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Plasma levels of DDT and their association with reproductive hormones in adult men from northern Thailand
Science of the Total Environment 355 (2006) 98 – 105 www.elsevier.com/locate/scitotenv
Plasma levels of DDT and their association with reproductive hormones in adult men from northern Thailand R. Asawasinsopona, T. Prapamontolb,T, O. Prakobvitayakitc, Y. Vaneesornd, A. Mangklabrukse, B. Hockf a
Environmental Science Ph.D. Program, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand b Pollution and Environmental Health Research Program, Research Institute for Health Sciences (RIHES), Chiang Mai University, P.O. Box 80 CMU, 50200 Chiang Mai, Thailand c Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand d Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand e Department of Internal Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand f Center of Life and Food Sciences Weihenstephan, Technical University of Munich, Alte Akademic12, 85350, Freising, Germany Received 13 December 2004; accepted 1 March 2005 Available online 17 June 2005
Abstract Historically, dichlorodiphenyltrichloroethane (DDT) was used in northern Thailand for malaria control and farming purposes. Several studies have investigated its effects on end points of adverse reproductive health outcomes. However, the few previous studies investigated hormonal effects in men and available data are inconclusive. The authors aimed to explore the main hypothesis that plasma DDT levels in adult men were associated with reproductive hormone levels. A cross-sectional study was performed of 97 adult men living in a highland village named Mae Sa Mai, 35 km north of Chiang Mai, Thailand. Venous blood samples were collected for measuring plasma levels of DDT and its metabolites and reproductive hormones, including 17h-estradiol (E2), testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH). 1,1-Dichloro-2,2di(4-chlorophenyl)ethylene (p,pV-DDE) and 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (p,pV-DDT) were detected in all plasma samples. p,pV-DDE had the highest level with a median of 4057.7 ng/g lipids and a relatively higher level compared with most other studies. Plasma p,pV-DDT levels were positively associated with years of residence (h + SE = 0.472 + 0.208, P = 0.028) and years of DDT usage for farming (h + SE = 0.177 + 0.084, P = 0.04). The remarkable findings were the negative association of plasma E2 levels with plasma p,pV-DDE levels (h + SE= 7.093 + 2.899, P = 0.016) and the positive association
T Corresponding author. Tel.: +66 53 221 465#235; fax: +66 53 221 849. E-mail addresses: [email protected], [email protected] (T. Prapamontol). 0048-9697/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2005.03.004
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with plasma 1,1-dichloro-2-(2-chlorophenyl)-2-(4-chlorophynyl)ethylene (o,pV-DDE) levels (h + SE = 16.381 + 5.596, P = 0.008) after adjusting for age and body mass index (BMI). However, these associations were rather weak. Our results suggest that these associations may reflect their different mechanisms of hormonal activities and they would be warrant further detail investigations. D 2005 Elsevier B.V. All rights reserved. Keywords: DDT; Persistent organic pollutants; Adult men; Endocrine disruption
1. Introduction In recent year, it has been shown that DDT and its metabolites are persistent organic pollutants and having endocrine disruption activities, acting as hormone receptor agonists and antagonists (Colborn et al., 1993; Kelce et al., 1997; Longnecker et al., 1997; Turusov et al., 2002). Because of their longterm adverse effects on living organisms, DDT which was of technical grade and contained mixed isomers has been banned in most developed countries since the 1970s. However, it is still used in some developing countries for essential public health purposes (Curtis and Lines, 2000; Turusov et al., 2002). Several studies in wildlife showed that exposure to DDT was linked to adverse reproductive effects in mammals, birds, amphibians, and fish. However, it was rather difficult to conclude particular causal agents were producing any effects (Colborn et al., 1993; Guillette et al., 1994, 1996; Vos et al., 2000). In epidemiological data on health effects in men, most studies used end points of adverse reproductive effects such as decreased sperm counts, reproductive tract abnormalities, infertilities, and reproductive cancers (Ayotte et al., 2001; Longnecker et al., 2002; Skakkebaek, 2002; Dalvie et al., 2004a,b). The few previous studies investigated effects of DDT on hormonal status in adult men, and available data are inconclusive. In fact, the hormonal actions of DDT could be expected to have some impact on the secretion of endocrine releasing factors, resulting in varying effects on reproductive hormonal metabolism. Further hormonal disturbance or imbalance could potentially occur in organs of the endocrine system (Jaga, 2000). In northern Thailand, and in Chiang Mai Province in particular, DDT was first introduced in 1949. Its use for malaria control was finally stopped altogether
in 1999, whereas the last spraying in Mae Sa Mai village which was the present study site was in 1989. Earlier, DDT was extensively used for farm activities in this village until it was banned for farming use in 1983, but most farmers illegally continued using in spite of its ban (Chareonviriyaphap et al., 2000; Stuetz et al., 2001). Stuetz et al. (2001) reported that total DDT levels in human milk of women from Mae Sa Mai village were still high with a median and a maximum level of 209 and 2012 ng/ml, respectively. In the present study, the authors aimed to assess whether plasma DDT levels in adult men residing in Mae Sa Mai village were associated with reproductive hormone levels.
2. Methods 2.1. Ethical approval The present study protocol was approved by the Human Experimentation Committee, Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand (Certificate of Ethical Clearance No.10/2003, 13 March 2003). 2.2. Study group, questionnaires, and anthropometric measurement A cross sectional study of 97 adult men, aged from 18 to 45 years old was conducted. All had resided inMae Sa Mai village in northern Thailand for at least 10 years. Written consent was obtained from individual subjects before collecting blood samples, interviewing for socio-demographic data, and measuring weight and height. Socio-demographic data were collected from individual participants by trained interviewers. Weight and height were also measured for BMI calculation.
