Mice carrying a humanized Foxp2 knock-in allele show region-specific shifts of striatal Foxp2 expression levels.

Genetic and clinical studies of speech and language disorders are providing starting points to unravel underlying neurobiological mechanisms. The gene encoding the transcription factor FOXP2 has been the first example of a gene involved in the development and evolution of this human-specific trait. A number of autosomal-dominant FOXP2 mutations are associated with developmental speech and language deficits indicating that gene dosage plays an important role in the disorder. Comparative genomics studies suggest that two human-specific amino acid substitutions in FOXP2 might have been positively selected during human evolution. A knock-in mouse model carrying these two amino acid changes in the endogenous mouse Foxp2 gene (Foxp2hum/hum) shows profound changes in striatum-dependent behaviour and neurophysiology, supporting a functional role for these changes. However, how this affects Foxp2 expression patterns in different striatal regions and compartments has not been assessed. Here, we characterized Foxp2 protein expression patterns in adult striatal tissue in Foxp2hum/hum mice. Consistent with prior reports in wildtype mice, we find that striatal neurons in Foxp2hum/hum mice and wildtype littermates express Foxp2 in a range from low to high levels. However, we observe a shift towards more cells with higher Foxp2 expression levels in Foxp2hum/hum mice, significantly depending on the striatal region and the compartment. As potential behavioural readout of these shifts in Foxp2 levels across striatal neurons, we employed a morphine sensitization assay. While we did not detect differences in morphine-induced hyperlocomotion during acute treatment, there was an attenuated hyperlocomotion plateau during sensitization in Foxp2hum/hum mice. Taken together, these results suggest that the humanized Foxp2 allele in a mouse background is associated with a shift in striatal Foxp2 protein expression pattern.

Chemical stabilization of polymers: Implications for dermal exposure to additives.

Technical benefits of additives in polymers stand in marked contrast to their associated health risks. Here, a multi-analyte method based on gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) was developed to quantify polymer additives in complex matrices such as low-density polyethylene (LDPE) and isolated human skin layers after dermal exposure ex vivo. That way both technical aspects and dermal exposure were investigated. The effects of polymer additivation on the material were studied using the example of LDPE. To this end, a tailor-made polymer was applied in aging studies that had been furnished with two different mixtures of phenol- and diarylamine-based antioxidants, plasticizers and processing aids. Upon accelerated thermo-oxidative aging of the material, the formation of LDPE degradation products was monitored with attenuated total reflectance-Fourier transformed infrared (ATR-FTIR) spectroscopy. Compared to pure LDPE, a protective effect of added antioxidants could be observed on the integrity of the polymer. Further, thermo-oxidative degradation of the additives and its kinetics were investigated using LDPE or squalane as matrix. The half-lives of additives in both matrices revealed significant differences between the tested additives as well as between LDPE and squalane. For instance, 2-tert-butyl-6-[(3-tert-butyl-2-hydroxy-5-methylphenyl)methyl]-4-methylphenol (Antioxidant 2246) showed a half-life 12 times lower when incorporated in LDPE as compared to squalane. As a model for dermal exposure of consumers, human skin was brought into contact with the tailor-made LDPE containing additives ex vivo in static Franz diffusion cells. The skin was then analyzed for additives and decomposition products. This study proved 10 polymer additives of diverse pysicochemical properties and functionalities to migrate out of the polymer and eventually overcome the intact human skin barrier during contact. Moreover, their individual distribution within distinct skin layers was demonstrated. This is exemplified by the penetration of the procarcinogenic antioxidant N-phenylnaphthalen-2-amine (Neozon D) into the viable epidermis and the permeation through the skin of the neurotoxic plasticizer N-butylbenzenesulfonamide (NBBS). In addition, the analyses of additive degradation products in the isolated skin layers revealed the presence of 2-tert-butyl-4-methylphenol in all layers after contact to a polymer with substances of origin like Antioxidant 2246. Thus, attention needs to be paid to absorption of polymer additives together with their degradation products when it comes to dermal exposure assessment.

Skin permeation of polycyclic aromatic hydrocarbons: A solvent-based in vitro approach to assess dermal exposures against benzo[a]pyrene and dibenzopyrenes.

Consumer products with high contents of polycyclic aromatic hydrocarbons (PAHs) were repeatedly identified by market surveillance authorities. Since several of the individual compounds have been identified as genotoxic carcinogens, there might be health risks associated with the usage of these items. It therefore becomes reasonable to argue to reduce PAH contents in consumer products to a level as low as possible. This study presents data on the migration of PAHs from consumer products into aqueous sweat simulant or aqueous ethanol and on its combined migration and penetration into human skin. Product specimens were either submerged in simulant, or placed directly on test skins in Franz cell chambers to simulate dermal contacts. Migration of hexacyclic dibenzopyrenes became detectable by using ethanolic simulant, but not in aqueous sweat simulant. Similarly, migration of the pentacyclic model carcinogen benzo[a]pyrene (B[a]P) into aqueous sweat simulant was significantly lower when compared with human skin or skin models. The results point to a gross underestimation (about two orders of magnitude) when using aqueous sweat simulant instead of human skin for assessing PAH migration. On the other side, the usage of 20% ethanol as simulant revealed good agreement to the actual exposure of human skin against B[a]P migrating out of contaminated products. Our results underline that aqueous sweat simulant is not suitable to study dermal migration of highly lipophilic compounds.

