Methylsulfonyl metabolites of PCBs and DDE in human tissues.

Methylsulfonyl metabolites of chlorinated biphenyls (MeSO2-CBs) and p,p'-DDE (MeSO2-DDEs) were determined in human adipose and liver tissues obtained at autopsy of seven Swedish individuals 47-80 years of age. Twenty MeSO2-CBs and two MeSO2-DDEs were found in the analyzed samples. In adipose tissue, most of the 4-MeSO2-CBs were found at higher concentrations than the corresponding 3-MeSO2-CBs and, in all samples of adipose tissue, 4-MeSO2-2,2',3',4',5-pentaCB (4-87) and 4-MeSO2-2,2',3,4',5',6-hexaCB (4-149) occurred at higher concentrations than other MeSO2-CBs. In the liver, 3-MeSO2-2,2',3',4',5,6-hexaCB (3-132) was by far the most abundant MeSO2-CB, contributing to 61-82% of the sum of MeSO2-CBs. In this tissue, most of the other 3-MeSO2-CBs were also found at higher concentrations than the corresponding 4-MeSO2-CBs. The ratios of the sum of MeSO2-CBs to the sum of determined chlorinated biphenyls (CBs) were 1/250 and 1/28 in adipose tissue and the liver, respectively, calculated from the median values. The concentration of 2-MeSO2-DDE was lower than that of 3-MeSO2-DDE in both adipose tissue and liver, except in the liver from one of the individuals. The concentration ratios of 2-MeSO2-DDE to 3-MeSO2-DDE were about 10 times higher in liver than in adipose tissue. The ratios of the sum of MeSO2-DDEs to p,p'-DDE were 1/455 and 1/61 in adipose tissue and liver, respectively, calculated from the median values. MeSO2-CBs and MeSO2-DDEs were also determined in lung tissue from one of the individuals. In this sample, the profiles of MeSO2-CBs and MeSO2-DDEs were similar to the profiles of these compounds in adipose tissue.

HJear Pt t 105:64649 (1997) Polychlorinated biphenyls (PCBs) and 1,1bis(4-chlorophenyl) -2,2-dichloroethene (p,p'-DDE), a metabolite of the pesticide DDT, are well-known pollutants that have been found in many environmental matrices and in humans (1). The industrial use of PCBs has ceased in most countries, but objects containing these compounds are still employed and pollution may continue for a long time, e.g., from electronic equipment and building materials. DDT was a commonly used insecticide in the 1940s-1960s. In Sweden and many other industrialized countries, the usage of DDT was banned in the early 1970s and, consequently, the levels have decreased in these areas. However, DDT is still used in large quantities in countries where malaria is a serious problem (2,3). Due to the persistency of DDT compounds and the long-distance atmospheric transportation, the residue levels of the pesticide in the Western countries will probably be long-lasting. In the metabolism of chlorinated biphenyls (CBs) and p,p'-DDE, corresponding methylsulfonyl (MeSO2) metabolites can be formed via the mercapturic acid pathway (MAP) (4,5). The CBs with nonchlorinated, adjacent 3 and 4 positions and chlorine atoms in the 2, 5 or 2, 3, 6 positions of the same ring have been shown to form metabolites with the MeSO2-group in position 3 or 4 (6). The MeSO2-group location in MeSO2-DDEs is limited to either the 2 or the 3 position. The formation of methylsulfonyl metabolites of, e.g., CBs with vicinal hydrogen atoms, is initiated by cytochrome P450-mediated oxidation to arene oxide intermediates. Arene oxides with chlorine atoms on both sides of the 3,4-positions readily react with glutathione in position 3 or 4. The glutathione conjugates are transformed via MAP to cystein conjugates, which are cleaved by C-S lyase to thiol-substituted CBs (4,5). These are methylated by adenosyl-methionine to methyl sulfides, which then are oxidized to methyl sulfones via an intermediate formation of methylsulfinyl-CBs (4,5). Methylsulfonyl metabolites of CBs and p,p'-DDE are persistent and, due to their lipophilic character, they accumulate in adipose tissue (6)(7)(8). They may also have specific protein-binding properties that cause them to be retained in the body, e.g., 3-MeSO2-DDE in adrenal tissue (9,10) and certain MeSO2-CBs in lung, kidney, and uterine fluid (11)(12)(13), as demonstrated in experimental animals.