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2.3. Blood sampling and storage Twelve millilliters of fasting venous blood samples were collected in heparin tubes (Vacutainer 366480, BD,USA). Plasma for DDT analysis was collected in glass tubes and frozen at 20 8C until analysis. Plasma for lipid and hormone analysis was collected in centrifuge tubes and frozen at 70 8C until analysis. 2.4. Analysis of DDT and its metabolites DDT and its metabolites included p,pV-DDE, p,pVDDT, 1,1-dichloro-2,2-di(4-chlorophenyl)ethane (p,pV-DDD), o,pV-DDE, and 1,1,1-trichloro-2-(2chlorophenyl)-2-(4-chlorophynyl)ethane (o,pV-DDT). All solvents for extraction were ultra resi-analysed grade for organic residue analysis (J.T.Baker, USA). The analytes were extracted by solid phase extraction using octadecyl (C18)-bonded silica cartridges (BondElut, Varian, USA). Extraction of these compounds from 2 ml plasma samples was performed according to the Prapamontol and Stevenson method (Prapamontol and Stevenson, 1991). The isooctane extracts were analysed for DDT and its metabolites using gas chromatography with electron capture detection (HP 5890 Series II equipped with a 63Ni electron capture dectector, an autosampler (HP7673), a fused silica capillary column (Ultra 2: 25 m 0.32 mm i.d. with 0.52 Am film thickness, J and W Scientific, USA), and computerized data handling system (HP3365 Series Chemstation). Recoveries of individual components of DDT ranged from 91.47% for o,pV-DDT to 108.87% for p,pV-DDT. The quantitation limit for individual compounds ranged from 0.5 ng/ml for p,pV-DDE to 2.63 ng/ml for p,pV-DDD. The intrabatch coefficient of variation (% CV) ranged from 4.3% for p,pV-DDT to 4.34 % for p,pV-DDE and interbatch % CV ranged from 3.4% for p,pV-DDE to 8.8% for p,pV-DDT. The determined mean levels for calculating CVs ranged from 0.99 ng/ml for o,pV-DDE to 15.07 ng/ml for p,pVDDE. 2.5. Lipid and reproductive hormone analysis Plasma triglycerides and total cholesterol levels were determined using an enzymatic method (Syn-
chron CX systems, Beckman, Germany). The intrabatch percent CV was 1.74% for triglycerides and 2.21% for cholesterol. The interbatch percent CV was 4.4% for triglycerides and 1.54 % for cholesterol. The determined mean levels for calculating CVs were 115 mg/dl for triglycerides and 158.4 mg/ dl for cholesterol. The following conversion formula was used for calculation of total lipids (Phillips et al., 1989): Plasmalipids; mg=dl ¼ 2:27ðtotalcholesterolÞ þ triglycerides þ 0:623 Plasma E2, testosterone, LH, and FSH were measured using chemiluminescent competitive immunoassay (Elecsys 1010, Roche, USA). The intrabatch percent CV ranged from 1.13% for LH to 3.1% for E2. The interbatch percent CV ranged from 10.56% for LH to 11.1% for E2. The determined mean levels for calculating CVs were 20.5 pg/ml for E2, 5 ng/ml for testosterone, 10.36 mIU/ml for LH, and 11.88 mIU/ml for FSH. 2.6. Statistical analysis SPSS version 11 was performed for data analysis. Descriptive parameters, including geometric mean, median, minimum (Min), maximum (Max), quartile (25th–75th percentile), and percentile were computed. The levels of DDT and its metabolites below the quantitation limit were not reported. DDT levels were adjusted for plasma lipids. To obtain the best linearity, natural logarithm transformation was applied for all variables before the parametric test. Pearson’s correlation coefficient was calculated for the association between plasma levels of DDT and its metabolites and reproductive hormones. Multiple linear regression analysis was conducted for the association of DDT and its metabolites with years of residence and DDT usage for farming. This regression analysis was also conducted for the associations of p,pV-DDE and o,pV-DDE with E2, adjusted for confounders including age and BMI. Beta (h) and standard error (SE) were calculated. Student’s t-test was used for testing differences in plasma DDT levels between persons who had used DDT for farming and those who had never used it.
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The level of significance was set at a P value b 0.05 (2-tailed).
Table 2 Multiple linear regression analysis for the association of DDT and its metabolites with years of residence and DDT usage for farming b
Variables
3. Results 3.1. Demographic characteristics of the study population The geometric mean age of 97 adult men was 29 years. All of them (100%) were farmers. The mean times of residence in this village and farm experience were 25.6 and 13 years, respectively. BMI ranged from 18.7 to 32.3 kg/m2 with a mean of 23 kg/m2. Sixty-two men (63.9%) had used DDT for farming, of whom 55 (88.7%) mixed DDT with plant seed before planting and 7 (11.3%) used it only for spraying. The mean duration of DDT usage for farming was 4 years (ranged from 1 to 20 years). By student’s t-test analysis, men who had used DDT for farming had higher mean plasma levels of p,pV-DDE, p,pV-DDT and o,pV-DDE than men who never used it ( P b 0.05). 3.2. The association of DDT and its metabolites with environmental and occupational exposures Plasma p,pV-DDE and p,pV-DDT were detected in all subjects of the study population. p,pV-DDE had the highest level with a median of 4057.7 ng/g lipids (Table 1). Plasma p,pV-DDE levels were positively associated with plasma p,pV-DDT levels (r = 0.675, P = 0.000). Table 2 shows the association of DDT and its metabolites with years of residence and DDT usage for farming, using multiple linear regression analysis. Plasma p,pV-DDT levels were positively associated with years of residence (h + SE = 0.472+0.208, P = 0.028) and years of DDT usage for farming (h + SE = 0.177 + 0.084, P = 0.04).
