This article was downloaded by: [North Dakota State University] On: 19 November 2014, At: 11:41 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Toxicology and Environmental Health, Part A: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh20
Residues of Selected Polychlorinated Biphenyls (PCB) and Organochlorine Pesticides (OCP) in Postmortem Lungs From Epirus, Northwestern Greece a
a
b
a
Georgios N. Rallis , Vassiliki A. Boumba , Vasilios A. Sakkas , Kleio Fragkouli , Georgios a
b
a
Siozios , Triantafyllos A. Albanis & Theodore Vougiouklakis a
Laboratory of Forensic Medicine & Toxicology, Department of Medicine, School of Health Sciences, University of Ioannina, Ioannina, Greece b
Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece Published online: 19 May 2014.
To cite this article: Georgios N. Rallis, Vassiliki A. Boumba, Vasilios A. Sakkas, Kleio Fragkouli, Georgios Siozios, Triantafyllos A. Albanis & Theodore Vougiouklakis (2014) Residues of Selected Polychlorinated Biphenyls (PCB) and Organochlorine Pesticides (OCP) in Postmortem Lungs From Epirus, Northwestern Greece, Journal of Toxicology and Environmental Health, Part A: Current Issues, 77:13, 767-775, DOI: 10.1080/15287394.2014.892447 To link to this article: http://dx.doi.org/10.1080/15287394.2014.892447
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Toxicology and Environmental Health, Part A, 77:767–775, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287394.2014.892447
RESIDUES OF SELECTED POLYCHLORINATED BIPHENYLS (PCB) AND ORGANOCHLORINE PESTICIDES (OCP) IN POSTMORTEM LUNGS FROM EPIRUS, NORTHWESTERN GREECE Georgios N. Rallis1, Vassiliki A. Boumba1, Vasilios A. Sakkas2, Kleio Fragkouli1, Georgios Siozios1, Triantafyllos A. Albanis2, Theodore Vougiouklakis1
Downloaded by [North Dakota State University] at 11:41 19 November 2014
1
Laboratory of Forensic Medicine & Toxicology, Department of Medicine, School of Health Sciences, University of Ioannina, Ioannina, Greece 2 Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece Organochlorine (OC) pesticides and polychlorinated biphenyls (PCB) are compounds characterized as persistent organic pollutants (POP) in the environment. These compounds are monitored globally since they enter the human body and accumulate in tissues, resulting in consequent adverse effects. In this study concentrations of selected OC compounds were determined in human autopsy lungs from Epirus, a relatively restricted region in northwestern Greece. This is the first epidemiologic study from Greece reporting on monitoring of environmental pollutants in human autopsy material. Thirty lungs collected from that number of autopsy cases were analyzed: 19 males and 11 females. The age range was 14–91 yr (mean ± SD = 61.8 ± 22.5 yr). Twenty-two cases (73%) were positive for at least one pollutant and eight cases were negative (27%). PCB were the most abundant class of contaminants, detected in 15 out of the 30 cases (50%). Dichlorodiphenyltrichloroethane (DDT) and hexachlorocyclohexanes (HCH) were second and third in abundance with 9 (30%) and 8 (27%) positive cases, respectively. The frequency of detection showed a tendency to increase with age of individuals. The patterns of OC found in human autopsy lungs were similar to those reported previously for other human specimens. Our results demonstrated a similar trend in contamination sources and distribution has occurred in western Greece as noted globally.
atmospheric transport (Lucena et al., 2007; U.S. EPA, 2013). Although industrialized nations have restricted or banned many OC pesticides, some of these chemicals such as endosulfans continue to be manufactured and used (Lucena et al., 2007). OC are lipophilic in nature, potentially toxic for organisms, and enter food chains, accumulating in considerable levels in top predators, particularly in the fatty tissues (Lucena et al., 2007; Yu et al., 2010). Humans may be exposed to OC through diet, occupational activity, or environmental exposure (Kumar, 2011).
