Transfer of preclinical study data on the influence of cimicifuga racemosaon functional changes in the hippocampus during menopause.

Menopausal transition in women involves complex neurobiochemical changes linked to ovarian dysfunction, resulting in symptoms like vasomotor symptoms (VMS), sleep disturbances, anxiety, and cognitive impairments. Hormone replacement therapy is the first-line treatment. However, many women are reluctant to use HRT or have contraindications toward HRT and seek for alternatives. Non-hormonal therapies with extracts of Cimicifuga racemosa rhizomes like the isopropanolic extract (iCR, black cohosh) offer a promising alternative. A preclinical pilot study exploring iCR's effects on gene expression in the hippocampus and hypothalamus of ovarectomized (OVX) rats mimicking menopausal conditions identified important signaling pathways and CNS-based contributions to the multitargeted modes of action of iCR. Especially in the hippocampus, iCR compensated effects of OVX on gene expression profiles. These changes are reflected by the genes AVPR1A, GAL, CALCA, HCRT, PNOC, ESR1, ESR2 and TAC3 contributing to the formation of hot flushes or thermoregulation as well as to secondary effects such as blood pressure, metabolism, hormonal regulation, homeostasis, mood regulation, neuroendocrine modulation, regulation of sleep and arousal, and in learning, memory and cognition. To understand the mechanisms in the brain of estrogen-depressed animals (OVX) and subsequent iCR treatment we combined the results of the pilot study with those of up-to-date literature and tried to transfer the current knowledge to humans during menopausal transition and adaptation. Focus was laid on changes in the hippocampal function, that is disturbed by hormonal fluctuations, but can also be brought back into balance by iCR.

Gene expression analyses on multi-target mode of action of black cohosh in menopausal complaints - a pilot study in rodents.

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.

Non-interventional observational study broadens positive benefit-risk assessment of an immunomodulating herbal remedy in the common cold.

Background: The unique extract of a mixture of Baptisiae tinctoriae radix, Echinaceae pallidae/purpureae radix and Thujae occidentalis herba alleviates the typical symptoms of the common cold and shortens the duration of the disease. Purpose: The risk-benefit ratio of a concentrated formulation of this herbal extract was investigated under everyday conditions. Study design: Pharmacy-based, non-interventional, multicenter, open, uncontrolled study registered at DRKS00011068. Methods: For 10 days, patients completed a diary questionnaire rating the severity of each common cold symptom on a 10-point scale. For evaluation, symptoms were combined into the scores "overall severity", "rhinitis", "bronchitis" and "general symptoms". Cox models were used to evaluate the influence of covariates on the time of stable improvement. Results: In total 955 patients (12 to 90 years) were analyzed; 85% assessed the efficacy as good or very good. Response (improvement of the overall severity by at least 50%) was reached at median day 5 (95% CImedian 5-5). General symptoms abated faster than the other complaints. The percentage of predominantly moderate or severe symptoms to predominantly mild or absent symptoms reversed on day 3.9 (interpolation). Results of adolescents and adults did not differ (p = .6013; HR = 0.918). Concomitant medication did not boost the effect of the herbal remedy. Early start of treatment of the cold accelerated the recovery (p = .0486; HR = 0.814). Thirty-four cases of adverse events were self-recorded in the diaries; none of them were serious. The tolerability was assessed as "good or very good" by 98% of the patients. Conclusion: The benefit-risk assessment of this herbal extract clearly remains positive. This non-interventional study accords with and shows transferability of the results of previous placebo-controlled studies with this extract in a real-life setting.

DNA damage response curtails detrimental replication stress and chromosomal instability induced by the dietary carcinogen PhIP.

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.

Methionine restriction inhibits chemically-induced malignant transformation in the BALB/c 3T3 cell transformation assay.

High consumption of red meat entails a higher risk of developing colorectal cancer. Methionine, which is more frequently a component of animal proteins, and folic acid are members of the one carbon cycle and as such important players in DNA methylation and cancer development. Therefore, dietary modifications involving altered methionine and folic acid content might inhibit colon cancer development. In the present study, the BALB/c 3T3 cell transformation assay was used to investigate whether methionine and folic acid are able to influence the malignant transformation of mouse fibroblasts after treatment with the known tumour initiator 3-methylcholanthrene. Three different methionine concentrations (representing a -40%, a "normal" and a +40% cell culture medium concentration, respectively) and two different folic acid concentrations (6 and 20 µM) were thereby investigated. Methionine restriction led to a decrease of type III foci, while enhancement of both methionine and folic acid did not significantly increase the cell transformation rate. Interestingly, the focus-lowering effect of methionine was only significant in conjunction with an elevated folic acid concentration. In summary, we conclude that the malignant transformation of mouse fibroblasts is influenced by methionine levels and that methionine restriction could be a possible approach to reduce cancer development.

Impact of dextran sulphate sodium-induced colitis on the intestinal transport of the colon carcinogen PhIP.