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SE
P
0.299 0.037
0.205 0.082
0.15 0.657
p,pV-DDT Years of residence Years of DDT usage for farming
0.472 0.177
0.208 0.084
0.028T 0.04T
p,pV-DDD Years of residence Years of DDT usage for farming
1.029 0.283
0.755 0.304
0.306 0.451
o,pV-DDE Years of residence Years of DDT usage for farming
0.199 0.223
0.464 0.187
0.678 0.263
o,pV-DDT Years of residence Years of DDT usage for farming
0.012 0.194
0.616 0.248
0.985 0.449
p,pV-DDE Years of residence Years of DDT usage for farming
T P b 0.05.
3.3. The association of DDT and its metabolites with reproductive hormones The median levels of plasma E2, testosterone, LH and FSH were 22.29 pg/ml, 4.67 ng/ml, 3.68 mIU/ml, and 5.53 mIU/ml, respectively. The value at 5th percentile of plasma E2, testosterone, and LH was below the reference value of the manufacturer, Roche, USA (Table 3). By Pearson’s correlation analysis, plasma E2 levels were negatively associated with plasma p,pV-DDE levels (r = 0.239, P = 0.018), but positively associated with plasma o,pV-DDE levels (r = 0.473, P = 0.02) (Table 4). These associations did not change after adjusting for age and BMI (h + SE = 7.093 + 2.899, P = 0.016 for the association between plasma E2 and p,pV-DDE and h + SE = 16.381 + 5.596, P = 0.008 for the association between
Table 1 Plasma levels of DDT and its metabolites adjusted for lipids (ng/g lipids) in 97 adult men DDT
Detected in samples, %
Geometric mean
Median
25th–75th percentile
Min–Max
p,pV-DDE p,pV-DDT p,pV-DDD o,pV-DDE o,pV-DDT
100 100 10.8 24.5 25.5
4013.0 628.7 390.5 75.8 97.8
4057.7 628.8 373.0 72.9 76.5
2989.5–5232.7 440.9–830.2 251.9–533.3 55.3–87.7 62.8–141.7
1325.1–12683.7 224.9–3085.1 211.7–1161.5 36.9–219.1 42.9–632.7
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Table 3 Plasma levels of reproductive hormones in 97 adult men Hormones
Min
E2, pg/ml Testosterone, ng/ml LH, mIU/ml FSH, mIU/ml a
Percentile
5.75 1.18 1.23 2.09
5th
25th
50th
75th
95th
8.72 2.18 1.55 3.27
15.38 3.45 2.59 4.18
22.29 4.67 3.68 5.53
28.23 5.8 5.02 8.57
42.21 7.99 7.02 13.88
Max
Reference Valuea (5th–9th)
114.1 15.0 12.9 46.55
11–43.9 2.8–8.0 1.7–8.6 1.5–12.4
Reference value from manufacturer, Roche, USA.
chain (Stuetz et al., 2001). In our data, p,pV-DDT was associated with years of residence and DDT usage for farming. In addition, men who had reportedly ever used DDT for farming had higher mean plasma levels of p,pV-DDE, p,pV-DDT and o,pV-DDE than men who never used it ( P b 0.05). It would seem that durations of environmental exposure and DDT usage for farming in the past were determinants of DDT levels. Our results therefore suggest that DDT residues in these group men were due to both environmental and occupational exposures. With regard to reproductive hormones, the values at the 5th percentile of plasma E2, testosterone, and LH were below the reference values of the manufacturer. Therefore, these reference values may be inappropriate for Thai populations. Therefore the most remarkable findings were the negative association of plasma E2 levels with plasma p,pV-DDE levels and the positive association with plasma o,pV-DDE levels, adjusted for age and BMI. The opposite direction of the associations for p,pVDDE and o,pV-DDE may reflect their different mechanisms of hormonal activities, since the p,pV isomers act as androgen antagonists whereas the o,pV isomers act as estrogen agonists (Bitman et al., 1968; Nelson, 1974; Kelce et al., 1995; Sohoni and Sumpter, 1998). However, these associations were rather weak.
plasma E2 and o,pV-DDE). No significant association was found when p,pV-DDE, o,pV-DDE, age, and BMI were all once included in multiple comparison (Table 5). Considering the association of DDT and its metabolites expressed on a fresh weight basis, only plasma E2 levels were negatively associated with plasma p,pV-DDE levels (data not shown).
4. Discussion The p,pV-isomers (i.e. p,pV-DDE and p,pV-DDT) were detected in all study subjects and the o,pVisomers (i.e. o,pV-DDE and o,pV-DDT) were also detected in a quarter of the study population. In general, the p,pV-isomers were present at much higher levels than o,pV-isomers, because technical grade DDT contains approximately 80% of p,pV-DDT and 20% of o,pV-DDT. The p,pV-isomers also have long half lives, which partly accounts for their higher levels in people. This population might have been exposed to these compounds by a continuing low level intake, presumably dietary, or an extremely high cumulative exposure in the years preceding the ban. DDT was applied for malaria control and farming purposes for 40 years in the study village. Data were collected, DDT and its metabolites are still detected in the food
Table 4 Pearson’s correlation coefficients (r) between plasma levels of DDT and its metabolites and reproductive hormones Variables p,pV-DDE p,pV-DDT p,pV-DDD o,pV-DDE o,pV-DDT T P b 0.05.
