Mono-n-hexyl phthalate: exposure estimation and assessment of health risks based on levels found in human urine samples.

Mono-n-hexyl phthalate (MnHexP) is a primary metabolite of di-n-hexyl phthalate (DnHexP) and other mixed side-chain phthalates that was recently detected in urine samples from adults and children in Germany. DnHexP is classified as toxic for reproduction category 1B in Annex VI of Regulation (EC) 1272/2008 and listed in Annex XIV of the European chemical legislation REACH; thereby, its use requires an authorisation. Health-based guidance values for DnHexP are lacking and a full-scale risk assessment has not been carried out under REACH. The detection of MnHexP in urine samples raises questions about the sources of exposure and concerns of consumer safety. Here, we propose the calculation of a provisional oral tolerable daily intake value (TDI) of 63 µg/kg body weight/day for DnHexP and compare it to intake levels corresponding to levels of MnHexP found in urine. The resulting mean intake levels correspond to less than 0.2% of the TDI, and maximum levels to less than 5%. The TDI was derived by means of an approximate probabilistic analysis using the credible interval from benchmark dose modelling of published ex vivo data on reduced foetal testosterone production in rats. Thus, for the dose associated to a 20% reduction in testosterone production, a lower and upper credible interval of 14.9 and 30.0 mg/kg bw/day, respectively, was used. This is considered a conservative approach, since apical developmental endpoints (e.g. changed anogenital distance) were only observed at higher doses. In addition, we modelled various scenarios of the exposure to the precursor substance DnHexP from different consumer products, taking measured contamination levels into account, and estimated systemic exposure doses. Of the modelled scenarios including the application of sunscreen (as a lotion or pump spray), the use of lip balm, and the wearing of plastic sandals, and considering conservative assumptions, the use of DnHexP-contaminated sunscreen was highlighted as a major contributing factor. A hypothetical calculation using conservative assumptions for the latter resulted in a margin of safety in relation to the lower credible interval of 3267 and 1007 for adults and young children, respectively. Most importantly, it was found that only a fraction of the TDI is reached in all studied exposure scenarios. Thus, with regard to the reported DnHexP exposure, a health risk can be considered very unlikely.

Mineral oil in food, cosmetic products, and in products regulated by other legislations.

For a few years, mineral oils and their potential adverse health effects have been a constant issue of concern in many regulatory areas such as food, cosmetics, other consumer products, and industrial chemicals. Analytically, two fractions can be distinguished: mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH). This paper aims at assessing the bioaccumulative potential and associated histopathological effects of MOSH as well as the carcinogenic potential of MOAH for consumer-relevant mineral oils. It also covers the absorption, distribution, metabolism, and excretion of MOSH and MOAH upon oral and dermal exposures. The use and occurrence of consumer-relevant, highly refined mineral oils in food, cosmetics and medicinal products are summarized, and estimates for the exposure of consumers are provided. Also addressed are the challenges in characterizing the substance identity of mineral oil products under REACH. Evidence from more recent autopsy and biopsy studies, along with information on decreasing food contamination levels, indicates a low risk for adverse hepatic lesions that may arise from the retention of MOSH in the liver. With respect to MOAH, at present there is no indication of any carcinogenic effects in animals dermally or orally exposed to highly refined mineral oils and waxes. Such products are used not only in cosmetics but also in medicinal products and as additives in food contact materials. The safety of these mineral oil-containing products is thus indirectly documented by their prevalent and long-term use, with a simultaneous lack of clinical and epidemiological evidence for adverse health effects.

The Hypothalamic-Pituitary-Adrenal Axis and Serotonin Metabolism in Individual Brain Nuclei of Mice with Genetic Disruption of the NK1 Receptor Exposed to Acute Stress.

Mice lacking the substance P (SP) neurokinin-1 (NK1) receptor (NK1R-/-mice) were used to investigate whether SP affects serotonin (5-HT) function in the brain and to assess the effects of acute immobilisation stress on the hypothalamic-pituitary-adrenocortical (HPA) axis and 5-HT turnover in individual brain nuclei. Basal HPA activity and the expression of hypothalamic corticotropin-releasing hormone (CRH) in wild-type (WT)- and NK1R-/- mice were identical. Stress-induced increases in plasma ACTH concentration were considerably higher in NK1R-/- mice than in WT mice while corticosterone concentrations were equally elevated in both mouse lines. Acute stress did not alter the expression of CRH. In the dorsal raphe nucleus (DRN), basal 5-HT turnover was increased in NK1R-/- mice and a 15 min stress further magnified 5-HT utilisation in this region. In the frontoparietal cortex, medial prefrontal cortex, central nucleus of amygdala, and the hippocampal CA1 region, stress increased 5-HT and/or 5-hydroxyindoleacetic acid (5-HIAA) concentrations to a similar extent in WT and NK1R-/- mice. 5-HT turnover in the hypothalamic paraventricular nucleus was not affected by stress, but stress induced similar increases in 5-HT and 5-HIAA in the ventromedial and dorsomedial hypothalamic nuclei in WT and NK1R-/- mice. Our findings indicate that NK1 receptor activation suppresses ACTH release during acute stress but does not exert sustained inhibition of the HPA axis. Genetic deletion of the NK1 receptor accelerates 5-HT turnover in DRN under basal and stress conditions. No differences between the responses of serotonergic system to acute stress in WT and NK1R-/- mice occur in forebrain nuclei linked to the regulation of anxiety and neuroendocrine stress responses.