In environmental samples, MeSO2-CBs and MeSO2-DDEs were first identified in seal blubber from the Baltic (14); since then, such metabolites have been found in several species of animals (6) and in humans (7,8,15). Recently, MeSO2-CBs and MeSO2-DDEs were determined in Swedish mother's milk (16) and MeSO2-CBs were determined in human blood plasma (17).
The toxicological significance of MeSO2-CBs has not yet been clarified, but recently certain 3-MeSO2-CBs were reported to be potent inducers of hepatic enzymes that are related to toxic effects (18,19). 3-MeSO2-DDE is a potent toxicant for the adrenal cortex, e.g., in mice (9) and probably also in humans, as suggested by in vitro studies, showing that 3-MeSO2-DDE is bioactivated to a tissue-binding metabolite by the human adrenal gland (10).
The occurrence of MeSO2-CBs and MeSO2-DDEs in wildlife and human samples and the demonstrated accumulation of certain MeSO2-CBs and MeSO2-DDEs in specific tissues of mammals have made the investigation of these compounds important. The knowledge of accumulation and distribution of methylsulfonyl metabolites of aromatic organochlorine pollutants in humans is scarce. Therefore, in the present study we have determined MeSO2-CBs and MeSO2-DDEs together with CBs and p,p'-DDE in adipose tissue and liver from human subjects.

Materials and Methods
Sample description. Samples of adipose tissue from the abdominal region and of liver were collected at autopsy of five men and two women (Swedish) who had suffered sudden death. The age of the men ranged from 53 to 80 years (mean 68 years, median 68 years) and the age of the women were 47 and 62 years. The samples were frozen immediately after collection, kept at -200C, and thawed only before analysis.
Extraction, lipid determination, and preliminary purification. Liver (-3g) or adipose tissue (-0.5 g) was weighed into a 100-ml test tube with a polytetrafluoroethylene (PTFE)-lined screw cap. Internal and extracted with hexane/2-propanol (20 ml, 3/2 v/v). The homogenat( trifuged at 3500 rpm for 10 m organic solvent phase was coll round-bottomed flask. The extr; cedure was repeated once with propanol (20 ml, 3/2 v/v) and hexane (20 ml). The pooled org; were evaporated under reduced 35°C. The residue was dried in a with silica gel at room tempei then weighed. The residue was d hexane and transferred to Erlenmeyer flask (samples conta than 0.3 g lipids were diluted M and an aliquot of the sample w; the Erlenmeyer flask). The volt sample solution was adjusted to hexane. 2-Propanol (8 ml) and f (0.5 ml) were mixed with the sa tion, and 6 g Lipidex 5000 Instruments, Downers Grove, II added. Lipids and lipophilic con the sample were transferred into  e was cena liquid-gel partitioning technique iin and the described previously (25). The gel with lected in a incorporated lipophilic compounds was action pro-poured into a glass column with an inner hexane/2diameter (ID) of 2 cm (26) and the solvent once with was drained. The gel was washed with 40 anic phases ml each of methanol-water 30:70 (v/v) and pressure at 50:50 (v/v). The chlorinated compounds i desiccator and some lipids were eluted with acetonirature and trile (80 ml for liver samples and 110 ml lissolved in for adipose tissue samples). Remaining a 1 00-ml lipids were eluted with methanol/trichloroining more methane/hexane (60 ml 1:1:1 by volume). vith hexane The two fractions containing lipids were as taken to taken to near dryness under reduced presame of the sure and dried to constant weight in a des-i3 ml with iccator with silica gel. The sum of the formic acid weights of the residues in the two fractions imple solu-defined the amount of lipids in the sample. Articles * Weistrand and Nor6n or 6 g Lipidex 5000 and with different mixtures of hexane, 2-propanol, and formic acid. The gel with sorbed lipophilic compounds was eluted with acetonitrile as described above. The volumes of the fractions from the Lipidex column were measured and 0.5 ml aliquots were taken for determination of radioactivity (24).