E2
Testosterone
LH
FSH
r
P
r
P
r
P
r
P
0.239 0.196 0.371 0.473 0.012
0.018T 0.054 0.291 0.02T 0.953
0.176 0.142 0.185 0.333 0.161
0.085 0.165 0.608 0.112 0.442
0.129 0.153 0.203 0.402 0.081
0.208 0.134 0.547 0.052 0.702
0.122 0.073 0.37 0.181 0.045
0.235 0.48 0.293 0.398 0.831
R. Asawasinsopon et al. / Science of the Total Environment 355 (2006) 98–105 Table 5 Multiple linear regression analysis for the association of plasma E2 with plasma p,pV-DDE and o,pV-DDE after adjusting for age and BMI Variables Model 1: E2 p,pV-DDE Age BMI E2 o,pV-DDE Age BMI Model 2 a: E2 p,pV-DDE o,pV-DDE Age BMI
b
SE
P
7.093 4.773 15.917
2.899 5.676 11.743
0.016* 0.403 0.179
16.381 4.043 36.110
5.596 10.566 23.061
0.008** 0.706 0.133
4.410 8.467 8.526 2.338
4.398 4.240 7.898 18.694
0.329 0.055 0.294 0.902
a
p,pV-DDE, o,pV-DDE, age, and BMI were all once included in multiple comparison, *P b 0.05, **P b 0.01.
It is also possible that a mixture of estrogenic and antiandrogenic effects may affect different pathway of hormonal responses. Another possibility is that the association for o,pV-DDE may be a chance finding due to multiple comparison, since plasma o,pV-DDE in the present study was detected in only 24.7% of the study population and only 24 tests of association were therefore analysed. Moreover, no significant association was found when p,pV-DDE, o,pV-DDE, age, and BMI were all once included in multiple comparison. Levels of DDT in the body might be an important factor affecting hormonal status in men (Martin et al., 2002; Cocco et al., 2004). High environmental
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exposures to DDT might result in increased binding of DDT with human receptors, although binding of DDT is much less potent than endogenous hormones (Kelce et al., 1995; Chen et al., 1997; Matthews et al., 2000). Higher doses of DDT entering the body would increase bioaccumulation. Hence the levels of DDT at receptors would increase until the estrogen receptors reach a point of saturation with DDT binding (Eaton and Klassen, 1989). Average of the p,pV-isomer levels in North Carolina farmers, in Latvian and Swedish men, and in Italian men were approximately 310 times less than that in the present study, and they found no association with hormonal status (Table 6) (Hagmar et al., 2001; Martin et al., 2002; Cocco et al., 2004). On the other hand, average of the p,pV-isomers levels in adult men from Mexico and male workers from South Africa, where DDT is still used for malaria control, were approximately 1619 times higher than that in the present study (Ayotte et al., 2001; Dalvie et al., 2004a,b). The study in Mexican men found a negative association of plasma p,pV-DDE levels with the ratio of bioavailable to total testosterone, semen volumes, and sperm counts (Ayotte et al., 2001). The study in South African male workers found positive associations of p,pV-DDT and p,pVDDD levels with E2 and testosterone levels; however, they suggested that these associations might be due to chance, since p,pV-DDT and p,pV-DDD were not known to be a strongly anti-androgenic or estrogenic (Dalvie et al., 2004a,b). Therefore, it could be hypothesized that plasma levels of DDT in the present study might be not high enough to compete completely at the receptor level, or to affect hormonal metabolism and transport (Cocco, 2002). In addition,
Table 6 Mean or median levels of DDT and its metabolites expressed on a lipid basis and their association with reproductive hormones in men from different countries Authors
Study population
Ayotte et al. (2001) Dalvie et al. (2004a,b) Our study (2004) Martin, Jr. et al. (2002) Hagmar et al. (2001) Cocco et al. (2004)
Mexican men (n = 24) South African workers (n = 47) Thai adult men (n = 97) North Carolina farmers (n = 137) Latvian and Swedish men (n = 110) Italian men (n = 107)
a
Mean levels of DDE,
b
n/r = not reported.
Mean or median of DDT-related compounds, ng/g lipids p,pV-DDE
p,pV-DDT
77,900 65,000 4013 1213a 828 396
n/rb 26,100 628.7 n/rb 50 47
Association
Decreased testosterone Increased E2 and testosterone Increased E2 No association No associaton No association
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low levels of exposure in adult might be compensated by normal homeostatic mechanisms and therefore result in a small or an insignificant hormonal response.