A medical-toxicological view of tattooing.

Long perceived as a form of exotic self-expression in some social fringe groups, tattoos have left their maverick image behind and become mainstream, particularly for young people. Historically, tattoo-related health and safety regulations have focused on rules of hygiene and prevention of infections. Meanwhile, the increasing popularity of tattooing has led to the development of many new colours, allowing tattoos to be more spectacular than ever before. However, little is known about the toxicological risks of the ingredients used. For risk assessment, safe intradermal application of these pigments needs data for toxicity and biokinetics and increased knowledge about the removal of tattoos. Other concerns are the potential for phototoxicity, substance migration, and the possible metabolic conversion of tattoo ink ingredients into toxic substances. Similar considerations apply to cleavage products that are formed during laser-assisted tattoo removal. In this Review, we summarise the issues of concern, putting them into context, and provide perspectives for the assessment of the acute and chronic health effects associated with tattooing.

Towards the limiting of health risks associated with tattooing: whitelists for tattoo pigments and preservatives.

The number of pigments that could potentially be used in tattoo inks is vast. However, pigments are generally not manufactured for the purpose of being injected into subepidermal layers of the skin. Assuming 100% bioavailability after injection means that pigments can be imminently hazardous to human health. Given the ever-increasing number of pigments being circulated on the market or through the internet, a 'negative list' ('black' list) containing pigments with known adverse effects will never be finalised. If incriminated, substances could easily be replaced by structurally similar pigments that might be even more deleterious to human health. Therefore, we and others suggest the establishment of a whitelist ('positive list') that would only contain pigments that had undergone a risk assessment specifically for their application into the dermis. Some of the problems associated with such a 'positive list' are discussed. Another important issue with regard to tattoo safety is related to the preservatives used in ink preparations. Notwithstanding the demand for sterile tattoo inks, a whitelist for these compounds would be beneficial. At present, many technical preservatives are being used, despite their known detrimental effects to human health. Criteria for the inclusion of preservatives in a 'positive list' are also discussed.

Prolactin induces MAPK signaling in neural progenitors without alleviating glucocorticoid-induced inhibition of in vitro neurogenesis.

We recently demonstrated that prolactin (PRL) prevents chronic stress-induced inhibition of adult hippocampal neurogenesis. It remained unsettled, however, whether PRL is acting directly on neural stem and progenitors cells (NPCs) or if neurogenesis is affected by an indirect mechanism, for example through the extensively described effects of PRL on the HPA axis. To address this point, we used neurosphere cultures derived from the adult rat hippocampus as an in vitro model for NPCs. Dexamethasone (DEX) was applied to stress the NPCs, and proliferation, survival and differentiation of cells were examined. DEX markedly inhibited proliferation of NPCs and cells entered the G(0) phase of cell cycle. Moreover, DEX reduced NPC survival and repressed astroglial differentiation, which is normally induced by serum or bone morphogenetic protein application. Even though we could demonstrate that NPCs express the PRL receptor and ERK1/2 signaling is induced by PRL, we did not observe any effect of PRL on NPCs proliferation, differentiation or survival, neither in the presence nor during absence of DEX. In summary, our results indicate that PRL action on NPCs and neurogenesis in vivo occurs via an indirect mechanism.

Prolactin induces Egr-1 gene expression in cultured hypothalamic cells and in the rat hypothalamus.

Prolactin (PRL), the major lactogenic hormone, acts also as neuromodulator and regulator of neuronal and glial plasticity in the brain. There is an increase in synthesis and release of PRL within the hypothalamus during peripartum and in response to stress. To identify mechanisms by which PRL induces neuroplasticity, we studied the ability of PRL to induce the transcription factor Egr-1 in the hypothalamic cell line, 4B, in vitro, and in specific neuronal cell types of the hypothalamus in vivo. PRL induced Egr-1 mRNA expression in 4B cells, an effect which was prevented by the MEK inhibitor, U0126. In vivo, intracerebroventricular PRL (1 microg) increased Egr-1 mRNA levels in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) of female rats. The increase in mRNA paralleled elevated Egr-1 protein expression in the PVN and SON. Double staining immunohistochemistry revealed Egr-1 localization in oxytocin neurons of the PVN and SON, but not in vasopressin neurons in these regions. In the dorsomedial PVN, a population of non-oxytocin or vasopressin cells localized in a region corresponding to corticotropin-releasing hormone neurons also showed marked Egr-1 immunoreactivity. The data suggest that PRL modulates plasticity in oxytocinergic neurons, through MAP kinase-dependent induction of Egr-1.

