Changes in milk proteome and metabolome associated with dry period length, energy balance, and lactation stage in postparturient dairy cows.

The early lactation period of dairy cows, which produce high quantities of milk, is normally characterized by an insufficient energy intake to cover milk production and maintenance requirements. Mobilization of body reserves occurs to compensate this negative energy balance (NEB), and probably as a consequence there is a higher susceptibility to diseases and metabolic disorders. There are several diagnostic methods to detect NEB, usually involving ketosis related parameters. Due to the easy availability of milk this is a preferred matrix, but simple and robust predictors of NEB level are missing. To better understand the physiological mechanism of NEB, milk of cows subjected to different dry period lengths, in different energy balance status and lactation stage, were analyzed by untargeted metabolomics and proteomics techniques. Milk of cows in severe NEB showed higher concentrations of acute phase response proteins, unsaturated fatty acids, and galactose-1-phosphate. Improved energy balance (EB) resulted in higher concentration of cholesterol, cholesterol synthesis related proteins, and stomatin. The presence of stomatin and galactose-1-phosphate in milk was strongly dependent on the EB of the cows. These novel and interesting findings warrant more in-depth research to assess their applicability as robust indicators of NEB in milk and to clarify the role of stomatin and galactose-1-phophate in milk of dairy cows in NEB.

Different responses of Caco-2 and MCF-7 cells to silver nanoparticles are based on highly similar mechanisms of action.

The mode of action of silver nanoparticles (AgNPs) is suggested to be exerted through both Ag+ and AgNP dependent mechanisms. Ingestion is one of the major NP exposure routes, and potential effects are often studied using Caco-2 cells, a well-established model for the gut epithelium. MCF-7 cells are epithelial breast cancer cells with extensive well-characterized toxicogenomics profiles. In the present study, we aimed to gain a deeper understanding of the cellular molecular responses in Caco-2 and MCF-7 cells after AgNP exposure in order to evaluate whether epithelial cells derived from different tissues demonstrated similar responses. These insights could possibly reduce the size of cell panels for NP hazard identification screening purposes. AgNPs of 20, 30, 60, and 110 nm, and AgNO3 were exposed for 6 h and 24 h. AgNPs were shown to be taken up and dissolve intracellularly. Compared with MCF-7 cells, Caco-2 cells showed a higher sensitivity to AgNPs, slower gene expression kinetics and absence of NP size-dependent responses. However, on a molecular level, no significant differences were observed between the two cell types. Transcriptomic analysis showed that Ag(NP) exposure caused (oxidative) stress responses, possibly leading to cell death in both cell lines. There was no indication for effects specifically induced by AgNPs. Responses to AgNPs appeared to be induced by silver ions released from the AgNPs. In conclusion, differences in mRNA responses to AgNPs between Caco-2 and MCF-7 cells were mainly related to timing and magnitude, but not to a different underlying mechanism.

Influence of different proteolytic strains of Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp. bulgaricus on the metabolite profile of set-yoghurt.

Proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is one of the key factors that determine the fermentation process and final quality of yoghurt. In this study, the interaction between different proteolytic strains of S. thermophilus and L. delbrueckii subsp. bulgaricus was investigated in terms of microbial growth, acidification and changes in the biochemical composition of milk during set-yoghurt fermentation. A complementary metabolomics approach was applied for global characterization of volatile and non-volatile polar metabolite profiles of yoghurt associated with proteolytic activity of the individual strains in the starter cultures. The results demonstrated that only non-proteolytic S. thermophilus (Prt-) strain performed proto-cooperation with L. delbrueckii subsp. bulgaricus. The proto-cooperation resulted in significant higher populations of the two species, faster milk acidification, significant abundance of aroma volatiles and non-volatile metabolites desirable for a good organoleptic quality of yoghurt. Headspace SPME-GC/MS and (1)H NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Furthermore, multivariate statistical analysis allows discriminating set-yoghurts fermented by different types of starter cultures according to their metabolite profiles. Our finding underlines that selection of suitable strain combinations in yoghurt starters is important for achieving the best technological performance regarding the quality of product.