5. Conclusion DDT residues detected in adult men in the present study were likely due to environmental and occupational exposures. The opposite direction of the association of plasma E2 with plasma p,pV-DDE and o,pV-DDE may reflect mechanistic differences in their reproductive hormonal activities. However, these associations were rather weak. Low levels of DDT exposure in the present study might be a factor limiting interpretation. Although low levels of DDT exposure in adults may produce a small or an insignificant hormonal response, exposure at the same levels during fetal development may result in a permanent change in the hormonal system. Our further research will be aimed at understanding hormonal effects of DDT exposure in neonates by transplacental transfer. Acknowledgement The study was financed by Royal Golden Jubilee Ph.D. program, the Thailand Research Fund (PHD/ 0108/2545). We are grateful to Dr. John McDermed for editing the manuscript, Assoc. Prof. Dr. Prasak Thavornyutikarn for advising, and all volunteer subjects for their cooperation. References Ayotte P, Giroux S, Dewailly E, Hernandez AM, Farias P, Danis R, et al. DDT spraying for malaria control and reproductive function in Mexican men. Epidemiology 2001;12:366 – 7. Bitman J, Cecil HC, Harris SJ, Fries GF. Estrogenic activity of o,pVDDT in the mammalian uterus and avian oviduct. Science 1968;162:371 – 2. Chareonviriyaphap T, Bangs MJ, Ratanatham S. Status of malaria in Thailand. Southeast Asian J Trop Med Public Health 2000;31:225 – 37. Chen CW, Hurd C, Vorojeikina DP, Arnold SF, Notides AC. Transcriptional activation of the human estrogen receptor by
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Plasma levels of DDT and their association with reproductive hormones in adult men from northern Thailand R. Asawasinsopona, T. Prapamontolb,T, O. Prakobvitayakitc, Y. Vaneesornd, A. Mangklabrukse, B. Hockf a
Environmental Science Ph.D. Program, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand b Pollution and Environmental Health Research Program, Research Institute for Health Sciences (RIHES), Chiang Mai University, P.O. Box 80 CMU, 50200 Chiang Mai, Thailand c Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand d Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand e Department of Internal Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand f Center of Life and Food Sciences Weihenstephan, Technical University of Munich, Alte Akademic12, 85350, Freising, Germany Received 13 December 2004; accepted 1 March 2005 Available online 17 June 2005
Abstract Historically, dichlorodiphenyltrichloroethane (DDT) was used in northern Thailand for malaria control and farming purposes. Several studies have investigated its effects on end points of adverse reproductive health outcomes. However, the few previous studies investigated hormonal effects in men and available data are inconclusive. The authors aimed to explore the main hypothesis that plasma DDT levels in adult men were associated with reproductive hormone levels. A cross-sectional study was performed of 97 adult men living in a highland village named Mae Sa Mai, 35 km north of Chiang Mai, Thailand. Venous blood samples were collected for measuring plasma levels of DDT and its metabolites and reproductive hormones, including 17h-estradiol (E2), testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH). 1,1-Dichloro-2,2di(4-chlorophenyl)ethylene (p,pV-DDE) and 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (p,pV-DDT) were detected in all plasma samples. p,pV-DDE had the highest level with a median of 4057.7 ng/g lipids and a relatively higher level compared with most other studies. Plasma p,pV-DDT levels were positively associated with years of residence (h + SE = 0.472 + 0.208, P = 0.028) and years of DDT usage for farming (h + SE = 0.177 + 0.084, P = 0.04). The remarkable findings were the negative association of plasma E2 levels with plasma p,pV-DDE levels (h + SE= 7.093 + 2.899, P = 0.016) and the positive association
T Corresponding author. Tel.: +66 53 221 465#235; fax: +66 53 221 849. E-mail addresses: [email protected], [email protected] (T. Prapamontol). 0048-9697/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2005.03.004
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with plasma 1,1-dichloro-2-(2-chlorophenyl)-2-(4-chlorophynyl)ethylene (o,pV-DDE) levels (h + SE = 16.381 + 5.596, P = 0.008) after adjusting for age and body mass index (BMI). However, these associations were rather weak. Our results suggest that these associations may reflect their different mechanisms of hormonal activities and they would be warrant further detail investigations. D 2005 Elsevier B.V. All rights reserved. Keywords: DDT; Persistent organic pollutants; Adult men; Endocrine disruption
1. Introduction In recent year, it has been shown that DDT and its metabolites are persistent organic pollutants and having endocrine disruption activities, acting as hormone receptor agonists and antagonists (Colborn et al., 1993; Kelce et al., 1997; Longnecker et al., 1997; Turusov et al., 2002). Because of their longterm adverse effects on living organisms, DDT which was of technical grade and contained mixed isomers has been banned in most developed countries since the 1970s. However, it is still used in some developing countries for essential public health purposes (Curtis and Lines, 2000; Turusov et al., 2002). Several studies in wildlife showed that exposure to DDT was linked to adverse reproductive effects in mammals, birds, amphibians, and fish. However, it was rather difficult to conclude particular causal agents were producing any effects (Colborn et al., 1993; Guillette et al., 1994, 1996; Vos et al., 2000). In epidemiological data on health effects in men, most studies used end points of adverse reproductive effects such as decreased sperm counts, reproductive tract abnormalities, infertilities, and reproductive cancers (Ayotte et al., 2001; Longnecker et al., 2002; Skakkebaek, 2002; Dalvie et al., 2004a,b). The few previous studies investigated effects of DDT on hormonal status in adult men, and available data are inconclusive. In fact, the hormonal actions of DDT could be expected to have some impact on the secretion of endocrine releasing factors, resulting in varying effects on reproductive hormonal metabolism. Further hormonal disturbance or imbalance could potentially occur in organs of the endocrine system (Jaga, 2000). In northern Thailand, and in Chiang Mai Province in particular, DDT was first introduced in 1949. Its use for malaria control was finally stopped altogether
in 1999, whereas the last spraying in Mae Sa Mai village which was the present study site was in 1989. Earlier, DDT was extensively used for farm activities in this village until it was banned for farming use in 1983, but most farmers illegally continued using in spite of its ban (Chareonviriyaphap et al., 2000; Stuetz et al., 2001). Stuetz et al. (2001) reported that total DDT levels in human milk of women from Mae Sa Mai village were still high with a median and a maximum level of 209 and 2012 ng/ml, respectively. In the present study, the authors aimed to assess whether plasma DDT levels in adult men residing in Mae Sa Mai village were associated with reproductive hormone levels.
