [24] Barch M, Lawce H, Arsham M. The ACT Cytogenetics Laboratory Manual. 2nd ed. New York: Raven Press, 1992, pp. 17–23. [25] Fillmore CM, Lessenger JE. A cholinesterase testing program for pesticide applicators. J Occup Environ Med 1993;35:61–70. [26] Yunis JJ, Soreng AL, Bowe AE. Fragile sites are targets of diverse mutagens and carcinogens. Oncogene 1987;1:59–69. [27] Mangelsdorf M, Reid K, Woollatt E, Dayan S, Eyre H, Finnis M, Hobson L, Nancarrow J, Venter D, Baker E, Richards RI. Chromosomal fragile site FRA16D and DNA instability in cancer. Cancer Res 2000;60:1683–9.
L.R. Webster et al. / Cancer Genetics and Cytogenetics 133 (2002) 112–117 [28] Sutherland GR, Baker E, Richards RI. Fragile sites still breaking. Trends Genet 1998;14:501–6. [29] Nesina IP, Polishchuk LZ, Oliinichenko PI. The determination of chromosome fragile sites in the peripheral blood lymphocytes of patients with colorectal cancer taking into account the cancer pathology predisposition in the familial anamnesis. Tsitol Genet 2000;34:3–9.
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[30] Schantz SP, Hsu TC. Mutagen-induced chromosome fragility within peripheral blood lymphocytes of head and neck cancer patients. Head Neck 1989;11:337–42. [31] Hsu TC, Spitz MR, Schantz SP. Mutagen sensitivity: a biological marker of cancer susceptibility. Cancer Epidemiol Biomarkers Prev 1991;1:83–9.
Organophosphate-based pesticides and genetic damage implicated in bladder cancer Lucy R. Webster, Geoff H. McKenzie, Helen T. Moriarty* School of Biomedical Sciences, Faculty of Health Studies, Charles Sturt University, Wagga Wagga, 2678 Australia Received 19 June 2001; received in revised form 10 August 2001; accepted 14 August 2001
Abstract
Organophosphate-based pesticides have been associated with pathology and chromosomal damage in humans. There are also epidemiologic links with cancer. The few screening tests for low-level occupational exposure are of doubtful sensitivity; this investigation evaluated four methods. Blood samples were studied from 10 farmers before and after occupational exposure to organophosphate-based pesticides and five unexposed controls. The standard cholinesterase test was insensitive to the exposure (P0.815). However, a significant increase in Howell-Jolly bodies within erythrocytes was observed (P0.001). Cytogenetic studies on routine and aphidicolin-induced blood cultures revealed that following organophosphate exposure the total number of gaps and breaks on human chromosomes was significantly increased (P0.004 and P0.0006, respectively). We concluded that Howell-Jolly body and fragile site analysis were sensitive indicators of nuclear damage resulting from low-level occupational exposure to organophosphate. Such nuclear damage could be implicated in carcinogenesis. The development of bladder cancer is one such example. © 2002 Elsevier Science Inc. All rights reserved.
1. Introduction Organophosphate-based chemicals acting as anticholinesterase substances are widely used throughout the world [1]. A variety of short-term and chronic effects may be experienced following repeated or prolonged exposure to organophosphate-based pesticides, which include nausea, headache, and confusion [1,2]. Little information has been gathered with respect to the long-term effects of sublethal doses over a period of time, but reports of depression, memory loss, and chronic fatigue syndrome have been presented [1–3]; a carcinogenic effect of organophosphate-based pesticides has also been postulated [4,5]. Considerable attention has been given to the use of indicators of genetic damage such as chromosomal aberrations, sister-chromatid exchanges, and micronuclei to observe human populations occupationally exposed to organophosphate-based pesticides [4,6–8]. The precise effects of these chromosomal aberrations induced by occupational exposure to organophosphate-based pesticides are not fully known, but the increase in the number of aberrations following ex* Corresponding author. Tel.:61-2-6933-2195; fax: 61-2-69332587. E-mail address: [email protected] (H.T. Moriarty).
