Development of MS Binding Assays targeting the binding site of MB327 at the nicotinic acetylcholine receptor.

The bispyridinium compound MB327 has been shown previously to have a positive pharmacological effect against poisoning with organophosphorous compounds (OPCs). The mechanism by which it exerts its therapeutic effect seems to be directly mediated by the nicotinic acetylcholine receptor (nAChR). In the present study, the development of mass spectrometry based binding assays (MS Binding Assays) for characterization of the binding site of MB327 at the nAChR from Torpedo californica is described. MS Binding Assays follow the principle of radioligand binding assays, but do not, in contrast to the latter, require a radiolabeled reporter ligand, as the readout is in this case based on mass spectrometric detection. For [2H6]MB327, a deuterated MB327 analogue employed as reporter ligand in the MS Binding Assays, an LC-ESI-MS/MS method was established allowing for its fast and reliable quantification in samples resulting from binding experiments. Using centrifugation for separation of non-bound [2H6]MB327 from target-bound [2H6]MB327 in saturation and autocompetition experiments (employing native MB327 as competitor) enabled reliable determination of specific binding. In this way, the affinities for [2H6]MB327 (Kd=15.5±0.9µmolL-1) and for MB327 (Ki=18.3±2.6µmolL-1) towards the nAChR could be determined for the first time. The almost exactly matching affinities for MB327 and [2H6]MB327 obtained in the MS Binding Assays are in agreement with potencies previously found in functional studies. In summary, our results demonstrate that the established MS Binding Assays represent a promising tool for affinity determination of test compounds towards the binding site of MB327 at the nAChR.

In vitro pharmacological characterization of the bispyridinium non-oxime compound MB327 and its 2- and 3-regioisomers.

The primary toxic mechanism of organophosphorus compounds, i.e. nerve agents or pesticides, is based on the irreversible inhibition of acetylcholinesterase. In consequence of the impaired hydrolysis, the neurotransmitter acetylcholine accumulates in cholinergic synapses and disturbs functional activity of nicotinic and muscarinic acetylcholine receptors by overstimulation and subsequent desensitization. The resulting cholinergic syndrome will become acute life-threatening, if not treated adequately. The current standard treatment, consisting of administration of a competitive mAChR antagonist (e.g. atropine) and an oxime (e.g. obidoxime, pralidoxime), is not sufficient in the case of soman or tabun intoxications. Consequently, alternative therapeutic options are necessary. An innovative approach comprises the use of compounds selectively targeting nAChRs, especially positive allosteric modulators, which increase the population of the conducting receptor state. MB327 (1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium) di(iodide)) is able to restore soman-blocked muscle-force in preparations of various species including human and was recently identified as "resensitizer". In contrast to the well-studied MB327, the pharmacological efficacy of the 2- and 3-tert-butylpyridinium propane regioisomers is unknown. As a first step, MB327 and its 3-regioisomer (PTM0001) and 2-regioisomer (PTM0002) were pharmacologically characterized using [3H]epibatidine binding assays, functional studies by solid supported membranes based electrophysiology, and in vitro muscle-force investigations of soman-poisoned rat hemidiaphragm preparations by indirect field stimulation technique. The results obtained from targets of different complexity (receptor, muscle tissue) showed that the pharmacological profiles of the 2- and 3-regioisomers were relatively similar to those of MB327. Furthermore, high concentrations showed inhibitory effects, which might critically influence the application as an antidote. Thus, more effective drugs have to be developed. Nevertheless, the combination of the methods presented is an effective tool for clarifying structure-activity relationships.

Functional analysis of Torpedo californica nicotinic acetylcholine receptors in multiple activation states by SSM-based electrophysiology.