2. Methods 2.1. Ethical approval The present study protocol was approved by the Human Experimentation Committee, Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand (Certificate of Ethical Clearance No.10/2003, 13 March 2003). 2.2. Study group, questionnaires, and anthropometric measurement A cross sectional study of 97 adult men, aged from 18 to 45 years old was conducted. All had resided inMae Sa Mai village in northern Thailand for at least 10 years. Written consent was obtained from individual subjects before collecting blood samples, interviewing for socio-demographic data, and measuring weight and height. Socio-demographic data were collected from individual participants by trained interviewers. Weight and height were also measured for BMI calculation.
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2.3. Blood sampling and storage Twelve millilliters of fasting venous blood samples were collected in heparin tubes (Vacutainer 366480, BD,USA). Plasma for DDT analysis was collected in glass tubes and frozen at 20 8C until analysis. Plasma for lipid and hormone analysis was collected in centrifuge tubes and frozen at 70 8C until analysis. 2.4. Analysis of DDT and its metabolites DDT and its metabolites included p,pV-DDE, p,pVDDT, 1,1-dichloro-2,2-di(4-chlorophenyl)ethane (p,pV-DDD), o,pV-DDE, and 1,1,1-trichloro-2-(2chlorophenyl)-2-(4-chlorophynyl)ethane (o,pV-DDT). All solvents for extraction were ultra resi-analysed grade for organic residue analysis (J.T.Baker, USA). The analytes were extracted by solid phase extraction using octadecyl (C18)-bonded silica cartridges (BondElut, Varian, USA). Extraction of these compounds from 2 ml plasma samples was performed according to the Prapamontol and Stevenson method (Prapamontol and Stevenson, 1991). The isooctane extracts were analysed for DDT and its metabolites using gas chromatography with electron capture detection (HP 5890 Series II equipped with a 63Ni electron capture dectector, an autosampler (HP7673), a fused silica capillary column (Ultra 2: 25 m 0.32 mm i.d. with 0.52 Am film thickness, J and W Scientific, USA), and computerized data handling system (HP3365 Series Chemstation). Recoveries of individual components of DDT ranged from 91.47% for o,pV-DDT to 108.87% for p,pV-DDT. The quantitation limit for individual compounds ranged from 0.5 ng/ml for p,pV-DDE to 2.63 ng/ml for p,pV-DDD. The intrabatch coefficient of variation (% CV) ranged from 4.3% for p,pV-DDT to 4.34 % for p,pV-DDE and interbatch % CV ranged from 3.4% for p,pV-DDE to 8.8% for p,pV-DDT. The determined mean levels for calculating CVs ranged from 0.99 ng/ml for o,pV-DDE to 15.07 ng/ml for p,pVDDE. 2.5. Lipid and reproductive hormone analysis Plasma triglycerides and total cholesterol levels were determined using an enzymatic method (Syn-
chron CX systems, Beckman, Germany). The intrabatch percent CV was 1.74% for triglycerides and 2.21% for cholesterol. The interbatch percent CV was 4.4% for triglycerides and 1.54 % for cholesterol. The determined mean levels for calculating CVs were 115 mg/dl for triglycerides and 158.4 mg/ dl for cholesterol. The following conversion formula was used for calculation of total lipids (Phillips et al., 1989): Plasmalipids; mg=dl ¼ 2:27ðtotalcholesterolÞ þ triglycerides þ 0:623 Plasma E2, testosterone, LH, and FSH were measured using chemiluminescent competitive immunoassay (Elecsys 1010, Roche, USA). The intrabatch percent CV ranged from 1.13% for LH to 3.1% for E2. The interbatch percent CV ranged from 10.56% for LH to 11.1% for E2. The determined mean levels for calculating CVs were 20.5 pg/ml for E2, 5 ng/ml for testosterone, 10.36 mIU/ml for LH, and 11.88 mIU/ml for FSH. 2.6. Statistical analysis SPSS version 11 was performed for data analysis. Descriptive parameters, including geometric mean, median, minimum (Min), maximum (Max), quartile (25th–75th percentile), and percentile were computed. The levels of DDT and its metabolites below the quantitation limit were not reported. DDT levels were adjusted for plasma lipids. To obtain the best linearity, natural logarithm transformation was applied for all variables before the parametric test. Pearson’s correlation coefficient was calculated for the association between plasma levels of DDT and its metabolites and reproductive hormones. Multiple linear regression analysis was conducted for the association of DDT and its metabolites with years of residence and DDT usage for farming. This regression analysis was also conducted for the associations of p,pV-DDE and o,pV-DDE with E2, adjusted for confounders including age and BMI. Beta (h) and standard error (SE) were calculated. Student’s t-test was used for testing differences in plasma DDT levels between persons who had used DDT for farming and those who had never used it.
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The level of significance was set at a P value b 0.05 (2-tailed).