posure suggests an increase in the likelihood of cancer development induced by chromosomal damage [5,7,9]. In 1995, Sbrana and Musio [10] reported an increase in the expression of aphidicolin-induced chromosomal fragile sites following occupational exposure to pesticides. Aphidicolin induces 79 (67%) of the recognized fragile sites in the human genome and is the usual inducing agent used for environmental studies [10–14]. The study by Sbrana and Musio [10] reinforced the existence of a link between exposure to clastogenic agents (e.g., organophosphate-based pesticides) and fragile site induction. Furthermore, the analysis of the breakage effects of different carcinogens on fragile site regions appeared to be significant in the carcinogenic process of many cancers. Enhanced expression of particular fragile sites was implicated in cancers such as the leukemias and non-Hodgkin lymphoma and those sites were also the locations of numerous tumor suppressor genes and oncogenes [10,14]. These authors also reported that fragile site expression was a reproducible cell response to human exposure to pesticides [10]. Farmers are one occupational group with a high usage of organophosphate-based pesticides, and thus form a group of potential high risk to the types of genetic damage described here. In this connection, a number of studies have shown an increase in cancer incidence of numerous tumor types among the farm-
0165-4608/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S0165-4608(01)00 5 7 6 - 3
L.R. Webster et al. / Cancer Genetics and Cytogenetics 133 (2002) 112–117
ing population. These include bladder cancer [15,16], prostate cancer [15,17–19], non-Hodgkin lymphoma [17,18,20], multiple myeloma [17,18], skin cancer [18,21], kidney cancer [15], and lung cancer [21]. Of these tumor types, the bladder is potentially at a greater risk than any other organ with respect to carcinogenesis resulting from occupational pesticide exposure and, therefore, is of particular relevance to this study. The aim of this study was to test the hypothesis that occupational exposure of farmers to organophosphate-based pesticides enhances genetic damage, which may be implicated in bladder cancer.
2. Methods and materials Ten male farmers were recruited from the Riverina region, Australia. These farmers ranged from small-scale hobby farmers to larger scale broad-acre and sheep farmers. A small number of fruit growers were also sampled. The subjects were all healthy and occupationally exposed to organophosphate-based pesticides. Blood samples were collected from each of the 10 farmers on two separate occasions. The first sample was collected when the subjects had not been exposed to pesticides for at least 1 month. It was expected that the pesticide-induced changes would be transient and any nuclear damage should have recovered or at least decreased during that time [22]. The second blood samples were collected within 1 week of occupational exposure to organophosphate-based pesticides. The samples were collected over a 6-month period, subject to the various agricultural requirements for pesticide use by the group. Five normal control male samples were also collected to allow a comparison between occupational exposure to pesticides and nonexposure. The mean age was 45 years for both test and control groups. These control subjects had no exposure to organophosphate-based pesticides during the study and lived in townships within the farming area studied. Samples were collected from these subjects on a single occasion over the same 6-month period as the farmers. Both the farmers and the control group completed a questionnaire indicating pesticide exposure, type of pesticide used, and safety precautions taken (if appropriate). The procedures followed were in accordance with the ethical standards of the Charles Sturt University Ethics in Human Research Committee. 2.1. Howell-Jolly body analysis An EDTA blood sample was used to prepare blood films using the standard wedge smear technique. These films were then fixed in 100% methanol, and stained with May-Grünwald-Giemsa (Fronine, Riverstone, NSW, Australia). The number of Howell-Jolly bodies observed per 1500 erythrocytes per sample was recorded. 2.2. Plasma cholinesterase test Venous blood was collected into a lithium heparin Vacutainer tube (Beckton Dickinson, Lane Cove, NSW, Austra-
113
lia), which was centrifuged within 3–8 hours of collection for 10 minutes at 1460 g. Plasma cholinesterase levels were determined by the method of Dietz, Rubinstein, and Lubrano [23], using the Cobas Mira analyzer (Roche, Frenchs Forest, NSW, Australia). 2.3. Stimulated peripheral blood cultures Routine blood culture was performed by incubating whole blood in complete RPMI 1640 blood culture media with phytohemagglutinin at 37C for 96 hours. Standard cytogenetic techniques were used for the harvesting and dropping of all blood cultures [24]. Slides were stained with 10% Giemsa solution. 2.4. Aphidicolin-induced blood cultures Identical techniques as used for the stimulated peripheral blood culture methods were utilized for this procedure except that aphidicolin was added to the culture media to a final concentration of 0.4 M 24 hours prior to harvest. 2.5. Analysis of routine and aphidicolin-induced blood cultures Two hundred metaphases from each of the farmers preand postexposure samples were examined for breakage— 100 aphidicolin-induced and 100 noninduced. Similarly 200 metaphases from each of the control samples were examined. The degree of breakage was evaluated by scoring each metaphase for the number of gaps and breaks in chromatids and chromosomes. A chromatid gap was scored as “1” where as a chromosome gap was scored “2” (as it involved both chromatid arms); chromosome gaps and breaks were scored similarly. The gaps and breaks per 100 cells were then added. 