Colorectal cancer is one of the most frequent cancers in Western countries. Chronic intestinal diseases such as Crohn's disease and ulcerative colitis, in which the intestinal barrier is massively disturbed, significantly raise the risk of developing a colorectal tumour. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a genotoxic heterocyclic aromatic amine that is formed after strongly heating fish and meat. In this study, the hypothesis that PhIP uptake in the gut is increased during chronic colitis was tested. Chronic colitis was induced by oral administration of dextran sulphate sodium (DSS) to Fischer 344 rats. The transport of PhIP in eight different rat intestinal segments was examined in Ussing chambers. The tissues were incubated with 10 µM PhIP for 90 min, and the concentration of PhIP was determined in the mucosal and serosal compartments of the Ussing chambers as well as in the clamped tissues by LC-MS. Although chronic colitis was clearly induced in the rats, no differences in the intestinal transport of PhIP were observed between control and DSS-treated animals. The hypothesis that in the course of chronic colitis more PhIP is taken up by the intestinal epithelium, thereby increasing the risk of developing colorectal cancer, could not be confirmed in the present report.

Intestinal absorption and cell transforming potential of PhIP-M1, a bacterial metabolite of the heterocyclic aromatic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

Previous studies have shown that in the rat, the colon carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is only absorbed to a limited extent in the small intestines and that a major fraction of unmetabolised PhIP reaches the colon. Moreover, PhIP is extensively metabolised when incubated with human stool samples to a major derivative, 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido [3',2':4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1). In the present study, the uptake and transport of PhIP-M1 in Ussing chamber experiments, its cytotoxicity in the different segments of the Fischer 344 rat gut and its transforming potential in the BALB/c 3T3 cell transformation assay were analysed. At the most, 10-20% of the PhIP-M1 amount added to the mucosal compartment of the Ussing chambers per segment were absorbed within 90min. Therefore, the amount of PhIP-M1 detected in the tissues as well as in the serosal compartment of the Ussing chambers was extremely low. Moreover, human-relevant concentrations of PhIP-M1 were not cytotoxic and did not induce the malignant transformation of BALB/c 3T3 cells. In conclusion, even if one would assume that 100% of the daily amount of PhIP ingested by a human being is converted into PhIP-M1 in the colon, this concentration most probably would not lead to cytotoxicity and/or carcinogenicity in the colorectal mucosa.

Effects of single pesticides and binary pesticide mixtures on estrone production in H295R cells.

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.

The colon carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is actively secreted in the distal colon of the rat: an integrated view on the role of PhIP transport and metabolism in PhIP-induced colon carcinogenesis.

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.

Influence of a fat-rich diet, folic acid supplementation and a human-relevant concentration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine on the induction of preneoplastic lesions in the rat colon.

In the present study, the effect of three controversially discussed risk factors for colorectal cancer, a fat-rich diet (16% raw fat content), dietary folic acid supplementation (50 mg folic acid/kg lab chow) and a human-relevant concentration (0.1 ppm) of the heterocyclic aromatic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), either alone or in combination, on the induction of aberrant crypt foci (ACF) in the colon of male Fischer 344 rats was analyzed. The mean number of ACF per rat in the case of the four groups fed a fat-rich diet tended to be higher than that of the four groups being fed a standard diet. However, the increase in the mean number of ACF per rat only reached statistical significance in the case of the rats receiving a fat-rich lab chow supplemented with 50 mg/kg folic acid. Moreover, a concentration of 0.1 ppm PhIP per se, either in the standard or in the fat-rich lab chow, did not lead to an increase in the mean number of ACF per rat. In conclusion, the present study provides additional evidence for a colon cancer promoting effect of folic acid supplementation when rodents are fed the compound in supraphysiological concentrations.

Uptake of the colon carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by different segments of the rat gastrointestinal tract: its implication in colorectal carcinogenesis.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a colon carcinogen in rats. In the present study the absorption of PhIP in the small and large intestine of Fischer 344 rats was determined, and the relevance of the differences in the degree of absorption of PhIP along the gastrointestinal axis for the PhIP-mediated colon carcinogenesis process is discussed. PhIP uptake was low in the different gut sections of Fischer 344 rats, the PhIP tissue levels varying in the following order: proximal jejunum>distal jejunum>proximal colon>distal colon=rectum. Furthermore, abcc2 was mainly expressed in the proximal parts of the small intestine, in particular in the proximal jejunum. Extremely low expression levels were observed in distal jejunum, ileum, caecum and proximal colon, whereas abcc2 was almost not detected in distal colon and rectum. These data, together with previously published results, lend support to the hypothesis that PhIP is taken up in the proximal segments of the small intestine and after being metabolically activated in the liver reaches the stem cell compartment of the colonic crypts via the blood circulation, the crypt cells in the distal colon and rectum being particularly at risk, since these almost do not express abcc2.

PRNP promoter polymorphisms are associated with BSE susceptibility in Swiss and German cattle.

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. RESULTS: Allele, genotype and haplotype frequencies of the two indel polymorphisms were determined in 449 BSE cattle and 431 unaffected cattle from Switzerland and Germany including all 43 German BSE and 16 German control animals from the original study. When breeds with similar allele and genotype distributions were compared, the 23 bp indel polymorphism again showed a significant association with susceptibility to BSE. However, some additional breed-specific allele and genotype distributions were identified, mainly related to the Brown breeds. CONCLUSION: Our study corroborated earlier findings that polymorphisms in the PRNP promoter region have an influence on susceptibility to BSE. However, breed-specific differences exist that need to be accounted for when analyzing such data.

Functional relevance of DNA polymorphisms within the promoter region of the prion protein gene and their association to BSE infection.

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.