Table 2 Multiple linear regression analysis for the association of DDT and its metabolites with years of residence and DDT usage for farming b
Variables
3. Results 3.1. Demographic characteristics of the study population The geometric mean age of 97 adult men was 29 years. All of them (100%) were farmers. The mean times of residence in this village and farm experience were 25.6 and 13 years, respectively. BMI ranged from 18.7 to 32.3 kg/m2 with a mean of 23 kg/m2. Sixty-two men (63.9%) had used DDT for farming, of whom 55 (88.7%) mixed DDT with plant seed before planting and 7 (11.3%) used it only for spraying. The mean duration of DDT usage for farming was 4 years (ranged from 1 to 20 years). By student’s t-test analysis, men who had used DDT for farming had higher mean plasma levels of p,pV-DDE, p,pV-DDT and o,pV-DDE than men who never used it ( P b 0.05). 3.2. The association of DDT and its metabolites with environmental and occupational exposures Plasma p,pV-DDE and p,pV-DDT were detected in all subjects of the study population. p,pV-DDE had the highest level with a median of 4057.7 ng/g lipids (Table 1). Plasma p,pV-DDE levels were positively associated with plasma p,pV-DDT levels (r = 0.675, P = 0.000). Table 2 shows the association of DDT and its metabolites with years of residence and DDT usage for farming, using multiple linear regression analysis. Plasma p,pV-DDT levels were positively associated with years of residence (h + SE = 0.472+0.208, P = 0.028) and years of DDT usage for farming (h + SE = 0.177 + 0.084, P = 0.04).
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SE
P
0.299 0.037
0.205 0.082
0.15 0.657
p,pV-DDT Years of residence Years of DDT usage for farming
0.472 0.177
0.208 0.084
0.028T 0.04T
p,pV-DDD Years of residence Years of DDT usage for farming
1.029 0.283
0.755 0.304
0.306 0.451
o,pV-DDE Years of residence Years of DDT usage for farming
0.199 0.223
0.464 0.187
0.678 0.263
o,pV-DDT Years of residence Years of DDT usage for farming
0.012 0.194
0.616 0.248
0.985 0.449
p,pV-DDE Years of residence Years of DDT usage for farming
T P b 0.05.
3.3. The association of DDT and its metabolites with reproductive hormones The median levels of plasma E2, testosterone, LH and FSH were 22.29 pg/ml, 4.67 ng/ml, 3.68 mIU/ml, and 5.53 mIU/ml, respectively. The value at 5th percentile of plasma E2, testosterone, and LH was below the reference value of the manufacturer, Roche, USA (Table 3). By Pearson’s correlation analysis, plasma E2 levels were negatively associated with plasma p,pV-DDE levels (r = 0.239, P = 0.018), but positively associated with plasma o,pV-DDE levels (r = 0.473, P = 0.02) (Table 4). These associations did not change after adjusting for age and BMI (h + SE = 7.093 + 2.899, P = 0.016 for the association between plasma E2 and p,pV-DDE and h + SE = 16.381 + 5.596, P = 0.008 for the association between
Table 1 Plasma levels of DDT and its metabolites adjusted for lipids (ng/g lipids) in 97 adult men DDT
Detected in samples, %
Geometric mean
Median
25th–75th percentile
Min–Max
p,pV-DDE p,pV-DDT p,pV-DDD o,pV-DDE o,pV-DDT
100 100 10.8 24.5 25.5
4013.0 628.7 390.5 75.8 97.8
4057.7 628.8 373.0 72.9 76.5
2989.5–5232.7 440.9–830.2 251.9–533.3 55.3–87.7 62.8–141.7
1325.1–12683.7 224.9–3085.1 211.7–1161.5 36.9–219.1 42.9–632.7
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Table 3 Plasma levels of reproductive hormones in 97 adult men Hormones
Min
E2, pg/ml Testosterone, ng/ml LH, mIU/ml FSH, mIU/ml a
Percentile
5.75 1.18 1.23 2.09
5th
25th
50th
75th
95th
8.72 2.18 1.55 3.27
15.38 3.45 2.59 4.18
22.29 4.67 3.68 5.53
28.23 5.8 5.02 8.57
42.21 7.99 7.02 13.88
Max
Reference Valuea (5th–9th)
114.1 15.0 12.9 46.55
11–43.9 2.8–8.0 1.7–8.6 1.5–12.4
Reference value from manufacturer, Roche, USA.
chain (Stuetz et al., 2001). In our data, p,pV-DDT was associated with years of residence and DDT usage for farming. In addition, men who had reportedly ever used DDT for farming had higher mean plasma levels of p,pV-DDE, p,pV-DDT and o,pV-DDE than men who never used it ( P b 0.05). It would seem that durations of environmental exposure and DDT usage for farming in the past were determinants of DDT levels. Our results therefore suggest that DDT residues in these group men were due to both environmental and occupational exposures. With regard to reproductive hormones, the values at the 5th percentile of plasma E2, testosterone, and LH were below the reference values of the manufacturer. Therefore, these reference values may be inappropriate for Thai populations. Therefore the most remarkable findings were the negative association of plasma E2 levels with plasma p,pV-DDE levels and the positive association with plasma o,pV-DDE levels, adjusted for age and BMI. The opposite direction of the associations for p,pVDDE and o,pV-DDE may reflect their different mechanisms of hormonal activities, since the p,pV isomers act as androgen antagonists whereas the o,pV isomers act as estrogen agonists (Bitman et al., 1968; Nelson, 1974; Kelce et al., 1995; Sohoni and Sumpter, 1998). However, these associations were rather weak.
plasma E2 and o,pV-DDE). No significant association was found when p,pV-DDE, o,pV-DDE, age, and BMI were all once included in multiple comparison (Table 5). Considering the association of DDT and its metabolites expressed on a fresh weight basis, only plasma E2 levels were negatively associated with plasma p,pV-DDE levels (data not shown).