2.6. Statistical techniques Farmer pre- and postexposure Howell-Jolly body examinations and cholinesterase data were analyzed by paired two sample t tests. A square root transformation was performed on all cytogenetics data as it followed an approximate Poisson distribution. To compare the number of gaps and/or breaks per 100 cells between pre- and postexposure farmer samples paired, two sample t tests were used. To compare results between control samples and farmer samples (where the sample size was different), two sample t tests, assuming unequal variances, were used. All statistical analyses were performed using Microsoft Excel software. 3. Results 3.1. Plasma cholinesterase Exposure to organophosphate-based pesticides that causes a depression in plasma cholinesterase levels by 60% is regarded as a marker to cease exposure [25]. The data obtained in this study indicated that no statistically significant depression in cholinesterase levels had occurred postexposure and, therefore, the chemical exposure experienced by
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L.R. Webster et al. / Cancer Genetics and Cytogenetics 133 (2002) 112–117
these farmers was not acute or significant (P0.815). The largest depression in baseline cholinesterase levels (Table 1) was 15.0% for farmer 9, while farmer 4 had a 27.6% increase in cholinesterase levels following pesticide exposure. No statistically significant difference was observed between the cholinesterase levels in the control group and the postexposure farmers (P0.765). 3.2. Howell-Jolly bodies Blood films were examined from each farmer and control subject to detect the presence of Howell-Jolly bodies within erythrocytes. A statistically significant increase in the number of Howell-Jolly bodies present was observed in the farmers following pesticide exposure (P0.001) (Fig. 1). The number of Howell-Jolly bodies observed in the control samples was one or nil. 3.3. Noninduced cultures Statistically significant increases in the total number of noninduced gaps and breaks per 100 lymphocytes were seen when unexposed populations (control and preexposure samples) were compared with the exposed population (postexposure sample) (P0.004 and P0.008, respectively). Figs. 2a, b, and c demonstrate the different total numbers of aberrations per 100 cells for each group tested. It can be seen from Fig. 2c that the majority of the postexposure samples had more than 3 aberrations per 100 cells while the majority of control and preexposure populations (Figs. 2a and b, respectively) had two or less aberrations per 100 cells.
Fig. 1. Comparison of the number of Howell-Jolly bodies per 1500 erythrocytes in the farmer pre- and postexposure blood films.
of gaps and breaks present in this farmer was consistently high suggesting some residual chemical damage or that other forms of exposure may have been present in the preexposure sample. For the nine other farmers significant increases in the total number of gaps and breaks in 100 lymphocytes were seen following pesticide exposure. A significant difference was also found between the control samples and the postexposure samples (P1.96 105). No significant difference was observed between the control and preexposure samples (P0.382 noninduced and P0.128 aphidicolin-induced).
3.4. Aphidicolin-induced blood cultures
4. Discussion
A statistically significant increase in the total number of aphidicolin-induced gaps and breaks was seen in the farmers’ pre- and postexposure blood samples (P0.0006; Fig. 3). Fig. 3 also shows that only farmer 2 had a preexposure breakage level close to the postexposure level. The number
The results presented in this article show that even at low levels of exposure to organophosphate-based pesticides there was a statistically significant increase in the number of chromosomal fragile sites and of Howell-Jolly bodies. This suggests that these tests may be better indicators of pathology at low levels of exposure than traditional standard tests for exposure to organophosphate-based pesticides. In this connection, the cholinesterase test was insensitive to the exposure events experienced by the farmers in this study. Numerous previous studies on the effect of organophosphate exposure on plasma cholinesterase have yielded conflicting results, with some studies showing little significant depression, and others showing considerable depression. In addition, the use of a baseline preexposure plasma cholinesterase measurement had little influence on whether depression was observed or not. Our results reinforce the notion that at present the plasma cholinesterase levels are not a reliable indicator of low-level occupational exposure to organophosphate-based pesticides. This is only the second worldwide study where the impact of pesticide exposure on induced fragile site expression was observed. This method has been proposed as a more sensitive test for chemical mutagenesis than standard noninduced cultures [26]. Clastogenic exposure too weak to produce aberrations in standard culture is amplified in aphid-
Table 1 Plasma cholinesterase results for farmer and control subjects Case no.
Preexposure (U/L)
Postexposure (U/L)
% Depression in cholinesterase
1 2 3 4 5 6 7 8 9 10
10590 11361 11620 8276 13390 13420 12300 9380 11850 11620
9688 10510 11070 10560 11950 13740 11550 10072 10072 13370
8.52% 7.49% 4.73% 27.60% 10.75% 2.38% 6.10% 7.38% 15.00% 15.06%
Case no.
Cholinesterase level (U/L)
1 2 3 4 5
10310 13070 9431 11350 10960
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Fig. 3. Comparison of the total number of gaps and breaks per 100 lymphocytes in the farmer pre- and postexposure aphidicolin-induced blood cultures.
