Multi-omics for studying and understanding polar life.

Polar ecosystems are experiencing amongst the most rapid rates of regional warming on Earth. Here, we discuss 'omics' approaches to investigate polar biodiversity, including the current state of the art, future perspectives and recommendations. We propose a community road map to generate and more fully exploit multi-omics data from polar organisms. These data are needed for the comprehensive evaluation of polar biodiversity and to reveal how life evolved and adapted to permanently cold environments with extreme seasonality. We argue that concerted action is required to mitigate the impact of warming on polar ecosystems via conservation efforts, to sustainably manage these unique habitats and their ecosystem services, and for the sustainable bioprospecting of novel genes and compounds for societal gain.

Reproductive isolation and patterns of genetic differentiation in a cryptic butterfly species complex.

Molecular studies of natural populations are often designed to detect and categorize hidden layers of cryptic diversity, and an emerging pattern suggests that cryptic species are more common and more widely distributed than previously thought. However, these studies are often decoupled from ecological and behavioural studies of species divergence. Thus, the mechanisms by which the cryptic diversity is distributed and maintained across large spatial scales are often unknown. In 1988, it was discovered that the common Eurasian Wood White butterfly consisted of two species (Leptidea sinapis and Leptidea reali), and the pair became an emerging model for the study of speciation and chromosomal evolution. In 2011, the existence of a third cryptic species (Leptidea juvernica) was proposed. This unexpected discovery raises questions about the mechanisms preventing gene flow and about the potential existence of additional species hidden in the complex. Here, we compare patterns of genetic divergence across western Eurasia in an extensive data set of mitochondrial and nuclear DNA sequences with behavioural data on inter- and intraspecific reproductive isolation in courtship experiments. We show that three species exist in accordance with both the phylogenetic and biological species concepts and that additional hidden diversity is unlikely to occur in Europe. The Leptidea species are now the best studied cryptic complex of butterflies in Europe and a promising model system for understanding the formation of cryptic species and the roles of local processes, colonization patterns and heterospecific interactions for ecological and evolutionary divergence.

Fatty acids modulate the effect of darglitazone on macrophage CD36 expression.

BACKGROUND: Scavenger receptor-mediated uptake of cholesterol by macrophages in the arterial wall is believed to be proatherogenic. Thiazolidinediones are peroxisome proliferator-activated receptor gamma (PPARgamma)-agonists, which are used in the treatment of type II diabetes. They reduce atherogenesis in LDL receptor deficient and ApoE knockout mice, but up-regulate CD36, which may contribute to foam cell formation. The dyslipidaemia in type II diabetes is characterized by high levels of nonesterified fatty acids. Therefore we tested the effect of fatty acids and how fatty acids and the thiazolidinedione darglitazone interact in their effect on CD36 expression in human monocytes and macrophages. MATERIALS AND METHODS: Flow cytometry and reverse transcription-polymerase chain reaction were used to study CD36 expression. Cellular lipids were analyzed with high performance liquid chromatography. RESULTS: Darglitazone increased CD36 mRNA and protein expression in human macrophage cells. In the presence of 5% human serum, darglitazone increased the accumulation of triglycerides, but did not affect cholesterol ester levels. In the presence of albumin-bound oleic or linoleic acid, darglitazone did not increase CD36 mRNA, cell-surface CD36 protein or triglyceride content. Fatty acids per se increased CD36 mRNA and protein. DISCUSSION: The increase in CD36 in macrophages suggests a role for fatty acids in the regulation of foam cell formation. The results also suggest that the potentially proatherogenic CD36 up-regulating effect of thiazolidinediones in macrophages might not be present when the cells have access to physiological levels of albumin-bound fatty acids.

Inhibitory effects of N-acetylcysteine on scavenger receptor class A expression in human macrophages.