Organochlorine (OC) compounds are a major and diverse group of synthetic chemicals. Among the numerous OC that have been produced and used worldwide, the most widespread are OC pesticides, such as dichlorodiphenyltrichloroethane (DDT), hexachlorocyclohexanes (HCH), and polychlorinated biphenyls (PCB), which are produced as mixtures. OC are considered to be persistent organic pollutants (POP) in the environment due to (1) chemical stability and resistance to environmental degradation, (2) ability to bioaccumulate and biomagnify in food chains, and (3) capacity for long-range
Received 4 February 2014; accepted 5 February 2014. Address correspondence to Vassiliki A. Boumba, Assistant Professor, BSc, PhD, Laboratory of Forensic Medicine & Toxicology, Department of Medicine, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece. E-mail: [email protected] 767
Downloaded by [North Dakota State University] at 11:41 19 November 2014
768
Human health effects after exposure to OC are not adequately understood, although it is well established that these substances exert detrimental effects on both humans and wildlife. Generally, chronic exposure to low levels of various POP adversely affects humans and disturbs the functions of different organs including nervous, endocrine, immune, reproductive, renal, cardiovascular, and respiratory systems. In this regard, there is mounting evidence on the link of pesticide exposure with incidence of chronic diseases, including cancer, Parkinson’s, Alzheimer’s, multiple sclerosis, diabetes, aging, cardiovascular, and kidney disease (Li et al., 2006a; Ropstad et al., 2006; Mostafalou and Abdollahi, 2013). Serum, breast milk, hair, and adipose tissue are the main human specimens that have been analyzed to demonstrate exposure to OC (Lucena et al., 2007). The OC levels in adipose tissue have been preferentially used as indicative of chronic human exposure, while levels in hair and serum are considered indicative of relative recent exposure. OC levels in tissues from living individuals, besides serum, breast milk, and hair, that could be obtained only by invasive procedures or during surgery (Esteban and Castano, 2009) have been reported in limited studies. It is worthwhile noting that most relevant studies performed on autopsy specimens (Williams et al., 1984; Mussalo-Rauhamma, 1991; Kemper, 1993; Galleli et al., 1995; Weistrand and Noren, 1998; Ben-Michael et al., 1999; Dewailly et al., 1999; Corrigan et al., 2000; Chu et al., 2003; Waliszewski et al., 2004), and levels of pollutants detected in the different tissues may be considered indicative of accumulation of compounds in humans. As regards Greece, the commercial disposal of PCB and other OC was banned before the mid-1970s. Nonetheless, these pollutants persist in the Greek environment and food (Golfinopoulos et al., 2003; Konstantinou et al., 2004; Sakellarides et al., 2006 Chrysikou et al., 2008; Christoforidis et al., 2008). Biomonitoring Greek studies reported OC levels in human serum (Costopoulou et al., 2006; Kalantzi et al., 2011), milk (Costopoulou et al.,
G. N. RALLIS ET AL.
2006), and hair (Tsatsakis et al., 2008a, 2008b) from residents of the central and southern areas of the country, respectively. This study aimed to determine the levels of selected OC pesticides and PCB in human lungs collected during autopsy from routinely autopsied bodies of residents of the region of Epirus, in northwestern Greece. This is the first study from Greece on the possible bioaccumulation of OC in humans performed with autopsy material.
MATERIALS AND METHODS Autopsy Material Postmortem lung tissue weighing about 10 g was sampled during autopsy. Tissues were divided in portions of 1 g, placed in plastic bags, and stored frozen at –25◦ C until analysis. The cases included in the study were subjected to autopsy, routinely, in a period that was estimated to be 10–18 h from death. However, some deceased bodies were kept occasionally at 4◦ C from identification of the body until autopsy.
Sample Preparation Sample preparation and gas chromatography with electron capture detector (GCECD) determination of analytes was performed according to the analytical procedure developed and validated previously (Rallis et al., 2012). Briefly, a portion of lung tissue of 0.5 g was placed in a mortar. The sample was blended with 2 g Florisil (sorbent) and 2 g Na2 SO4 (dehydrating agent). The mixture was transferred into a column containing a 0.22-μm membrane filter and 1.54 g of Florisil (co-column). A portion of glass wool was placed at the top of the column; then 15.45 mL n-hexane:dichloromethane (11:89, v/v) was added as elution solvent. The eluent was collected, evaporated, and re-dissolved with 100 μl isooctane containing PCB 118 (I.S.). A 1.5 μl extract was injected into the GC-ECD.