Organophosphorus compounds (OPC), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). Inhibited AChE results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nAChR) in the postsynaptic membrane is provoked. Direct targeting of nAChR to reduce receptor desensitisation might be an alternative therapeutic approach. For drug discovery, functional properties of potent therapeutic candidates need to be investigated in addition to affinity properties. Solid supported membrane (SSM)-based electrophysiology is useful for functional characterisation of ligand-gated ion channels like nAChRs, as charge translocations via capacitive coupling of the supporting membrane can be measured. By varying the agonist (carbamoylcholine) concentration, different functional states of the nAChR were initiated. Using plasma membrane preparations obtained from Torpedo californica electric organ, functional properties of selected nAChR ligands and non-oxime bispyridinium compounds were investigated. Depending on overall-size, the bispyridinium compounds enhanced or inhibited cholinergic signals induced by 100 µM carbamoylcholine. Applying excessive concentrations of the agonist carbamoylcholine provoked desensitisation of the nAChRs, whereas addition of bispyridinium compounds bearing short alkyl linkers exhibited functional recovery of previously desensitised nAChRs. The results suggest that these non-oxime bispyridinium compounds possibly interacted with nAChR subtypes in a manner of a positive allosteric modulator (PAM). The described newly developed functional assay is a valuable tool for the assessment of functional properties of potential compounds such as nAChR modulating ligands, which might be a promising approach in the therapeutically treatment of OPC-poisonings.

Affinities of bispyridinium non-oxime compounds to [(3)H]epibatidine binding sites of Torpedo californica nicotinic acetylcholine receptors depend on linker length.

The toxicity of organophosphorus nerve agents or pesticides arises from accumulation of acetylcholine and overstimulation of both muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs) due to inhibition of acetylcholinesterase (AChE). Standard treatment by administration of atropine and oximes, e.g., obidoxime or pralidoxime, focuses on antagonism of mAChRs and reactivation of AChE, whereas nicotinic malfunction is not directly treated. An alternative approach would be to use nAChR active substances to counteract the effects of accumulated acetylcholine. Promising in vitro and in vivo results were obtained with the bispyridinium compounds SAD-128 (1,1'-oxydimethylene bis(4-tert-butylpyridinium) dichloride) and MB327 (1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium) di(iodide)), which were partly attributed to their interaction with nAChRs. In this study, a homologous series of unsubstituted and 4-tert-butyl-substituted bispyridinium compounds with different alkane linker lengths was investigated in competition binding experiments using [(3)H]epibatidine as a reporter ligand. Additionally, the effect of the well-characterised MB327 on the [(3)H]epibatidine equilibrium dissociation (KD) constant in different buffers was determined. This study demonstrated that divalent cations increased the affinity of [(3)H]epibatidine. Since quaternary ammonium molecules are known to inhibit AChE, the obtained affinity constants of the tested bispyridinium compounds were compared with the inhibition of human AChE. In competition experiments, bispyridinium derivatives of longer linker length displaced [(3)H]epibatidine and inhibited AChE strongly. Bispyridinium compounds with short linkers, at most, have an allosteric interaction with the [(3)H]epibatidine binding sites and barely inhibited AChE. In dependence on alkane linker length, the bispyridinium compounds seemed to interact at different binding sites. However, the exact binding sites of the bispyridinium compounds responsible for the positive pharmacological effects have still not been identified, making predictive drug design difficult.

Restoration of soman-blocked neuromuscular transmission in human and rat muscle by the bispyridinium non-oxime MB327 in vitro.

The standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes is not sufficiently effective against all types of nerve agents. Alternative therapeutic strategies are required and bispyridinium non-oximes, acting as nicotinic antagonists, were identified as promising compounds. A previous study showed that the di(methanesulfonate) salt of the bispyridinium compound MB327 could restore soman-impaired neuromuscular function in vitro and improve survival of sarin, soman and tabun poisoned guinea pigs in vivo. Here, by using the indirect field stimulation technique, the ability of MB327 to counteract soman-impaired neuromuscular transmission was investigated in human intercostal muscle and rat diaphragm preparations. MB327 restored muscle force in a concentration-dependent manner in both species without reactivating soman-inhibited acetylcholinesterase. The therapeutic effect of MB327 could be washed out, indicating a direct effect at the nicotinic receptor level. Also the ability of MB327 to restore respiratory muscle function could be demonstrated for the first time in rat and human tissue. In combination with previous in vitro and in vivo findings MB327 may be considered a promising compound for the treatment of nerve agent poisoning and further studies are needed to identify optimized drug combinations, concentrations and dosing intervals to provide an effective therapy for OP poisoning.

Competition radioligand binding assays for the investigation of bispyridinium compound affinities to the human muscarinic acetylcholine receptor subtype 5 (hM(5) ).

Standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes lacks efficacy with some nerve agents. Promising in vitro and in vivo results were obtained with the bispyridinium compound SAD-128 which was partly attributed to its interaction with nicotinic acetylcholine receptors. Previous studies indicate that bispyridinium compounds interact with muscarinic acetylcholine receptors as well. The muscarinic M(5) receptor is not well investigated compared to other subtypes, but could be important in the search for new drugs for treating nerve agent poisoning. A set of bispyridinium compounds structurally related to SAD-128 were tested in competition binding experiments with recombinant human M(5) muscarinic acetylcholine receptors. Five of the six investigated bispyridinium compounds interacted with the orthosteric binding site, with affinities in the low micromolar range. These data indicate that interaction of bispyridinium compounds with muscarinic receptors may contribute to their therapeutic efficacy.

Restoration of nerve agent inhibited muscle force production in human intercostal muscle strips with HI 6.

An important factor for successful therapy of poisoning with organophosphorus compounds (OP) is the rapid restoration of blocked respiratory muscle function. To achieve this goal, oximes are administered for reactivation of inhibited acetylcholinesterase (AChE). Unfortunately, clinically used oximes, e.g. obidoxime and pralidoxime, are of limited effectiveness in poisoning with different OP nerve agents requiring the search for alternative oximes, e.g. HI 6. In view of substantial species differences regarding reactivation properties of oximes, the effect of HI 6 was investigated with sarin, tabun and soman exposed human intercostal muscle. Muscle force production by indirect field stimulation and the activity of the human muscle AChE was assessed. 30 µM HI 6 resulted in an almost complete recovery of sarin blocked muscle force and in an increase of completely inhibited muscle AChE activity to approx. 30% of control. In soman or tabun exposed human intercostal muscle HI 6 (50 and 100 µM) had no effect on blocked muscle force or on inhibited human muscle AChE activity. In addition, HI 6 up to 1000 µM had no effect on soman blocked muscle force indicating that this oxime has no direct, pharmacological effect in human tissue. These results emphasize that sufficient reactivation of AChE is necessary for a beneficial therapeutic effect on nerve agent blocked neuromuscular transmission.

Interaction of bispyridinium compounds with the orthosteric binding site of human α7 and Torpedo californica nicotinic acetylcholine receptors (nAChRs).

Standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes lacks efficacy with different nerve agents. A direct pharmacologic intervention at the nicotinic acetylcholine receptor (nAChR) was proposed as an alternative therapeutic approach and promising in vitro and in vivo results were obtained with the bispyridinium compound SAD-128. In addition, a number of SAD-128 analogues improved neuromuscular transmission of soman-poisoned diaphragms in vitro. We investigated the interaction of six of these SAD-128 analogues with the orthosteric binding site of the human α7 nAChR and Torpedo californica nAChR with a high-throughput assay using radioactive ligands. The determined affinity constants indicate a weak interaction of three test compounds (K(i) in the micromolar range) with both receptors, but no interaction could be recorded with the other three test compounds. The six SAD-128 analogues showed a low intrinsic inhibitory potency with human acetylcholinesterase (IC50 > 400 µM). In conclusion, the results of the present study do not indicate a correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission and it is assumed that other mechanisms contribute to the therapeutic effect of the tested compounds.

[Congenital pulmonary fibrosarcoma. Differential diagnosis of infantile pulmonary spindle cell tumors]. / Kongenitales pulmonales Fibrosarkom. Differenzialdiagnose infantiler pulmonaler spindelzelliger Tumoren.

Primary pulmonary mesenchymal tumors are rare causes of intrathoracic lesions in newborns. We describe a case of pulmonary spindle-cell tumor with features of infantile fibrosarcoma and discuss the differential diagnosis of spindle-cell lesions in this location. In view of further case reports of the literature, this neoplasia can best be categorized in a spectrum of fibroblastic/myofibroblastic differentiated spindle-cell tumors, with excellent prognosis. Especially in congenital lesions a favorable clinical course is to be expected after complete surgical resection. Additional radio- and/or chemotherapy is not recommended.

Polyvinylchloride infusion lines expose infants to large amounts of toxic plasticizers.