OBJECTIVE: The formation of foam cells from monocyte-derived macrophages involves the uptake of modified lipoproteins by scavenger receptors. Antioxidants inhibit lipoprotein oxidation and may also modulate gene expression. We investigated the effect of the antioxidant N-acetylcysteine on the expression of the class A scavenger receptor (SR-A) types I and II in human macrophages. DESIGN: Monocytes and macrophages from healthy blood donors and plaque-derived macrophages from patients undergoing carotid endarterectomy were used for experiments. SR-A mRNA was analysed with quantitative and semiquantitative reverse transcription-polymerase chain reaction, and ligand binding and uptake were assessed with 125I-labelled acetylated low-density lipoprotein (LDL). RESULTS: Incubation of monocytes and monocyte-derived macrophages with N-acetylcysteine decreased both SR-A I and II mRNA expression. N-Acetylcysteine also reduced SR-A mRNA in lesion-derived cells. Binding and uptake of 125I-acetylated LDL was decreased after brief incubation with N-acetylcysteine. After longer periods of incubation with N-acetylcysteine we observed an increased degradation of lipoproteins. CONCLUSIONS: Our results imply that N-acetylcysteine leads to a decrease in SR-A mRNA and initially also to an attenuated uptake of modified lipoproteins. This adds more to the knowledge about the cellular actions of this drug.

Oxysterols induce interleukin-1beta production in human macrophages.

BACKGROUND: Oxysterols are biologically active molecules generated during the oxidation of low-density lipoprotein or formed enzymatically in vivo. In the atherosclerotic plaque newly recruited macrophages may be exposed to oxysterols present in the plaque. How these oxysterols affect the expression and secretion of inflammatory cytokines such as interleukin-1beta (IL-1beta) in macrophages is not known. Therefore the aim of the present study was to investigate how oxysterols regulate the expression and secretion of IL-1beta in human monocyte-derived macrophages. METHODS: The IL-1beta messenger RNA (mRNA) expression was analysed by reverse transcription-polymerase chain reaction, and the IL-1beta protein secretion was measured by enzyme-linked immunosorbent assay. RESULTS: A significant, dose-dependent increase in the secretion of IL-1beta was given by 25-hydroxycholesterol without the addition of lipopolysaccharide (LPS). At a concentration of 2.5 microg mL(-1) this increase was similar to that obtained by endotoxin (LPS, 1 microg mL(-1)). A transient increase in IL-1beta mRNA expression was found in macrophages incubated with 25-hydroxycholesterol compared with untreated controls. In addition, 25-hydroxycholesterol dramatically increased the IL-1beta secretion induced by LPS. At a concentration of 5 microg mL(-1) of 25-hydroxycholesterol the LPS-induced IL-1beta secretion was increased by about 25-fold. A similar tendency, but not so consistent, was found for 27-hydroxycholesterol. CONCLUSIONS: Our results show that oxysterols, and 25-hydroxycholesterol in particular, may modulate the inflammatory response in human macrophages. Consequently the presence of oxysterols in atherosclerotic tissue may dramatically influence the effect of inflammation.

Polybrominated diphenyl ethers in Swedish human liver and adipose tissue.

Paired samples of human liver and adipose tissue were analyzed for polybrominated diphenyl ethers (PBDEs) containing 3-6 bromine atoms. The samples were obtained at autopsy from one woman and four men at the age of 47 and 66-83 years, respectively. PBDEs were found in all samples. The sum of nine PBDE congeners ranged 5-18 ng/g lipids and 4-8 ng/g lipids in liver and adipose tissue, respectively. In three paired samples the concentrations were similar in liver and adipose tissue, while in two of the pairs the concentrations were higher in liver than in adipose tissue. The PBDE congeners 2,2',4,4'-tetraBDE (BDE-47), 2,2',4,4',5-pentaBDE (BDE-99), and 2,2',4,4',5,5'-hexaBDE (BDE-153) occurred at highest levels and constituted together 87-96% and 84-94% of the total sum of PBDEs in liver and adipose tissue, respectively. The levels of PBDEs were compared to those of polychlorinated biphenyls (PCBs), polychlorinated naphthalenes (PCNs), 1,1-bis(4-chlorophenyl)-2,2-dichloroethene (p,p'-DDE), and hexachlorobenzene (HCB).