ORGANOCHLORINE COMPOUNDS IN AUTOPSY LUNGS
Downloaded by [North Dakota State University] at 11:41 19 November 2014
GC-ECD Conditions Separation of analytes was achieved on a DB-5 column, 30 m × 0.25 mm ID, containing 5% phenylmethylpolysiloxane with a phase thickness of 0.25 μm. GC conditions were as follows: injector at 250◦ C in splitless mode; helium at 1.5 mL/min as carrier gas; nitrogen at 35 mL/min as makeup gas; temperature program: 150◦ C for 2 min; inceased to 180◦ C, rate 2◦ C/min; 184◦ C, rate 0.5 ◦ C/min, hold 2 min; 200◦ C, rate 2◦ C/min, hold 200 min; 270◦ C, rate 20◦ C/min, hold 2 min. The detector was an 63 Ni ECD operated at 300◦ C. Statistical Analysis For statistical significance, nonparametric methods were performed using the program Winstat 9.01 version (R. Fitch Software, Chicago, IL). Spearman correlation analyses were applied for relationships between different OC levels and other variables. A p value < .05 was considered significant. RESULTS AND DISCUSSION Classification of Analytes and Cases The selection of monitored analytes was based upon their abundance in the environment and extensive use in Europe. In particular, PCB congeners monitored in this study were those usually monitored to assess environmental exposure in Europe (termed “ICES7” or “indicator PCBs”), which were found to be correlated with congeners of toxicological relevance, and encompass a wide range of chlorination degrees (tri- to heptachlorination) and boiling points. Analytes were classified into six chemical classes as follows: chlordanes (γ-chlordane), DDT (p,p’-DDT, p,p’-DDE, p,p’DDD), endosulfans (endosulfan I, ensosulfan sulfate), endrins (endrin ketone), HCH (β-, γ-isomers), and PCB (138-,153-, and 180congeners). Lung tissues were collected during autopsy from 30 routinely autopsied deceased subjects, of both genders and different ages, during the period 2010–2012. Autopsies were performed
769
in the Laboratory of Forensic Medicine and Toxicology, University of Ioannina. The lab is legally authorized to perform all medico-legal autopsies and investigate all obvious or suspected unnatural deaths in the four prefectures of the Region of Epirus. All cases included in the study were residents of the region of Epirus. Pesticide or drug poisoning cases (Boumba et al., 2012), drowning deaths, and cases presented with obvious signs of putrefaction at autopsy were excluded from the study. Thirty lungs collected from respective number of autopsy cases were analyzed: 19 males and 11 females. In Table 1 are presented the age, gender, and area distribution of the deceased. According to the age of the deceased, cases were divided in three groups: group I (6 cases) including deceased aged up to 45 yr; group II (7 cases) including deceased aged 46–65 yr; and group III (17 cases), including deceased aged more than 66 yr. As regards the area distribution, 17 cases were from urban or semi-urban areas and 13 cases from rural areas. Patterns of OC in Human Lungs The frequencies of detection of each class of analytes in each age group are presented in Figure 1. Twenty-two cases (22/30, 73%) were found positive for at least one pollutant. In age group I, two out of the six total cases (33%) were positive for OC pesticides residues (HCH were detected in 2/2 positive cases and DDT in one case). Within age group II, five out of the seven total cases (71%) were positive for PCB or OC pesticides. PCB were the most abundant pollutants among the positive cases in the age group II (4/5 positive cases, 80%). Within age group III, 15 out of the 17 total cases (88%) were found positive. PCB were the most abundant pollutants detected among the positive cases in the older age group III (11/15, 73%), followed by DDT (7/15, 47%) and HCH (5/15, 33%). Overall, PCB were the most abundant contaminants quantified in 15/30 cases (50%). All other OC had lower detection frequencies, ranging from 30% (9/30 cases) and 27%
770
G. N. RALLIS ET AL.
TABLE 1. Characteristics of Autopsy Cases Age range
Area distribution
Gender (n)
Age range, yr
Mean age, yr ± SD
Group I (≤45 yr)
Group II (46–65 yr)
Group III (≥66 yr)
Urban/ semiurban
Rural
Males (19) Females (11) Total (30)
14–84 21–91 14–91
57.5 ± 23.0 69.1 ± 20.8 61.8 ± 22.5
5 1 6
5 2 7
9 8 17
9 8 17
10 3 13
Detection frequencies of OCs per age group 15
14 12
11 Chlordanes
No of Cases
Downloaded by [North Dakota State University] at 11:41 19 November 2014
16
10
DDTs Endosulfans Endrins
8
HCHs
7
PCBs Positives
6
5 4
4 2
2 0
0
4 2 1 1
0
0
0
< 46 y (Group I)
1 0
Negatives
4
2
1
5
2
2
1
0
46–65 y (Group II)
> 65 y (Group III)
Age Range, y FIGURE 1. Detection frequencies of OC per age group.
