Background: Non-synonymous polymorphisms within the prion protein gene (PRNP) influence the susceptibility and incubation time for transmissible spongiform encephalopathies (TSE) in some species such as sheep and humans. In cattle, none of the known polymorphisms within the PRNP coding region has a major influence on susceptibility to bovine spongiform encephalopathy (BSE).Recently, however, we demonstrated an association between susceptibility to BSE and a 23 bp insertion/deletion (indel) polymorphism and a 12 bp indel polymorphism within the putative PRNP promoter region using 43 German BSE cases and 48 German control cattle. The objective of this study was to extend this work by including a larger number of BSE cases and control cattle of German and Swiss origin.

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases that can occur spontaneously or can be caused by infection or mutations within the prion protein gene PRNP. Nonsynonymous DNA polymorphisms within the PRNP gene have been shown to influence susceptibility/resistance to infection in sheep and humans. Analysis of DNA polymorphisms within the core promoter region of the PRNP gene in four major German bovine breeds resulted in the identification of both SNPs and insertion/deletion (indel) polymorphisms. Comparative genotyping of both controls and animals that tested positive for bovine spongiform encephalopathy (BSE) revealed a significantly different distribution of two indel polymorphisms and two SNPs within Braunvieh animals, suggesting an association of these polymorphisms with BSE susceptibility. The functional relevance of these polymorphisms was analyzed using reporter gene constructs in neuronal cells. A specific haplotype near exon 1 was identified that exhibited a significantly lower expression level. Genotyping of nine polymorphisms within the promoter region and haplotype calculation revealed that the haplotype associated with the lowest expression level was underrepresented in the BSE group of all breeds compared to control animals, indicating a correlation of reduced PRNP expression and increased resistance to BSE.

PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PKcs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs.

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Epidemiological studies show that a positive correlation exists between the consumption of strongly heated meat and fish and the development of colorectal tumours. In this context, it has been postulated that the uptake of toxic substances formed during meat and fish processing such as heterocyclic aromatic amines (HCAs) may be causally related to colon carcinogenesis. In a previous study, we have shown that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundantly formed HCA in the above-mentioned food items, is mainly absorbed in the small intestine (i.e. proximal jejunum) of the rat. In the present study, we analysed whether PhIP can actively be secreted by enterocytes in the rat proximal jejunum and distal colon. Unidirectional PhIP flux rates from the mucosal-to-the serosal compartment (J ms ) and in the opposite direction (J sm ) were examined in Ussing chambers with (14)C-PhIP as radiotracer and in the absence of electrochemical gradients. Under these experimental conditions, significant negative net flux rates (J net  = J ms  - J sm ) can only be explained by an active secretion of PhIP into the luminal compartment, and such an effect was observed in the rat distal colon, but not in the proximal jejunum. Moreover, the data obtained suggest that the breast cancer resistance protein, the multidrug resistance protein 4 and P-glycoprotein are not involved in the active secretion of PhIP in the rat distal colon. The potential role of PhIP transport in colon carcinogenesis is discussed.

The aim of the present study was to determine whether the human adrenocortical carcinoma cell line H295R can be used as an in vitro test system to investigate the effects of binary pesticide combinations on estrone production as biological endpoint. In the first step ten pesticides selected according to a tiered approach were tested individually. The anilinopyrimidines cyprodinil and pyrimethanil as well as the dicarboximides iprodione and procymidone increased estrone concentration, while the triazoles myclobutanil and tebuconazole as well as the strobilurins azoxystrobin and kresoxim-methyl decreased estrone concentration in the supernatant of H295R cells. The N-methylcarbamate methomyl did not show any effects, and the phthalimide captan reduced estrone concentration unspecifically due to its detrimental impact on cellular viability. When cyprodinil and pyrimethanil, which belong to the same chemical group and increase estrone production, were combined, in most of the cases the overall effect was solely determined by the most potent compound in the mixture (i.e., cyprodinil). When cyprodinil and procymidone, which belong to different chemical groups but increase estrone production, were combined, in most cases an additive effect was observed. When cyprodinil, which increased estrone production, was combined with either myclobutanil or azoxystrobin, which decreased estrone production, the overall effect of the mixture was in most cases either entirely determined by myclobutanil or at least partially modulated by azoxystrobin. In conclusion, H295R cells appear to be an adequate in vitro test system to study the effect of combining two pesticides affecting estrone production.

Purpose This study aimed at assessing gene expression profiles in hippocampus and hypothalamus of ovariectomized (OVX) rats with or without treatment with an isopropanolic extract of Cimicifuga racemosa rhizomes (iCR) in comparison to intact rats. Methods Exploration of hippocampal (Hi) and hypothalamic (Hy) tissue from Sprague Dawley rats: without OVX (NHi = NHy = 4), tissues 3 months after OVX (NHi = 4, NHy = 3), or tissues of rats after their treatment with iCR for 3 months after OVX (NHi = NHy = 2). Gene expression profiles in these tissues were investigated by RNA-microarray-analysis and subsequent verification by qPCR. Results 4812 genes were differentially regulated when comparing the three groups in hippocampus and hypothalamus. iCR compensated the effects of OVX in 518 genes. This compensatory effect was most prominent in hippocampal signalling pathways, thereof genes (GAL, CALCA, HCRT, AVPR1A, PNOC, etc.) involved in thermoregulation, regulation of sleep and arousal, blood pressure regulation, metabolism, nociception, hormonal regulation, homeostasis, learning and cognition, mood regulation, neuroendocrine modulation, etc.. In the hypothalamus, iCR compensated OVX-effects at TAC3 and OPRM1 but not at KISS1. These genes are involved in the pathophysiology of hot flashes. Conclusions Our pilot study findings support a multifaceted mode of action of iCR in menopausal complaints on a tissue-specific brain gene expression level.