Results
In the preliminary study of transfer of lipophilic compounds into Lipidex, the recoveries of 14C-cholesterol and 14C-phosphatidylcholine added to samples of adipose tissue and liver from human subjects prior to homogenization were 92-105% and 97-98%, respectively. The concentration of lipids in adipose tissue and liver from the seven subjects ranged from 52 to 99% and from 3 to 23%, respectively. The recoveries of the internal standard, MeSO2-IS, added to all samples prior to homogenization ranged from 67 to 103% (mean 83%; n = 14). The recoveries of MeSO2-IS have been shown to correlate well with the recoveries of MeSO2-CBs (16,1?) and 3-MeSO2-DDE (16).
The concentrations of MeSO2-CBs and MeSO2-DDEs varied in the different subjects (Table 1); therefore, mean values were not calculated for the individual compounds. However, the profiles of MeSO2-CBs and MeSO2-DDEs (Figs. 1, 2, and 3) were similar in all seven subjects.
In adipose tissue and liver, most of the penta-and hexachloro-substituted congeners were found at higher concentrations than the other MeSO2-CBs (Table 1, Fig.  1). In adipose tissue, the pentaCB methyl sulfone 4-87 and the hexachloro compound 4-149 were found at highest concentrations (Fig. 2). In the liver, compound 3-132 was found at a higher concentration than any of the other aryl methyl sulfones; the contribution of this compound to the sum of MeSO2-CBs ranged from 61% to 82% in the individual samples. In adipose tissue, the concentration of 3-132 was low, only 0-4% of the sum of MeSO2-CBs. In the liver, the sum of 3-MeSO2-CBs was higher than the sum of 4-MeSO2-CBs. The ratios of the sums of 3-MeSO2-CBs to 4-MeSO2-CBs ranged from 4 to 21 in the liver samples. Only compounds 4-49, 4-87, 4-101, and 4-141 were found at higher concentrations than the corresponding 3-MeSO2substituted congeners. In adipose tissue, the ratios of the sums of 3-MeSO2-CB to 4-MeSO2-CBs were below 1 (0.2-0.5) in all subjects, and only compounds 3-64, 3-70, and 3-174 were found in larger amounts than the corresponding 4-MeSO2 -CBs.
The sum of the determined CBs was similar in adipose tissue and liver in paired 19. samples (Weistrand and Noren, unpublished data). In contrast, the sum of the concentrations of MeSO2-CBs was significantly higher in the liver than in adipose tissue in all cases. The ratios of the sum MeSO2-CBs to the sum of CBs were 8-34 times higher in the liver than in adipose tissue as calculated from the individual values. Two methyl sulfone metabolites ofp,p'-DDE were found in the samples (Figs. 1, 3, Table 1). We lacked authentic 2-MeSO2-DDE, but comparison with studies where this compound has been identified (6) suggests that the peak appearing ahead of 3-MeSO2-DDE in the selected ion current chromatogram represents 2-MeSO2-DDE (Fig. 3). Assuming that 2-MeSO2-DDE has the same response as 3-MeSO2-DDE, the concentrations of 2-MeSO2-DDE were lower than those of 3-MeSO2-DDE in all samples, except in the liver sample from one of the female subjects (47 years old). The ratios of 2-MeSO2-DDE to 3-MeSO2-DDEs were about 10 times higher in liver than in adipose tissue ( Table 1).
The concentrations of p,p'-DDE were similar in liver and adipose tissue in the individual subjects. The concentrations of the methyl sulfone metabolites ofp,p'-DDE were higher in liver than in adipose tissue in all individuals. The ratios of the sum of MeSO2-DDEs to p,p'-DDE were 2-28 times higher in the liver than in adipose tissue as calculated from individual values.
In addition, a sample of lung tissue (2 g) was collected from one of the male subjects (68 years old) and was treated and analyzed using the same procedure as described for the liver samples. The ratios of the sum of MeSO2-CBs and MeSO2-DDEs to the sum of CBs and p,p'-DDE, respectively, were very similar in lung and adipose tissue from this individual. Also, the profile of MeSO2-CBs and MeSO2-DDEs in lung resembled the profile of these compounds in adipose tissue. However, 2-MeSO2-DDE was not detected in the lung.