(8/30 cases) for DDT and HCH, respectively, to 13% (4/30 cases) and 6.7% (2/30 cases) for endosulfans, endrins, and chlordanes respectively. Lungs from six cases contained at least three different classes of pollutants, where the combination of PCB, DDT, and HCH was the most abundant profile, observed in 5/6 cases (not shown). Frequencies of detection of each specific analyte in each one of the three age groups are illustrated in Figure 2. Higher frequencies of detection were observed for PCB 138 (10/30, 33%) and PCB 153 (9/30, 30%), p,p’DDE (9/30, 30%), and γ-HCH (8/30, 27%). The distribution of pollutants did not demonstrate significant differences between males and females or between different residential areas
(urban/semi-urban/rural areas). This observation may be attributed to a greater rural area in Epirus, where 33% of inhabitants reside in an urban/semi-urban environment while the rest, 77%, live in rural areas. In addition, there are no large urban centers in the region and the vast majority of the population carries out to some degree agricultural practices regardless of their residential area. Levels of OC in Human Lungs The concentration ranges of each analyte, their contribution to the total burden of the respective class of analytes (% range), and the numbers of positive cases and the age ranges of individuals are presented in Table 2. The compounds p,p’-DDT, p,p’-DDE, γ-HCH, PCB 153,
ORGANOCHLORINE COMPOUNDS IN AUTOPSY LUNGS
771
Frequency of each analyte per age group PCB 180 PCB 138 PCB 153 γ-HCH β-HCH < 46 y
Endrin ketone
46–65 y
Endos. sulfate
> 65 y
Endosulfan I p,p´-DDE p,p´-DDD
Downloaded by [North Dakota State University] at 11:41 19 November 2014
p,p´-DDT γ-Chlordane 0
2
4
6 8 No of cases
10
12
FIGURE 2. Frequency of each analyte per age group.
PCB138, and PCB180 were the main pollutants detected. Our results demonstrated that certain OC accumulated in human lungs of residents of Epirus as a result of their persistence in northwestern and other areas of Greece (Golfinopoulos et al., 2003; Konstantinou et al., 2006; Sakellarides et al., 2006 Chrysikou et al., 2008; Christoforidis et al., 2008). The patterns of OC found in lungs were similar to those reported in previous studies for other human specimens from Greece (Costopoulou et al., 2006; Kalantzi et al., 2011; Tsatsakis et al., 2008a, 2008b) and other European countries
(Lucena et al., 2007). Nevertheless, the levels reported herein may not be compared directly to those observed previously, because the latter were expressed as weight of pollutant per lipid content of the examined specimen. The main contributor to DDTs was p,p’DDE in five cases and p,p’-DDT in two cases. The metabolite p,p’-DDD was the minor contributor to DDT in the two cases where it was detected. p,p’-DDT was detected simultaneously with p,p’-DDE and the concentrations of p,p’-DDT and p,p’-DDE were significantly correlated. It is known that once inside the body, p,p’-DDT is degraded to p,p’-DDE or p,p’DDD. The compound p,p’-DDD may be further metabolized, while p,p’-DDE shows high bioaccumulation potential with a reported half life up to 15 yr (U.S. Environmental Protection Agency [EPA], 2013). Therefore, p,p’-DDE is usually the predominant variant of DDT in humans (Jaga and Dharmani, 2003), as was the case here. In consideration of the following facts, (i) commercial DDT product was banned in Greece since the mid-1970s, (ii) p,p’-DDT and its metabolite p,p’-DDE are highly persistent in the Greek environment and living organisms (Sakellarides et al., 2006; Chrysikou et al., 2008; Christoforidis et al., 2008), (iii) the possible transfer of DDT through rivers and aquatic biota from neighboring countries
TABLE 2. Concentrations of Analytes (Ranges, Means, Percent Range), Number of Positive Cases for Each Analyte, and the Age of Individuals Where Each Analyte Was Detected Class of analytes DDTs
HCHs
Endosulfans
Chlordanes Endrins PCBs
Analytes
C range, ng/g
Mean C ± SD, ng/g
Percent C range
Positive cases, N
Age range, yr
p,p -DDT p,p -DDD p,p -DDE DDTs β-HCH γ-HCH HCHs Endosulfan I Endos. sulfate Endosulfans γ-Chlordane Endrin ketone PCB 138 PCB 153 PCB 180 PCBs
2.15–4.89 1.42–1.46
Journal of Toxicology and Environmental Health, Part A: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh20
Residues of Selected Polychlorinated Biphenyls (PCB) and Organochlorine Pesticides (OCP) in Postmortem Lungs From Epirus, Northwestern Greece a
a
b
a
Georgios N. Rallis , Vassiliki A. Boumba , Vasilios A. Sakkas , Kleio Fragkouli , Georgios a
b
a
Siozios , Triantafyllos A. Albanis & Theodore Vougiouklakis a
Laboratory of Forensic Medicine & Toxicology, Department of Medicine, School of Health Sciences, University of Ioannina, Ioannina, Greece b
Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece Published online: 19 May 2014.