[Occurrence and relevance to health of persistent organic substances and phthalates in breast milk]. / Vorkommen und gesundheitliche Bedeutung von persistenten organischen Substanzen und Phthalaten in der Muttermilch.

The aim of this study is to give an overview of the concentrations of persistent organic pollutants like the polychlorinated dibenzo- P-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), polychlorinated biphenyls (PCB), polybrominated diphenyl ether (PBDE), perfluorinated compounds (PFC) and of phthalates in breast milk. On the basis of median and 95 (th) percentile values an "average" and a "high" intake were calculated for a 3-month-old infant exclusively breast-fed. Moreover, the actual daily intake was compared with tolerable daily intakes (TDI) recommended by scientific institutions. On this basis, we found an "average" ("high") daily intake of 70 (140) pg TEQ/kg body weight (b. w.) for PCDD/F and dioxin-like PCB (dl-PCB), 10 (20) ng/kg b. w. for PFOS (perfluorooctanesulfonate), 20 (50) ng/kg b. w. for PFOA (perfluorooctanoate), 1.7 (7.5) ng/kg b. w. for BDE 47, and 0.6 (2.1) ng/kg b. w. for BDE 99. For di-2-ethylhexyl phthalate (DEHP) and di- N-butyl phthalate (DnBP) an "average" and "high" intake of 400 ng/kg b. w. and 2,000 ng/kg b. w. and of 100 and 500 ng/kg b.w. were assumed, respectively. For all of these substances we found a daily intake via breast milk below the TDI, established on a livelong basis. On contrary, the daily intake for the sum of the PCDD/F and dl-PCB considerably exceeded the recommended TDI value. Even with regard to the "high" daily intake values the share of PBDE, PFC, and phthalates on the TDI was only in the lower percentage. Scientific organisations assume that an exceeding of the PCDD/F and dl-PCB intake in relation to the TDI value is acceptable only on the basis of the still declining levels in breast milk and the fact that this high exposure only occurs during some months of the entire life when breast milk is consumed. On the basis of the recent exposure situation mothers can exclusively breast-feed their infants for 6 months without any hesitation. The well established health benefits for mothers and infants when exclusively breast-feeding should be utilised. There is also no health concern if the mother decides to breast-feed the baby for longer than 6 months when the infant also receives additional food.

Exposition to and health effects of residues in human milk.

A great variety of drugs, cosmetics, food ingredients as well as environmental contaminants are secreted with human milk as a result of actual exposure or the accumulated body burden of the mother. Of great concern and least amenable to short-term intervention are persistent substances in the environment with long half-lives in the body due to their lipophilic properties and minimal degradation. Polyhalogenated aromatic hydrocarbons, namely organochlorine pesticides, polychlorinated biphenyls (PCB) and polychlorinated dibenzodioxins (PCDD) and dibenzofurans (PCDF) are fetotoxic, neurotoxic, immunotoxic, some are promoting carcinogens and/or interfere with hormonal receptors. They pass the placenta and equilibrate among the lipid compartments of the body including breast milk lipids. Transplacental exposure is more relevant with regard to physical development and cognitive functioning of the child than postnatal exposure via breastmilk. Restrictions for production, use and release have been successful in decreasing exposure as shown by a downward trend of their contents both in human milk and serum lipids for the last 15 to 20 years. It is difficult to evaluate the potentially late effects of the exposure via breastmilk which is 10 to 100 times higher in industrialised countries than the tolerable daily intake (TDI) of 1 to 4 toxic equivalents (WHO-TEQ) pg/kg/day established in 1998 by WHO for dioxins and dioxin-like PCBs but which lasts for 0.6% of the expected life span only. Carefully conducted long-term follow-up of cohorts with defined exposure levels, with consideration of numerous biological and psychological parameters, is expected to provide the answer.

CYP2E1 expression in bone marrow and its intra- and interspecies variability: approaches for a more reliable extrapolation from one species to another in the risk assessment of chemicals.