Early postnatal depletion of NMDA receptor development affects behaviour and NMDA receptor expression until later adulthood in rats--a possible model for schizophrenia.

There is increasing evidence that a dysfunction of the N-methyl-d-aspartate (NMDA) receptor system plays a key role in the pathophysiology of schizophrenia. Non-competitive NMDA-antagonists induce schizophrenia-like symptoms and cognitive impairment in healthy humans as well as rodents. As receptor dysfunction precedes clinical disorder manifestation, the present study investigated whether transient perinatal NMDA antagonism constitutes a suitable long-term animal model for schizophrenia. Male Wistar rats were treated from postnatal days 6-21 with the NMDA receptor antagonist MK-801, and then subjected to behavioural analysis up to an age of 180d. Alterations in cortical NMDA receptor expression and lymphocyte cAMP-response-element-binding-protein (CREB) were assessed. In comparison to controls, MK-801-treated animals showed differences in behaviour up to an age of 180d. Western blot analysis revealed that transient perinatal application of MK-801 caused a persistent increase in cortical NMDA-R1 protein in combination with a persistent disturbance of CREB phosphorylation, a downstream target of NMDA signalling. This animal model demonstrates that early postnatal NMDA receptor blockade leads to schizophrenia-like symptoms with persistent behavioural and neurochemical disturbances throughout life. Therefore, it might provide a basis for further understanding of the disease and evaluation of new therapeutic strategies.

Prolactin prevents chronic stress-induced decrease of adult hippocampal neurogenesis and promotes neuronal fate.

Chronic exposure to stress results in a reduction of hippocampal neurogenesis and of hippocampal volume. We examined whether prolactin (PRL), a regulator of the stress response and stimulator of neurogenesis in the subventricular zone, influences neurogenesis in the hippocampal dentate gyrus (DG) of chronically stressed adult C57BL/6 male mice. Chronically stressed (4 h daily immobilization for 21 d) or nonstressed mice were treated with either ovine PRL or vehicle between days 1-14. BrdU was injected daily between days 1-7 to evaluate cell survival and fate, or twice on day 21 to evaluate cell proliferation. Hippocampal cell proliferation was unchanged by either stress exposure or PRL at the end of the treatments. In contrast, the number of cells in the DG that incorporated BrdU during the first phase of the experiment and survived to the end of the experiment was decreased in vehicle-treated stressed mice compared with PRL- or vehicle-treated nonstressed control mice. Stressed animals receiving PRL had significantly more BrdU-labeled cells than vehicle-treated stressed mice at this time point. Cell fate analysis revealed a higher percentage of neurons in PRL- compared with vehicle-treated stressed mice. The results demonstrate that PRL protects neurogenesis in the DG of chronically stressed mice and promotes neuronal fate.

Prolactin activates mitogen-activated protein kinase signaling and corticotropin releasing hormone transcription in rat hypothalamic neurons.

Prolactin (PRL) modulates maternal behavior and mediates hypothalamic pituitary adrenal axis inhibition during lactation via PRL receptors in the brain. To identify mechanisms mediating these effects, we examined the effects of PRL on signaling and CRH transcription in hypothalamic neurons in vivo and in vitro. Western blot of hypothalamic proteins from rats receiving intracerebroventricular PRL injection revealed increases in phosphorylation of the MAPK and ERK. Double-staining immunohistochemistry demonstrated phosphorylated ERK localization in parvocellular CRH neurons as well as magnocellular vasopressin and oxytocin neurons of the hypothalamic paraventricular (PVN) and supraoptic nuclei. PRL also induced ERK phosphorylation in vitro in the hypothalamic cell line, 4B, which expresses PRL receptors, and in primary hypothalamic neuronal cultures. Using reporter gene assays in 4B cells, or quantitative RT-PCR for primary transcript in hypothalamic cell cultures, PRL potentiated forskolin-stimulated CRH transcription through activation of the ERK/MAPK pathway. The effect of PRL in hypothalamic cell cultures was unaffected by tetrodotoxin, suggesting a direct effect on CRH neurons. The data show that PRL activates the ERK/MAPK pathway and facilitates CRH transcription in CRH neurons, suggesting that the inhibitory effect of PRL on hypothalamo-pituitary-adrenal axis activity reported in vivo is indirect and probably mediated through modulation of afferent pathways to the PVN. In addition, the prominent stimulatory action of PRL on the ERK/MAPK pathway in the hypothalamic PVN and supraoptic nucleus is likely to mediate neuroplasticity of the neuroendocrine system during lactation.