To cite this article: Georgios N. Rallis, Vassiliki A. Boumba, Vasilios A. Sakkas, Kleio Fragkouli, Georgios Siozios, Triantafyllos A. Albanis & Theodore Vougiouklakis (2014) Residues of Selected Polychlorinated Biphenyls (PCB) and Organochlorine Pesticides (OCP) in Postmortem Lungs From Epirus, Northwestern Greece, Journal of Toxicology and Environmental Health, Part A: Current Issues, 77:13, 767-775, DOI: 10.1080/15287394.2014.892447 To link to this article: http://dx.doi.org/10.1080/15287394.2014.892447
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Toxicology and Environmental Health, Part A, 77:767–775, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287394.2014.892447
RESIDUES OF SELECTED POLYCHLORINATED BIPHENYLS (PCB) AND ORGANOCHLORINE PESTICIDES (OCP) IN POSTMORTEM LUNGS FROM EPIRUS, NORTHWESTERN GREECE Georgios N. Rallis1, Vassiliki A. Boumba1, Vasilios A. Sakkas2, Kleio Fragkouli1, Georgios Siozios1, Triantafyllos A. Albanis2, Theodore Vougiouklakis1
Downloaded by [North Dakota State University] at 11:41 19 November 2014
1
Laboratory of Forensic Medicine & Toxicology, Department of Medicine, School of Health Sciences, University of Ioannina, Ioannina, Greece 2 Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece Organochlorine (OC) pesticides and polychlorinated biphenyls (PCB) are compounds characterized as persistent organic pollutants (POP) in the environment. These compounds are monitored globally since they enter the human body and accumulate in tissues, resulting in consequent adverse effects. In this study concentrations of selected OC compounds were determined in human autopsy lungs from Epirus, a relatively restricted region in northwestern Greece. This is the first epidemiologic study from Greece reporting on monitoring of environmental pollutants in human autopsy material. Thirty lungs collected from that number of autopsy cases were analyzed: 19 males and 11 females. The age range was 14–91 yr (mean ± SD = 61.8 ± 22.5 yr). Twenty-two cases (73%) were positive for at least one pollutant and eight cases were negative (27%). PCB were the most abundant class of contaminants, detected in 15 out of the 30 cases (50%). Dichlorodiphenyltrichloroethane (DDT) and hexachlorocyclohexanes (HCH) were second and third in abundance with 9 (30%) and 8 (27%) positive cases, respectively. The frequency of detection showed a tendency to increase with age of individuals. The patterns of OC found in human autopsy lungs were similar to those reported previously for other human specimens. Our results demonstrated a similar trend in contamination sources and distribution has occurred in western Greece as noted globally.
atmospheric transport (Lucena et al., 2007; U.S. EPA, 2013). Although industrialized nations have restricted or banned many OC pesticides, some of these chemicals such as endosulfans continue to be manufactured and used (Lucena et al., 2007). OC are lipophilic in nature, potentially toxic for organisms, and enter food chains, accumulating in considerable levels in top predators, particularly in the fatty tissues (Lucena et al., 2007; Yu et al., 2010). Humans may be exposed to OC through diet, occupational activity, or environmental exposure (Kumar, 2011).