When characterizing the health risks for man by exposure to chemicals, species-specific differences have to be taken into consideration, otherwise extrapolation from animal data to the human situation would be inadequate. The site-specific toxicity of chemicals may be explained by the following alternatives: (1) reactive metabolites are generated in the liver and subsequently transported to the target tissue(s); (2) metabolism of the parent compound occurs in the target tissue, a pathway by which the enzymes necessary for activation must be expressed in the target tissue. Cytochrome P450 2E1 (CYP2E1) is an important phase-I enzyme activating several chemicals. In the study described in this paper, myeloid intra- and interspecies variability in the expression of CYP2E1 has been investigated in rats, rabbits and man, because the bone marrow represents an important target organ for toxic effects of several chemicals, e.g. benzene. CYP2E1 at the protein level was detected by Western blotting and enzyme activities were determined by CYP2E1-dependent hydroxylation of chlorzoxazone (CLX). In the bone marrow of Wistar rats, the CLX hydroxylase activities were within the same order of magnitude (range: 0.1-0.4 pmol/mg protein per min) as previously described for mice (range 0.2-0.8 pmol/mg protein per min), whereas the CYP2E1 activities in two strains of rabbits were significantly higher (range: 1.7-4.7 pmol/mg protein per min) than in the rodents (P < 0.05). In human CD34+ bone marrow stem cells, CYP2E1 could also be detected on the protein level by Western blotting. The data demonstrate a presence of CYP2E1 in the bone marrow of all species investigated, thus supporting the hypothesis of CYP2E1-dependent local metabolism of several chemicals as a factor possibly contributing to their myelotoxicity and haematotoxicity. The data show that intraspecies/intrastrain variability of CYP2E1 activity in rodents is small. However, CYP2E1 activity between rodents and a non-rodent species was quite different indicating considerable interspecies variability.

CYP2E1-dependent benzene toxicity: the role of extrahepatic benzene metabolism.

Benzene, a ubiquitous environmental pollutant, is haematotoxic and myelotoxic. As has been shown earlier, cytochrome P450 2E1 (CYP2E1)-dependent metabolism is a prerequisite for the cytotoxic and genotoxic effects of benzene, but which of the benzene metabolites produces toxicity is still unknown. The observed differences between the toxicity of benzene and that of phenol, a major metabolite of benzene, could be explained by alternative hypotheses. That is, whether (1) toxic benzene effects are caused by metabolites not derived from phenol (e.g. benzene epoxide, muconaldehyde). which are formed in the liver and are able to reach the target organ(s); or (2) benzene penetrates into the bone marrow, where local metabolism takes place, whereas phenol does not reach the target tissue because of its polarity. To further investigate hypothesis 2, we used various strains of mice (AKR, B6C3F1, CBA/Ca, CD-1 and C57B1/6), for which different toxic responses have been reported in the haematopoietic system after chronic benzene exposure. In these strains, CYP2E1 expression in bone marrow was investigated and compared with CYP2E1 expression in liver by means of two independent methods. Quantification of CYP2E1-dependent hydroxylation of chlorzoxazone (CLX) by high-performance liquid chromatography (HPLC; functional analysis) was used to characterize specific enzymatic activities. Protein identification was performed by Western blotting using CYP2E1-specific antibodies. In liver microsomes of all strains investigated, considerable amounts of CYP2E1-specific protein and correspondingly high CYP2E1 hydroxylase activities could be detected. No significant differences in CYP2E1-dependent enzyme activities were found between the five strains (range of medians, 4.6 12.0 nmol 6-OH-CLX/[mg protein x min]) in hepatic tissue. In the bone marrow, CYP2E1 could also be detected in all strains investigated. However, chlorzoxazone hydroxylase activities were considerably lower (range of medians, 0.2-0.8x10(-3) nmol 6-OH-CLX/[mg protein x min]) compared with those obtained from liver microsomes. No significant (P>0.05) interstrain differences in CYP2E1 expression in liver and/or bone marrow could be observed in the mouse strains investigated. The data obtained thus far from our investigations suggest that strain-specific differences in the tumour response of the haematopoietic system of mice chronically exposed to benzene cannot be explained by differences in either hepatic or in myeloid CYP2E1-dependent metabolism of benzene.

[Determination of the by-product 10-bromocarbamazepine in the drug carbamazepine with DC- and differential pulse polarography]. / Bestimmung des Nebenproduktes 10-Bromcarbamazepin im Wirkstoff Carbamazepin durch Gleichstrom- und Differenzpulspolarografie.

The quantitative determination of the impurity 10-bromocarbamazepine (caused by manufacturing method) in the drug carbamazepine is possible by using their cathodic two-electron debromination at a dropping mercury electrode in tetraethylammonium perchlorate/acetonitrile or 80% aqueous methanol as supporting electrolyte. Direct current polarographic (dcp) and differential pulse polarographic (dpp) methods are described which can be used in process control and quality control of the drug production. These analytic methods allow to determine 10-bromocarbamazepine in carbamazepine up to a limiting concentration of 3 X 10(-5) mol/l (100 ppm bromine; dcp) and of 3 X 10(-6) mol/l (10 ppm bromine; dpp). On the basis of electroanalytical results the mechanism of the polarographic reduction of 10-bromocarbamazepine is discussed.