PURPOSE: The purpose of this study was to evaluate whether infusion lines are able to leach plasticizers in substantial amounts and thus be a candidate substance for hepatotoxic effects during long-term total parenteral nutrition (TPN). METHODS: TPN solutions, blood products, and selected drugs typical for preterm infants concerning amount, content, and infusion time were perfused through common polyvinylchloride (PVC) infusion lines. Concentration of diethylhexyl-phthalate (DEHP) before and after perfusion was determined by gas chromatography/mass spectrometry. RESULTS: Daily quantities of DEHP by 24-hour infusions were Lipid emulsion 20%: 10185.6 microg; aminoacid/glucose-solution: 116.2 microg; midazolaminfusion for sedation: 26.4 microg; fentanyl for sedation: 132.5 microg; propofol for sedation: 6561.0 microg. The amount of DEHP by single doses of blood products (20 mL) were packed red blood cells: 144-608 microg; platelet rich plasma: 928 microg; and fresh frozen plasma: 552-8108 microg. The dose of DEHP for a typical preterm neonate requiring TPN and additional therapy like sedation or blood products is at minimum 10 mg and can easily reach 20 mg/d. CONCLUSION: This large amount of DEHP is especially disturbing, because it effects the most vulnerable patients (neonates). Whether there is a relation to TPN-induced hepatobiliary dysfunction remains to be elucidated and is under investigation. With respect to recent literature, a biological effect of these doses must be assumed.

Toxaphenes and chlorinated naphthalenes in adipose tissue of children.

BACKGROUND: Chlorinated hydrocarbons are ingested by humans in food and accumulate in adipose tissue. At the University Kinderklinik, Mannheim, previously unknown substances have been found in children (e.g., the pesticide toxaphene and chlorinated naphthalenes). These substances have been widely used for industrial purposes in the past. Samples from West and East Germany; Saratov, Russia; and Almaty, Kazakhstan were examined to determine whether these substances are ubiquitous. METHODS: After Soxhlet extraction, the extracts were cleaned up using a liquid chromatographic technique. Measurement was performed by gas chromatography-mass spectrometry using negative chemical ionization in the single-ion-monitoring mode. RESULT: In specimens from all cities, toxaphene congeners Parlar 26 and Parlar 50 and six chlorinated naphthalenes were traced. Highest median load of toxaphene was 1.97 microg/kg for Parlar 26 and 2.36 microg/kg for Parlar 50 in Stralsund, East Germany. For chlorinated naphthalenes, the median was highest in Mannheim, West Germany, with 12.0 microg/kg. CONCLUSION: These findings show that monitoring these toxic substances remains necessary. Even though the use and as a consequence the amount of chlorinated hydrocarbons were reduced, these substances have by no means disappeared from the environment.

Is there a correlation between organochlorine compounds and undescended testes?

Some pesticides and synthetic chemicals are known to act as hormonal modulators, often possessing oestrogenic activity (xenooestrogens). They are persistent and accumulate in fatty tissue. Aim of our study is to address the question, whether a selection of such compounds is to be found in the fatty tissue of children undergoing surgical procedures and whether there are differences in values obtained from patients with or without undescended testes. Fat samples of 48 patients, 18 of whom had undescended testes, were examined by high-resolution gas chromatography and mass spectrometry for DDT and metabolites, polychlorinated biphenyls (PCB), toxaphenes, hexachlorocyclohexane (HCH), chlorinated cyclodienes and chlorinated benzenes. We were able to find accumulation of all substances in every patient. Statistical analysis revealed a highly significant difference between patients from the control group and those from the undescended testes group for two compounds, namely heptachloroepoxide (HCE) and hexachlorobenzene (HCB), increased values being found in the patients with undescended testes. Since the aetiology of this entity is unknown in most of the cases, prenatal exposure to exogenous oestrogens is an attractive and plausible hypothesis. In order to confirm this, some questions will have to be answered in further studies: effect of exposure to xenooestrogens during a specific period of development, probable role of other substances with proven or suspected hormonal activity, potential synergism of such compounds and differences in individual susceptibility.

Fibrinogen heterogeneity in homozygous plasminogen deficiency type I: further evidence that plasmin is not involved in formation of LMW- and LMW'-fibrinogen.