Organochlorine (OC) compounds are a major and diverse group of synthetic chemicals. Among the numerous OC that have been produced and used worldwide, the most widespread are OC pesticides, such as dichlorodiphenyltrichloroethane (DDT), hexachlorocyclohexanes (HCH), and polychlorinated biphenyls (PCB), which are produced as mixtures. OC are considered to be persistent organic pollutants (POP) in the environment due to (1) chemical stability and resistance to environmental degradation, (2) ability to bioaccumulate and biomagnify in food chains, and (3) capacity for long-range
Received 4 February 2014; accepted 5 February 2014. Address correspondence to Vassiliki A. Boumba, Assistant Professor, BSc, PhD, Laboratory of Forensic Medicine & Toxicology, Department of Medicine, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece. E-mail: [email protected] 767
Downloaded by [North Dakota State University] at 11:41 19 November 2014
768
Human health effects after exposure to OC are not adequately understood, although it is well established that these substances exert detrimental effects on both humans and wildlife. Generally, chronic exposure to low levels of various POP adversely affects humans and disturbs the functions of different organs including nervous, endocrine, immune, reproductive, renal, cardiovascular, and respiratory systems. In this regard, there is mounting evidence on the link of pesticide exposure with incidence of chronic diseases, including cancer, Parkinson’s, Alzheimer’s, multiple sclerosis, diabetes, aging, cardiovascular, and kidney disease (Li et al., 2006a; Ropstad et al., 2006; Mostafalou and Abdollahi, 2013). Serum, breast milk, hair, and adipose tissue are the main human specimens that have been analyzed to demonstrate exposure to OC (Lucena et al., 2007). The OC levels in adipose tissue have been preferentially used as indicative of chronic human exposure, while levels in hair and serum are considered indicative of relative recent exposure. OC levels in tissues from living individuals, besides serum, breast milk, and hair, that could be obtained only by invasive procedures or during surgery (Esteban and Castano, 2009) have been reported in limited studies. It is worthwhile noting that most relevant studies performed on autopsy specimens (Williams et al., 1984; Mussalo-Rauhamma, 1991; Kemper, 1993; Galleli et al., 1995; Weistrand and Noren, 1998; Ben-Michael et al., 1999; Dewailly et al., 1999; Corrigan et al., 2000; Chu et al., 2003; Waliszewski et al., 2004), and levels of pollutants detected in the different tissues may be considered indicative of accumulation of compounds in humans. As regards Greece, the commercial disposal of PCB and other OC was banned before the mid-1970s. Nonetheless, these pollutants persist in the Greek environment and food (Golfinopoulos et al., 2003; Konstantinou et al., 2004; Sakellarides et al., 2006 Chrysikou et al., 2008; Christoforidis et al., 2008). Biomonitoring Greek studies reported OC levels in human serum (Costopoulou et al., 2006; Kalantzi et al., 2011), milk (Costopoulou et al.,
G. N. RALLIS ET AL.
2006), and hair (Tsatsakis et al., 2008a, 2008b) from residents of the central and southern areas of the country, respectively. This study aimed to determine the levels of selected OC pesticides and PCB in human lungs collected during autopsy from routinely autopsied bodies of residents of the region of Epirus, in northwestern Greece. This is the first study from Greece on the possible bioaccumulation of OC in humans performed with autopsy material.
MATERIALS AND METHODS Autopsy Material Postmortem lung tissue weighing about 10 g was sampled during autopsy. Tissues were divided in portions of 1 g, placed in plastic bags, and stored frozen at –25◦ C until analysis. The cases included in the study were subjected to autopsy, routinely, in a period that was estimated to be 10–18 h from death. However, some deceased bodies were kept occasionally at 4◦ C from identification of the body until autopsy.
Sample Preparation Sample preparation and gas chromatography with electron capture detector (GCECD) determination of analytes was performed according to the analytical procedure developed and validated previously (Rallis et al., 2012). Briefly, a portion of lung tissue of 0.5 g was placed in a mortar. The sample was blended with 2 g Florisil (sorbent) and 2 g Na2 SO4 (dehydrating agent). The mixture was transferred into a column containing a 0.22-μm membrane filter and 1.54 g of Florisil (co-column). A portion of glass wool was placed at the top of the column; then 15.45 mL n-hexane:dichloromethane (11:89, v/v) was added as elution solvent. The eluent was collected, evaporated, and re-dissolved with 100 μl isooctane containing PCB 118 (I.S.). A 1.5 μl extract was injected into the GC-ECD.