Construction of high-complexity combinatorial phage display peptide libraries.

Random peptide libraries displayed on the surface of filamentous bacteriophage are widely used as tools for the discovery of ligands for biologically relevant macromolecules, including antibodies, enzymes, and cell surface receptors. Phage display results in linkage of an affinity-selectable function (the displayed peptide) to the DNA encoding that function, allowing selection of individual binding clones by iterative cycles of in vitro panning and in vivo amplification. Critical to the success of a panning experiment is the complexity of the library: the greater the diversity of clones within the library, the more likely the library contains sequences that will bind a given target with useful affinity. A method for construction of high-complexity (> or = 10(9) independent clones) random peptide libraries is presented. The key steps are highly efficient binary ligation under conditions where the vector is relatively dilute, with only a modest molar excess of insert, followed by efficient electrotransformation into Escherichia coli. Library design strategies and a protocol for rapid sequence characterization are also presented.

Certain organochlorine and organobromine contaminants in Swedish human milk in perspective of past 20-30 years.

The investigations of organochlorine compounds in breast milk from women living in the Stockholm region started in 1967. The present study summarises the investigations of polychlorinated biphenyls (PCBs), naphthalenes (PCNs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), polybrominated diphenyl ethers (PBDEs) and pesticides (DDT, DDE, hexachlorobenzene, dieldrin) as well as methylsulfonyl metabolites of PCBs and DDE in human milk sampled during different periods up to 1997. During the course of 20-30 yr the levels of organochlorine compounds in human milk have decreased to various extent. A decrease to the half of the original concentration was attained in the range of 4-17 yr periods. On the contrary to the organochlorine compounds, the concentrations of PBDEs have increased during the period 1972-1997, indicating a doubling of the levels by 5 yr. The levels reflect the environmental contamination and background levels in the population. The accumulation and ongoing increase in the levels of PBDEs calls for immediate measures to stop the environmental pollution and human exposure to PBDEs.

Analysis of polybrominated diphenyl ethers in Swedish human milk. A time-related trend study, 1972-1997.

A previously described method for analysis of organochlorine compounds in human milk was adopted for analysis of brominated diphenyl ethers (BDEs) substituted with three to six bromine atoms. Analytes were extracted from human milk with the lipophilic gel Lipidex 5000. Further purifications were performed on partly deactivated aluminum oxide and silica gel columns, followed by gel permeation chromatography. The concentrations of BDEs were determined by gas chromatography/mass spectrometry (GC/MS). The average recoveries of 2,2',4-triBDE (BDE-17), 2,4,4'-triBDE (BDE-28), 2,2',4,4'-tetraBDE (BDE-47), 2,3',4,4'-tetraBDE (BDE-66), 2,2,3,4,4'-pentaBDE (BDE-85), 2,2',4,4',5-pentaBDE (BDE-99), 2,2',4,4',6-pentaBDE (BDE-100), 2,2',4,4',5,5'-hexaBDE (BDE-153), and 2,2',4,4',5,6'-hexaBDE (BDE-154) added to the samples before extraction ranged from 86% to 102%. Pooled samples of breast milk, collected at eight time periods between 1972 and 1997, were analyzed for PBDEs. BDE-47 was the most abundant PBDE congener in all samples. In total, eight PBDE congeners were identified in the milk. The sum of the concentrations of BDE congeners in human milk increased from 0.07 to 4.02 ng/g lipids during the 25-yr period studied.

Distribution of PCB congeners, DDE, hexachlorobenzene, and methylsulfonyl metabolites of PCB and DDE among various fractions of human blood plasma.