Human plasma fibrinogen is heterogeneous in SDS-polyacrylamide gel electrophoresis and other methods for separation of proteins by molecular size. A high molecular weight fraction (HMW-fibrinogen, 340 kD) contributes approximately 50% of total fibrinogen antigen. Low molecular weight fibrinogen (LMW-fibrinogen, 300 kD) adds another 40%. The residual amount is LMW'-fibrinogen with a molecular weight of 280 kD, and a small amount of very high molecular weight fibrinogen (Fib420), the product of alternative splicing of the A alpha-chain genetic information, resulting in an extended A alpha-chain C-terminus. Fibrinogen was detected by specific immunostaining of nonreduced SDS-PAGE gel immunoblots with antibodies against fibrinopeptide A. Using densitometric scans of the immunoblots we found a ratio of HMW-, LMW- and LMW'-fibrinogen in a patient with homozygous plasminogen deficiency that was similar to the ratio found in immunoblots of plasma from healthy blood donors. Treatment with plasminogen concentrate resulted in a slight decrease of the proportion of HMW-fibrinogen, followed by an increase to 78%. The LMW'-fibrinogen band gained intensity initially, increasing to 11.9% of fibrinogen antigen 6 h after starting plasminogen infusion, but then dropped to levels below detection limit of the immunoblotting assay. LMW-fibrinogen remained constant during the initial 72 h of plasminogen treatment, then dropping to values in the range of 22-25% afterwards. The proportion of HMW-, LMW-, and LMW'-fibrinogen again reached the initial levels two weeks after starting treatment with plasminogen concentrate. We conclude that plasminogen is not involved in the limited proteolysis leading to formation of LMW-fibrinogen and LMW'-fibrinogen in the absence of a generalized fibrinolytic condition. Fibrinolytic activation may lead to the formation of fibrinogen degradation product X, which appears in a similar position as LMW'-fibrinogen in SDS-PAGE.

[Chlorinated carbohydrate content of fetal and pediatric organs and tissues]. / Gehalt fetaler und kindlicher Organe und Gewebe an chlorierten Kohlenwasserstoffen.

Organic halogens are used as pesticides and in chemical industries. They are secreted with breast milk and accumulated in fat tissue of infants. Organic halogens can be found already in newborns. We analysed polychlorinated biphenyl (PCB), DDT, hexachlorocyclohexane (HCH), and heptachlor in subcutaneous fat tissue and other tissues (placenta, liver, kidney, lung, brain, thymus, muscle, heart) of 34 fetuses and dead children. These substances were found regularly in placenta, in fetal subcutaneous fat tissue and in fetal organs. They therefore can influence possibly early and sensitive stages of intrauterine development. The average concentrations found in fetal fat tissue were: PCB 0.7 mg/kg fat tissue, DDT 0.7 mg/kg, HCH 0.14 mg/kg, and heptachlor 0.03 mg/kg.

Chlorinated hydrocarbons in human bone marrow of healthy individuals and leukemia patients.

In this pilot study, concentrations of chlorinated hydrocarbons in bone marrow were determined by capillary-column gas chromatography. Bone marrow was obtained from a total of 29 healthy adults and from patients with leukemia or lymphoma. The chlorinated hydrocarbons in adults that occurred in the highest concentrations were dichlorodiphenyltrichloroethane and its derivatives (mean = 129 mg/g DNA). Hexachlorobenzene, the hexachlorohexane isomers, and dieldrin were also found, but in reduced concentrations (i.e., mean concentrations: 49.5 mg/g DNA, 14.9 mg/g DNA, and 5.9 mg/g DNA, respectively). Patients who had leukemia and lymphoma did not have significantly increased concentrations of chlorinated hydrocarbons.

A comparison of the concentrations of certain chlorinated hydrocarbons and polychlorinated biphenyls in bone marrow and fat tissue of children and their concentrations in breast milk.

Chlorinated hydrocarbon (CHC) and polychlorinated biphenyl (PCB) concentrations in the bone marrow of 57 children were compared with the concentrations in adipose tissue of 50 children and the concentrations in breast milk in the Federal Republic of Germany from 1984 to 1991. The concentrations of hexachlorobenzene (HCB), the dichlorodiphenyltrichloroethane (DDT)-metabolites, and polychlorinated biphenyl (PCB) congeners no. 138 and no. 153 were increased threefold, while the concentrations of several hexachloro-cyclohexane (HCH)-isomers and PCB congener no. 180 were only increased twofold. Because breast feeding is the primary source of CHC and PCB in toddlers and infants we also compared the concentrations in bone marrow of children with the concentrations in breast milk and found approximately fourfold higher concentrations for the most highly chlorinated PCB congener no. 180, but only threefold higher concentration for PCB 138 and 153 and the DDT-metabolites. The concentrations of beta-HCH and HCB were only slightly higher in bone marrow.