ORGANOCHLORINE COMPOUNDS IN AUTOPSY LUNGS
Downloaded by [North Dakota State University] at 11:41 19 November 2014
GC-ECD Conditions Separation of analytes was achieved on a DB-5 column, 30 m × 0.25 mm ID, containing 5% phenylmethylpolysiloxane with a phase thickness of 0.25 μm. GC conditions were as follows: injector at 250◦ C in splitless mode; helium at 1.5 mL/min as carrier gas; nitrogen at 35 mL/min as makeup gas; temperature program: 150◦ C for 2 min; inceased to 180◦ C, rate 2◦ C/min; 184◦ C, rate 0.5 ◦ C/min, hold 2 min; 200◦ C, rate 2◦ C/min, hold 200 min; 270◦ C, rate 20◦ C/min, hold 2 min. The detector was an 63 Ni ECD operated at 300◦ C. Statistical Analysis For statistical significance, nonparametric methods were performed using the program Winstat 9.01 version (R. Fitch Software, Chicago, IL). Spearman correlation analyses were applied for relationships between different OC levels and other variables. A p value < .05 was considered significant. RESULTS AND DISCUSSION Classification of Analytes and Cases The selection of monitored analytes was based upon their abundance in the environment and extensive use in Europe. In particular, PCB congeners monitored in this study were those usually monitored to assess environmental exposure in Europe (termed “ICES7” or “indicator PCBs”), which were found to be correlated with congeners of toxicological relevance, and encompass a wide range of chlorination degrees (tri- to heptachlorination) and boiling points. Analytes were classified into six chemical classes as follows: chlordanes (γ-chlordane), DDT (p,p’-DDT, p,p’-DDE, p,p’DDD), endosulfans (endosulfan I, ensosulfan sulfate), endrins (endrin ketone), HCH (β-, γ-isomers), and PCB (138-,153-, and 180congeners). Lung tissues were collected during autopsy from 30 routinely autopsied deceased subjects, of both genders and different ages, during the period 2010–2012. Autopsies were performed
769
in the Laboratory of Forensic Medicine and Toxicology, University of Ioannina. The lab is legally authorized to perform all medico-legal autopsies and investigate all obvious or suspected unnatural deaths in the four prefectures of the Region of Epirus. All cases included in the study were residents of the region of Epirus. Pesticide or drug poisoning cases (Boumba et al., 2012), drowning deaths, and cases presented with obvious signs of putrefaction at autopsy were excluded from the study. Thirty lungs collected from respective number of autopsy cases were analyzed: 19 males and 11 females. In Table 1 are presented the age, gender, and area distribution of the deceased. According to the age of the deceased, cases were divided in three groups: group I (6 cases) including deceased aged up to 45 yr; group II (7 cases) including deceased aged 46–65 yr; and group III (17 cases), including deceased aged more than 66 yr. As regards the area distribution, 17 cases were from urban or semi-urban areas and 13 cases from rural areas. Patterns of OC in Human Lungs The frequencies of detection of each class of analytes in each age group are presented in Figure 1. Twenty-two cases (22/30, 73%) were found positive for at least one pollutant. In age group I, two out of the six total cases (33%) were positive for OC pesticides residues (HCH were detected in 2/2 positive cases and DDT in one case). Within age group II, five out of the seven total cases (71%) were positive for PCB or OC pesticides. PCB were the most abundant pollutants among the positive cases in the age group II (4/5 positive cases, 80%). Within age group III, 15 out of the 17 total cases (88%) were found positive. PCB were the most abundant pollutants detected among the positive cases in the older age group III (11/15, 73%), followed by DDT (7/15, 47%) and HCH (5/15, 33%). Overall, PCB were the most abundant contaminants quantified in 15/30 cases (50%). All other OC had lower detection frequencies, ranging from 30% (9/30 cases) and 27%
770
G. N. RALLIS ET AL.
TABLE 1. Characteristics of Autopsy Cases Age range
Area distribution
Gender (n)
Age range, yr
Mean age, yr ± SD
Group I (≤45 yr)
Group II (46–65 yr)
Group III (≥66 yr)
Urban/ semiurban
Rural
Males (19) Females (11) Total (30)
14–84 21–91 14–91
57.5 ± 23.0 69.1 ± 20.8 61.8 ± 22.5
5 1 6
5 2 7
9 8 17
9 8 17
10 3 13
Detection frequencies of OCs per age group 15
14 12
11 Chlordanes
No of Cases
Downloaded by [North Dakota State University] at 11:41 19 November 2014
16
10
DDTs Endosulfans Endrins
8
HCHs
7
PCBs Positives
6
5 4
4 2
2 0
0
4 2 1 1
0
0
0
< 46 y (Group I)
1 0
Negatives
4
2
1
5
2
2
1
0
46–65 y (Group II)
> 65 y (Group III)
Age Range, y FIGURE 1. Detection frequencies of OC per age group.