Fig. 2. The number of gaps and breaks present in control, pre- and postexposure subjects per 100 lymphocytes.
icolin treated cultures, as a result of aphidicolin’s ability to inhibit effective DNA synthesis, and consequently even mild exposure to clastogenic agents can be detected [10]. The data obtained strongly supports the findings by Sbrana and Musio [10] where statistically significantly increased numbers of aphidicolin-induced gaps and breaks were found. It should be noted that this statistical significance was achieved between the farmers’ pre- and postexposure populations as well as the nonexposed control and postexposure farmer populations. The use of a preexposure farmer population is perhaps a more accurate measure of damage relating to an environmental mutagen than a nonexposed control group as any baseline damage and individual variation is accounted for
(providing that a period of at least 1 month of nonexposure can be achieved). The observation of blood films for Howell-Jolly bodies (DNA fragments) was performed in this study as an additional indicator of chromosomal damage resulting from pesticide exposure. The increase of these micronuclei within red blood cells of the farmers complements the findings from the lymphocytes as it confirms that the chromosomes were being broken and pieces subsequently detached following organophosphate pesticide exposure. One farmer did not have an increase in Howell-Jolly bodies and this coincided with virtually no increase in the number of breaks and gaps in the aphidicolin-induced postexposure sample (332 preexposure compared to 337 postexposure). However, the persistent high numbers of breaks and gaps recorded for this farmer suggest that he was in fact experiencing exposure of which he was unaware (according to his questionnaire response). Apart from their use as a general indicator of environmental damage, cytogenetic breakpoint studies were performed in this project to determine whether there might be an association between fragile sites and bladder cancer. Indeed, increasing importance is being placed on such a link. The three fragile sites studied in detail so far, FRA3B, FRA7G, and FRA16D, have demonstrated strong correlations with carcinogenesis, suggesting that perhaps this is a universal feature of all common fragile sites [27]. Other positive discoveries include FRA11B being located within the CBL2 oncogene and FRA3B being within the FHIT tumor suppressor gene [28]. Other studies that support the theory of fragile site involvement in carcinogenic processes include those of Ribas et al. [12] and Fundia et al. [11]. The latter authors reported that 73% of induced common fragile sites coincided with cancer breakpoints; 81% of those breakpoints were especially associated with bands involved in structural rearrangements in chronic lymphocytic leukemia. Further suggestions that increased fragile site expression may be a marker for genetic predisposition to particular cancer types have been proposed [13,29].
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Focusing specifically on bladder cancer, the link between fragile sites and chromosome aberrations seen in that cancer has yet to be established. However, preliminary studies by our group (Webster, McKenzie, Moriarty; unpublished observations) indicate a likely link. Of the total 118 common and rare fragile sites, 77 (65%) were implicated in one or more cases of bladder cancer. More than half of those implicated fragile sites were found to be the location of one or more genes that are involved in cell regulation and have been associated with human cancer. Aphidicolin-induced fragile sites were implicated in 60% of sites involved in bladder cancer and 52% of these were associated with cancer genes. This preliminary evidence, together with the study by Sbrana and Musio [10] favor fragile sites being involved in at least a proportion of bladder cancer cases. Sustained chromosomal breakages and ineffective DNA repair mechanisms resulting from continued exposure to the damaging agent could result in carcinogenesis through gene disruption and loss of heterozygosity. Schantz and Hsu [30] explored chromosome instability with an in vitro bleomycin-induced lymphocyte assay and have postulated a similar theory. Hsu, Spitz, and Schantz [31] further suggested that variable host susceptibility might be explained by the range of chromosomal lesions between individuals. The cytogenetics of bladder cancer is highly heterogeneous with no specific aberrations reported. It is postulated here that there are a variety of combinations of genetic changes that separately result in a bladder cancer phenotype. Further analysis is being undertaken in our laboratory to identify the breakpoints from the subjects in this study to reveal consistent sites that have been damaged (i.e., particular gene involvement). Those studies may also reveal a particular template of chromosomal breakage sites consistent with each individual’s Mendelian pattern of inheritance of fragile sites and thereby expose potential hidden fingerprints of carcinogenic mechanisms. Acknowledgments From CSU: Kerry Cullis, School of Information Studies; John Kent, Farrer Centre; Todd Walker, School of Biomedical Sciences. John Kemp, Cytogenetics Unit, The New Children’s Hospital, Westmead, Sydney. Sources of support: The School of Biomedical Sciences, Faculty of Health Studies, Charles Sturt University; CSU Small Grant Scheme. References [1] Gordon CJ, Rowsey PJ. Poisons and fever. Clin Exp Pharmacol Physiol 1998;25:145–9. [2] O’Malley M. Clinical evaluation of pesticide exposure and poisonings. Lancet 1997;349:1161–5. [3] Marrs TC. Organophosphate poisoning. Pharmacol Ther 1993;58:51–66. [4] Rupa DS, Reddy PP, Reddi OS. Chromosomal aberrations in peripheral lymphocytes of cotton field workers exposed to pesticides. Environ Res 1989;49:1–6.
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