[Simultaneous determination of inorganic anions in body fluids. A new method in pediatric laboratory diagnosis--results of a pilot study]. / Simultane Bestimmung anorganischer Anionen in Körperflüssigkeiten. Eine neue Methode in der pädiatrischen Labordiagnostik--Ergebnisse einer Pilotstudie.

The anions analysis was a methodical problem up to now. This was the reason for low interest. Biological fluids like saliva and urine which could easily receive without any stress for the children, are little investigated for its capacity on nitrite, nitrate, bromide and sulfate. In this performance there will presented an ion-chromatographic method to determine inorganic anions in the following body-fluids: serum saliva, liquor and urine. The anions chloride, nitrite, bromide, nitrate, phosphate and sulfate was determined quantitatively. The method was proved in a pilot-study on children's body-fluids serum, liquor and saliva. The objects was to get a landmark in expectation from anion concentrations. Bromide was detected as a constant part in all body fluids. The origin and importance is not clear till now. Also was found nitrate in all investigated body fluids. There seems to be a connection between diarrhea and an increase in serum levels from nitrate. We found considerable amounts of nitrate in saliva by babies and infants. The method is distinguished by little fluctuation in measurement and high specificity. Short time in analysis and simple handling will do the method for a qualified one in pediatrics.

A pilot study on polychlorinated biphenyl levels in the bone marrow of healthy individuals and leukemia patients.

In this pilot study the concentrations of polychlorinated biphenyls (PCBs) was determined by capillary column gas chromatography in bone marrow from 29 adults. The highest concentration in all adult individuals was detected for PCB no. 180 (mean = 0.991) followed by two other highly chlorinated PCBs, no. 153 (mean = 0.918) and no. 138 (mean = 0.927). The less chlorinated PCBs, no. 101 (mean = 0.255), no. 52 (mean = 0.161), and no. 28 (mean = 0.324) contributed to a lesser extent. Additional samples from children (N = 19) were used to assess the dependence of PCB concentrations on patient age (Scheele et al. Eur J Pediatr 1992; 151:802-805). When comparing the data of adult leukemia and lymphoma patients with a reference group of healthy adult individuals, no significant increase in the leukemia patients was found.

Chlorinated hydrocarbons in the bone marrow of children: studies on their association with leukaemia.

Concentrations of chlorinated hydrocarbons and polychlorinated biphenyls (PCB) were determined by capillary column gas chromatography in samples of bone marrow from 38 children with leukaemia (16 samples/pools) and 15 control (5 pools). The highest mean and median concentrations were detected for total PCB (mean = 3.568 mg/kg fat basis/median 2.904 mg/kg) followed by the sum of the dichlorodiphenyltrichloroethane metabolites (1.775/1.059 mg/kg), hexachlorobenzene 0.354/0.260 mg/kg), the sum of the hexachlorocyclohexane isomers (0.133/0.093 mg/kg) and dieldrin (0.109/0.063 mg/kg). The CHC and PCB concentrations in bone marrow were two- to threefold higher than in fat tissue. Comparing children with and without leukaemia similar concentrations of CHC and PCB were found.

[Exposure of our children to pesticides, PCB and potentially critical anions]. / Belastung unserer Kinder mit Pestiziden, PCB und potentiell kritischen Anionen.

Among environmental pollutants the organohalogens still play an important role since they accumulate in human fat tissue and are secreted with mother's milk during lactation. Fortunately DDT-, HCH- and HCB-levels decreased in breast milk during the last years. In contrast, the PCB concentrations were still three-to five-fold above the permitted limits for cow's milk. Fat tissue of 262 newborns, infants and children was already as severely loaded with organohalogens as human milk. However, children with malignant tumors or congenital malformations did not show elevated concentrations in fat tissue. The significance of the potentially critical anions such as nitrate, nitrite, bromide, sulfate for the health of our children needs further clarification.