(8/30 cases) for DDT and HCH, respectively, to 13% (4/30 cases) and 6.7% (2/30 cases) for endosulfans, endrins, and chlordanes respectively. Lungs from six cases contained at least three different classes of pollutants, where the combination of PCB, DDT, and HCH was the most abundant profile, observed in 5/6 cases (not shown). Frequencies of detection of each specific analyte in each one of the three age groups are illustrated in Figure 2. Higher frequencies of detection were observed for PCB 138 (10/30, 33%) and PCB 153 (9/30, 30%), p,p’DDE (9/30, 30%), and γ-HCH (8/30, 27%). The distribution of pollutants did not demonstrate significant differences between males and females or between different residential areas
(urban/semi-urban/rural areas). This observation may be attributed to a greater rural area in Epirus, where 33% of inhabitants reside in an urban/semi-urban environment while the rest, 77%, live in rural areas. In addition, there are no large urban centers in the region and the vast majority of the population carries out to some degree agricultural practices regardless of their residential area. Levels of OC in Human Lungs The concentration ranges of each analyte, their contribution to the total burden of the respective class of analytes (% range), and the numbers of positive cases and the age ranges of individuals are presented in Table 2. The compounds p,p’-DDT, p,p’-DDE, γ-HCH, PCB 153,
ORGANOCHLORINE COMPOUNDS IN AUTOPSY LUNGS
771
Frequency of each analyte per age group PCB 180 PCB 138 PCB 153 γ-HCH β-HCH < 46 y
Endrin ketone
46–65 y
Endos. sulfate
> 65 y
Endosulfan I p,p´-DDE p,p´-DDD
Downloaded by [North Dakota State University] at 11:41 19 November 2014
p,p´-DDT γ-Chlordane 0
2
4
6 8 No of cases
10
12
FIGURE 2. Frequency of each analyte per age group.
PCB138, and PCB180 were the main pollutants detected. Our results demonstrated that certain OC accumulated in human lungs of residents of Epirus as a result of their persistence in northwestern and other areas of Greece (Golfinopoulos et al., 2003; Konstantinou et al., 2006; Sakellarides et al., 2006 Chrysikou et al., 2008; Christoforidis et al., 2008). The patterns of OC found in lungs were similar to those reported in previous studies for other human specimens from Greece (Costopoulou et al., 2006; Kalantzi et al., 2011; Tsatsakis et al., 2008a, 2008b) and other European countries
(Lucena et al., 2007). Nevertheless, the levels reported herein may not be compared directly to those observed previously, because the latter were expressed as weight of pollutant per lipid content of the examined specimen. The main contributor to DDTs was p,p’DDE in five cases and p,p’-DDT in two cases. The metabolite p,p’-DDD was the minor contributor to DDT in the two cases where it was detected. p,p’-DDT was detected simultaneously with p,p’-DDE and the concentrations of p,p’-DDT and p,p’-DDE were significantly correlated. It is known that once inside the body, p,p’-DDT is degraded to p,p’-DDE or p,p’DDD. The compound p,p’-DDD may be further metabolized, while p,p’-DDE shows high bioaccumulation potential with a reported half life up to 15 yr (U.S. Environmental Protection Agency [EPA], 2013). Therefore, p,p’-DDE is usually the predominant variant of DDT in humans (Jaga and Dharmani, 2003), as was the case here. In consideration of the following facts, (i) commercial DDT product was banned in Greece since the mid-1970s, (ii) p,p’-DDT and its metabolite p,p’-DDE are highly persistent in the Greek environment and living organisms (Sakellarides et al., 2006; Chrysikou et al., 2008; Christoforidis et al., 2008), (iii) the possible transfer of DDT through rivers and aquatic biota from neighboring countries
TABLE 2. Concentrations of Analytes (Ranges, Means, Percent Range), Number of Positive Cases for Each Analyte, and the Age of Individuals Where Each Analyte Was Detected Class of analytes DDTs
HCHs
Endosulfans
Chlordanes Endrins PCBs
Analytes
C range, ng/g
Mean C ± SD, ng/g
Percent C range
Positive cases, N
Age range, yr
p,p -DDT p,p -DDD p,p -DDE DDTs β-HCH γ-HCH HCHs Endosulfan I Endos. sulfate Endosulfans γ-Chlordane Endrin ketone PCB 138 PCB 153 PCB 180 PCBs
2.15–4.89 1.42–1.46