4. Discussion The p,pV-isomers (i.e. p,pV-DDE and p,pV-DDT) were detected in all study subjects and the o,pVisomers (i.e. o,pV-DDE and o,pV-DDT) were also detected in a quarter of the study population. In general, the p,pV-isomers were present at much higher levels than o,pV-isomers, because technical grade DDT contains approximately 80% of p,pV-DDT and 20% of o,pV-DDT. The p,pV-isomers also have long half lives, which partly accounts for their higher levels in people. This population might have been exposed to these compounds by a continuing low level intake, presumably dietary, or an extremely high cumulative exposure in the years preceding the ban. DDT was applied for malaria control and farming purposes for 40 years in the study village. Data were collected, DDT and its metabolites are still detected in the food
Table 4 Pearson’s correlation coefficients (r) between plasma levels of DDT and its metabolites and reproductive hormones Variables p,pV-DDE p,pV-DDT p,pV-DDD o,pV-DDE o,pV-DDT T P b 0.05.
E2
Testosterone
LH
FSH
r
P
r
P
r
P
r
P
0.239 0.196 0.371 0.473 0.012
0.018T 0.054 0.291 0.02T 0.953
0.176 0.142 0.185 0.333 0.161
0.085 0.165 0.608 0.112 0.442
0.129 0.153 0.203 0.402 0.081
0.208 0.134 0.547 0.052 0.702
0.122 0.073 0.37 0.181 0.045
0.235 0.48 0.293 0.398 0.831
R. Asawasinsopon et al. / Science of the Total Environment 355 (2006) 98–105 Table 5 Multiple linear regression analysis for the association of plasma E2 with plasma p,pV-DDE and o,pV-DDE after adjusting for age and BMI Variables Model 1: E2 p,pV-DDE Age BMI E2 o,pV-DDE Age BMI Model 2 a: E2 p,pV-DDE o,pV-DDE Age BMI
b
SE
P
7.093 4.773 15.917
2.899 5.676 11.743
0.016* 0.403 0.179
16.381 4.043 36.110
5.596 10.566 23.061
0.008** 0.706 0.133
4.410 8.467 8.526 2.338
4.398 4.240 7.898 18.694
0.329 0.055 0.294 0.902
a
p,pV-DDE, o,pV-DDE, age, and BMI were all once included in multiple comparison, *P b 0.05, **P b 0.01.
It is also possible that a mixture of estrogenic and antiandrogenic effects may affect different pathway of hormonal responses. Another possibility is that the association for o,pV-DDE may be a chance finding due to multiple comparison, since plasma o,pV-DDE in the present study was detected in only 24.7% of the study population and only 24 tests of association were therefore analysed. Moreover, no significant association was found when p,pV-DDE, o,pV-DDE, age, and BMI were all once included in multiple comparison. Levels of DDT in the body might be an important factor affecting hormonal status in men (Martin et al., 2002; Cocco et al., 2004). High environmental
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exposures to DDT might result in increased binding of DDT with human receptors, although binding of DDT is much less potent than endogenous hormones (Kelce et al., 1995; Chen et al., 1997; Matthews et al., 2000). Higher doses of DDT entering the body would increase bioaccumulation. Hence the levels of DDT at receptors would increase until the estrogen receptors reach a point of saturation with DDT binding (Eaton and Klassen, 1989). Average of the p,pV-isomer levels in North Carolina farmers, in Latvian and Swedish men, and in Italian men were approximately 310 times less than that in the present study, and they found no association with hormonal status (Table 6) (Hagmar et al., 2001; Martin et al., 2002; Cocco et al., 2004). On the other hand, average of the p,pV-isomers levels in adult men from Mexico and male workers from South Africa, where DDT is still used for malaria control, were approximately 1619 times higher than that in the present study (Ayotte et al., 2001; Dalvie et al., 2004a,b). The study in Mexican men found a negative association of plasma p,pV-DDE levels with the ratio of bioavailable to total testosterone, semen volumes, and sperm counts (Ayotte et al., 2001). The study in South African male workers found positive associations of p,pV-DDT and p,pVDDD levels with E2 and testosterone levels; however, they suggested that these associations might be due to chance, since p,pV-DDT and p,pV-DDD were not known to be a strongly anti-androgenic or estrogenic (Dalvie et al., 2004a,b). Therefore, it could be hypothesized that plasma levels of DDT in the present study might be not high enough to compete completely at the receptor level, or to affect hormonal metabolism and transport (Cocco, 2002). In addition,
Table 6 Mean or median levels of DDT and its metabolites expressed on a lipid basis and their association with reproductive hormones in men from different countries Authors
Study population
Ayotte et al. (2001) Dalvie et al. (2004a,b) Our study (2004) Martin, Jr. et al. (2002) Hagmar et al. (2001) Cocco et al. (2004)
Mexican men (n = 24) South African workers (n = 47) Thai adult men (n = 97) North Carolina farmers (n = 137) Latvian and Swedish men (n = 110) Italian men (n = 107)
a
Mean levels of DDE,
b
n/r = not reported.
Mean or median of DDT-related compounds, ng/g lipids p,pV-DDE
p,pV-DDT
77,900 65,000 4013 1213a 828 396
n/rb 26,100 628.7 n/rb 50 47
Association
Decreased testosterone Increased E2 and testosterone Increased E2 No association No associaton No association
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low levels of exposure in adult might be compensated by normal homeostatic mechanisms and therefore result in a small or an insignificant hormonal response.
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