The concentrations of chlorinated biphenyls (CBs), 1, 1-bis(4-chlorophenyl)-2,2-dichloroethene (DDE), hexachlorobenzene (HCB), and the methylsulfonyl metabolites of CBs (MeSO(2)-CBs) and DDE (MeSO(2)-DDE) were determined in human plasma samples and in the fractions obtained by ultracentrifugation of plasma into very-low-density (VLDL), low-density (LDL), high-density (HDL) lipoprotein and lipoprotein depleted (LPDP) fractions (containing primarily albumin). The concentrations of triacylglycerols, cholesterol, phospholipids, and apolipoprotein B (apoB) were determined. The organochlorine compounds were associated with all fractions, but predominantly with the LPDP fraction. On an average 44% of CBs, 61% of MeSO(2)-CBs, 73% of DDE, 77% of MeSO(2)-DDE, and 45% of HCB were distributed in the LPDP fraction. A tendency to greater association of 3-methylsulfonyl substituted than of corresponding 4-methylsulfonyl substituted chlorobiphenyls to the LPDP fraction was noticed. Among the lipoprotein fractions, LDL was the main carrier of HCB, DDE and CBs. MeSO(2)-DDE was predominantly found in HDL and MeSO(2)-CBs were distributed equally among the LDL and HDL fractions. Calculating the concentrations of organochlorine compounds in relation to the content of apoB, the levels were about 10 times higher in VLDL than in LDL.

The maltose-binding protein as a scaffold for monovalent display of peptides derived from phage libraries.

Random peptide libraries are displayed on filamentous bacteriophage as fusions to either the minor coat protein, pIII, or the major coat protein, pVIII. We have devised a means of isolating the peptide displayed on a phage clone by transferring it to the N-terminus of the maltose-binding protein (MBP) of Escherichia coli encoded by malE. Transfer of a peptide sequence to monomeric MBP eliminates phage-encoded amino acids downstream of the insert peptide as well as avidity effects caused by multivalent display on phage. Peptide:MBP fusions are also easily affinity purified on amylose columns. The pMal-p2 vector was engineered to accept phage DNA encoding pIII- and pVIII-displayed peptides fused to their respective leader sequences. Both types of leader sequence were shown to target the peptide:MBP fusions to the periplasm of E. coli. A streamlined procedure for transferring peptides to MBP was applied to clones that had been isolated from a panel of pVIII-displayed peptide libraries by screening with an HIV-1-specific monoclonal antibody (Ab). By enzyme-linked immunosorbent assay, the Ab bound each of the peptide:MBP fusions and required the presence of a disulfide bridge within each peptide. Some of the peptide:MBP fusions were also analyzed using surface plasmon resonance. Thus, our study shows the value of malE fusion vectors in characterizing phage-displayed peptides.

Genetic association of Ctla-4 to myasthenia gravis with thymoma.

Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) plays a pivotal role in downregulating both the cellular and the humoral response by suppressing ongoing responses of activated T cells. Our earlier study showed that genetic variations in interleukin-1 genes confer susceptibility to myasthenia gravis, especially in patients having the lowest risk from major histocompatibility complex genes. Here we describe an association of Ctla-4 gene to the disease with thymoma and a higher prevalence of CTLA-4 gene polymorphism allele 104 in patients positive for IL-1beta TaqI allele 2, an IL-1beta 'high secretor' phenotype. There was no association in patients with hyperplasia and normal thymic histology. These results further advocate that MG is a polygenetic disease and suggest that co-stimulators such as CTLA-4 and CD28 might have an important role in the pathogenesis of the disease.

Polychlorinated naphthalenes and other organochlorine contaminants in Swedish human milk, 1972-1992.