Discussion
In a previous study using Lipidex for extraction of PCBs and lipids in fish oil (25), the lipids mainly consisted of triacylglycerols. To validate the method for cholesterol and more polar phospholipids occurring in the liver, recovery studies were performed with 14C-cholesterol and '4Cphosphatidylcholine added to liver and adipose tissue from human subjects. Slight modifications of the previous method were made to achieve satisfactory recoveries. The ratio of hexane to 2-propanol was changed, formic acid added, and the amount of Lipidex increased.
In adipose tissue, most of the 4-MeSO2-CBs were present at higher concentrations than the corresponding 3-MeSO2-CBs; congeners 4-87 and 4-149 were found at the highest concentrations. These were also the major congeners in Swedish human milk (16) and blood plasma (17). In addition, the concentration ratios of MeSO2-CBs to CB-153, which is a stable and predominant CB in mammals, were similar in Articles * PCB/DDE metabolites in human tissues    (16), and from the Swedish subjects. Compound 3-methyl sulfone and was found at about one-)02-0.03) (17). 132 was by far the predominant MeSO2-CB third of the concentration of 3-MeSO2of MeSO2-CBs in the in the liver samples from Swedish subjects; DDE. The sum of the concentrations of (human milk, blood plas-also, most of the other identified 3-MeSO2-MeSO2-DDEs and the ratio of 2-MeSO2-, lung, liver) differed from CBs were found at higher concentrations DDE to 3-MeSO2-DDE were higher in the se investigation (7). In the than the corresponding 4-MeSO2-CBs in liver than in adipose tissue, which indicates 4-87 was the most abun-this tissue. The differences between the selective binding in the liver. In wild mamin adipose and lung tissue Japanese and Swedish profiles of MeSO2mals, the ratio of 2-MeSO2-DDE to 3ient, but not in the samples CBs may be due to different sources of expo-MeSO2-DDE in these tissues, has been ubject. 4-MeSO2-2,4',5sure and/or metabolism of CBs. found to vary with species and tissue, and st abundant MeSO2-CB in The profiles of MeSO2-CBs in wild ani-the results indicate differences in exposure n the control subject, while mals differ from those in humans and have and/or metabolism of DDT/DDE (6). triCB was found at highest also been found to vary in differer the liver from this individspecies (6). In muscle or blubb t, MeSO2-CBs with two or Swedish wildlife (otter, mink, grey ims were not found in the 101 was the predominant MeSO2. nt animal zer from r seal), 3  with CBs and p,p'-DDE in tissues from seven Swedish human subjects. MeSO2-CBs and MeSO2-DDEs were found in all samples showing a general occurrence of these metabolites together with CBs and p,p'-DDE. The profiles of MeSO2-CBs and MeSO2-DDEs were similar in adipose tissue and lung. A different pattern was found in the liver. The concentrations of CBs were similar in adipose tissue and the liver. This was also the case for p,p'-DDE. However, the methyl sulfonyl metabolites of these compounds were significantly higher in liver than in adipose tissue (lipid weight basis). In the human liver, 3-MeSO2-2,2',3',4',5,6-hexaCB  was by far the predominant MeSO2-CB. Certain 3-MeSO2-CBs have previously been found to be selectively retained in the livers of different wild mammals. This indicates that certain structures of MeSO2-CBs facilitate noncovalent binding in the liver, most probably to proteins. Also, the metabolites may have toxic effects. However, little is known about the toxicity and possible effects in humans; therefore this subject requires further study. The main themes of the symposium are: *Management, organization, and control in the field of safety and health in the workplace (programs, risk analysis, assessment, normalization, quality...) *Specific problems of occupational safety and health in smalland mediumsized enterprises as well as possibilities for counseling and support *Current problems of occupational hygiene (dust, mineral fibers, surface treatments) Furthermore, the symposium will discuss other current problems and solutions, such as the transposition of European Directives or outsourcing.

Target groups
The symposium is aimed at safety engineers, occupational physicians, and hygenists, human resources managers, management representatives, experts in various fields from the industry, representatives of the social partners, social insurance institutions, and the authorities.