The concentrations of polychlorinated naphthalenes (PCNs) were determined together with polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), 1,1-bis-(4-chlorophenyl)-2,2,2-trichloroethane (p,p'-DDT), 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE) and hexachlorobenzene (HCB) in milk, sampled in the course of 1972-92 from mothers living in Stockholm. A previously developed method for multicomponent analysis of organochlorine environmental contaminants was adapted for simultaneous analysis of PCNs. The mean recoveries of seven chlorinated naphthalene (CN) congeners added to milk prior to extraction were 76-99%. Similar recoveries were obtained for the commercial PCN product Halowax 1014. The pattern of PCNs in milk differed to a great extent from that in the commercial PCN products. The dominating congeners in breast milk were 1,2,3,5,7-pentachloronaphthalene (CN-52), 1,2,3,4,6,7- and/or 1,2,3,5,6,7-hexachloronaphthalene (CN-66/ CN-67) and one unidentified tetrachloronaphthalene. There was a notable decrease in the concentrations of PCNs as was of the other organochlorine contaminants in milk from 1972 to 1992. During this time period the sum of CN congeners decreased from 3,081 to 483 pg/g milk fat and the sum of toxic equivalents of dioxin and dioxin-like compounds decreased from 100 to 39 pg/g milk fat.

Polychlorinated naphthalenes and other organochlorine contaminants in human adipose and liver tissue.

Chlorinated naphthalenes (CNs) and chlorinated biphenyls (CBs), hexachlorobenzene (HCB), and p,p'-DDE were determined in human adipose and liver tissues collected at autopsy of 5 men and 2 women (Swedish), 47-80 yr of age. In paired adipose tissue and liver samples, the differences of the distribution of CNs, CBs, HCB, and p,p'-DDE were small, but the concentrations of the compounds (lipid weight basis) varied between the subjects. Generally, the profiles of the contaminants were similar in the subjects. However, in one of the male subjects (80 yr of age) the concentrations of 1,2,3,5,7/1,2,4,6,7-pentaCN and 1,2,3,4,6,7/1,2,3,5,6,7-hexaCN were much higher in the liver: 6 and 20 times higher than in adipose tissue, respectively. The concentration of the toxic coplanar 3,3',4,4',5-pentaCB (CB-126) was also high in the liver from this individual, three times higher concentration than in adipose tissue. It is suggested that the multiplicity of contaminants found in humans should be taken into consideration in risk assessment and that further studies are needed on the distribution of contaminants in different tissues.

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.

Defectively N-glycosylated and non-O-glycosylated aminopeptidase N (CD13) is normally expressed at the cell surface and has full enzymatic activity.

In order to study the effects of the absence of O-glycosylation and modifications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the ldl(D) cell line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficiency which blocks the conversion of glucose into galactose derivatives. Thus it is possible in the ldl(D) cell line to selectively block O-glycosylation by the omission of N-acetylgalactoseamine from the culture medium and to alter N-glycosylation by the omission of galactose. In this way selectively altered glycosylated forms of the glycoprotein aminopeptidase N can be synthesized and the effects of altered glycosylation can be studied. It is demonstrated that aminopeptidase N carries "mucin-type" O-glycans and that this is predominantly located in the stalk, which connects the catalytic headgroup to the membrane anchor. Normally glycosylated aminopeptidase N is present in the plasma membrane of the ldl(D) cells. This is also the case for the non-O-glycosylated and defectively N-glycosylated forms. This is in line with the finding that the intracellular transport APN is unaffected by the absence of O-glycosylation or by changes in N-glycosylation as the various glycosylated forms of aminopeptidase N are normally converted from the high-mannose form to the complex glycosylated form. Enzymatic activity is not influenced by the changes in glycosylation.

Occupational exposure: Organochlorine compounds in blood plasma from potentially exposed workers. PCB, PCN, PCDD/PCDF, HCB and methylsulphonyl metabolites of PCB.

Concentrations of polychlorinated biphenyls (PCB), polychlorinated naphthalenes (PCN), polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), hexachlorobenzene and methylsulphonyl metabolites of PCB were determined in blood plasma from potentially exposed workers and controls. Three of the potentially exposed subjects had worked with cable incineration and two were electricians. Extraction of the organochlorine compounds and lipids were performed using the lipophilic gel Lipidex. Different adsorbents and gel permeation chromatography were applied for further purification of the samples and separation of analytes. Determinations of the chlorinated compounds were made by using gas chromatography with electron-capture detection and gas chromatography-mass spectrometry. Only small differences in the concentrations of organochlorine compounds were found in the plasma from the three subject groups. Thus, specific exposure of the workers could not be confirmed.

Methylsulfonyl metabolites of PCBs and DDE in human milk in Sweden, 1972-1992.

A multicomponent method used for analysis of organochlorine pesticides, polychlorinated biphenyls (PCBs), naphthalenes, dibenzo-p-dioxins, and dibenzofurans was adapted for the analysis of methylsulfonyl metabolites of chlorinated biphenyls (MeSO2-CBs) and of p,p'-DDE (MeSO2-DDE) in human milk. The extraction and initial purification was made by liquid-gel partitioning. Additional purification and separation steps were achieved by adsorption and gel permeation chromatography. The mean recoveries of 23 MeSO2-CBs and MeSO2-DDE standards, added to the milk before extraction, were 80-97%. Human milk sampled in Stockholm during 1972, 1976, 1980, 1984/85, 1990, 1991, and 1992 was analyzed by GC-MS. During the time course studied, the concentrations of MeSO2-CBs decreased from approximately 9 to 2 ng/g lipids and of MeSO2-DDE from 5 to 0.4 ng/g lipids. The concentrations of MeSO2-CBs and MeSO2-DDE correlated to the levels of total PCB and p,p'-DDE, respectively. 3-MeSO2-DDE was the major isomer of the aryl methyl sulfones studied in the milk. PCB methyl sulfones with five and six chlorine atoms in the molecule were predominant among the PCB methyl sulfones Generally, the concentrations of 4-MeSO2-CBs were higher than the corresponding 3-MeSO2-CB compound. The major MeSO2-CBs in the milk were 4-MeSO2-2,5,2',3',4'-pentaCB (4-87) and 4-MeSO2-2,3,6,2',4',5'-hexaCB (4-149).

Liquid-gel partitioning using Lipidex in the determination of polychlorinated biphenyls, naphthalenes, dibenzo-p-dioxins and dibenzofurans in blood plasma.

A method was developed for the transfer of fat, polychlorinated biphenyls (PCBs), naphthalenes (PCNs), dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) from blood plasma into the lipophilic gel Lipidex 5000. Subsequent elution of the gel separated about 70% of the fat from the analytes. Different adsorbents and activated charcoal were applied for further purification of the sample and separation of analytes. Identification and determination of the chlorinated compounds were made by gas chromatography with electron-capture detection (GC-ECD) or gas chromatography-mass spectrometry (GC-MS). Recoveries were studied by addition of Halowax 1014 and different congeners of PCBs, PCNs, PCDDs and PCDFs to 50 ml of plasma. The mean recoveries of the individual compounds studied were 72-99%. By using the liquid-gel partitioning technique emulsions were avoided. Concentrations of lipids in plasma obtained by the present method agreed well with the concentrations obtained using liquid-liquid partitioning with chloroform-methanol.

Contemporary and retrospective investigations of human milk in the trend studies of organochlorine contaminants in Sweden.

Milk from the Mothers' Milk Centre in Stockholm has been analysed for organochlorine contaminants in different time periods between 1967 and 1989. The contemporary investigations showed a decrease of the levels of certain pesticides and polychlorinated biphenyls (PCBs). The changes were related to the prohibitions and restrictions applied on the usage of the compounds. Milk samples, remaining from the contemporary studies, were archived from 1972 and onward. These samples were later reanalyzed and the investigations were extended to also include polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and specific congeners of PCBs, including non-ortho and mono-ortho coplanar PCBs. These retrospective investigations, of the same milk, showed a time related decrease in the concentrations also for these compounds. Calculating the levels by toxic equivalency factors relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) it was found that PCBs contributed the major part of the toxic equivalents in milk.