Some OH-PCBs are more potent inhibitors of aromatase activity and (anti-) glucocorticoids than non-dioxin like (NDL)-PCBs and MeSO2-PCBs.

Traditional risk assessment of potential endocrine-disruptive pollutants, including PCBs, focus mainly on the effects of parent compounds. Still, biotransformation results in systemic exposure to PCBs and their bioactive metabolites. In the present paper, the effects of twenty ultra-pure non-dioxin-like (NDL) PCBs and their environmentally relevant hydroxy- (OH-) and methylsulfonyl- (MeSO(2)-) metabolites on aromatase activity and their glucocorticoid properties were investigated. Although most NDL-PCBs were inactive, PCB28 inhibited aromatase activity in human placenta microsomes with an IC(50) of 2.2µM. Most of these NDL-PCBs were weak (ant-)agonist of the glucocorticoid receptor (GR). Interestingly, four OH-metabolites of the commonly found NDL-PCB180 were able to inhibit aromatase activity (LOECs in the low µM range) and showed anti-glucocorticoid properties (LOECs in the low nM range), in a concentration-dependent manner. Further, four MeSO(2)-PCBs slightly inhibited aromatase activity and showed anti-glucocorticoid properties. Although, these effects were also associated with cytotoxicity, they were dependent on the position of the MeSO(2)-group on the biphenyl ring. Our results are the first to show that OH-PCBs are both anti-glucocorticoids and aromatase inhibitors. Taken together, these results for PCBs again support the common idea that risk assessment of the endocrine disruptive potential of PCBs should also include their metabolites.

PCB-47, PBDE-47, and 6-OH-PBDE-47 differentially modulate human GABAA and alpha4beta2 nicotinic acetylcholine receptors.

Polychlorinated biphenyls (PCBs) and the structurally related polybrominated diphenyl ethers (PBDEs) are abundant persistent organic pollutants that exert several comparable neurotoxic effects. Importantly, hydroxylated metabolites of PCBs and PBDEs have an increased neurotoxic potency. Recently, we demonstrated that PCBs can act as (partial) agonist on GABA(A) neurotransmitter receptors, with PCB-47 being the most potent congener. It is, however, unknown whether PBDE-47 and its metabolite 6-OH-PBDE-47 exert similar effects and if these effects are limited to GABA(A) receptors only. We therefore investigated effects of PCB-47, PBDE-47, and 6-OH-PBDE-47 on the inhibitory GABA(A) and excitatory α(4)ß(2) nicotinic acetylcholine (nACh) receptor expressed in Xenopus oocytes using the two-electrode voltage-clamp technique. Since human exposure is generally not limited to individual compounds, experiments with binary mixtures were also performed. The results demonstrate that PCB-47 and 6-OH-PBDE-47 act as full and partial agonist on the GABA(A) receptor. However, both congeners act as antagonist on the nACh receptor. PBDE-47 does not affect either type of receptor. Binary mixtures of PCB-47 and 6-OH-PBDE-47 induced an additive activation as well as potentiation of GABA(A) receptors, whereas this mixture resulted in an additive inhibition of nACh receptors. Binary mixtures of PBDE-47 and 6-OH-PBDE-47 yielded similar effects as 6-OH-PBDE-47 alone. These findings demonstrate that GABA(A) and nACh receptors are affected differently by PCB-47 and 6-OH-PBDE-47, with inhibitory GABA(A)-mediated signaling being potentiated and excitatory α(4)ß(2) nACh-mediated signaling being inhibited. Considering these opposite actions and the additive interaction of the congeners, these effects are likely to be augmented in vivo.

Bioassay directed identification of natural aryl hydrocarbon-receptor agonists in marmalade.

Citrus fruit and citrus fruit products, like grapefruit, lemon and marmalade were shown to contain aryl hydrocarbon receptor (AhR) agonists, as detected with the DR CALUX bioassay. This is of interest regarding the role of the Ah-receptor pathway in the adverse effects of dioxins, PCBs and other aromatic hydrocarbons. So far it is unclear which compounds in citrus fruit are responsible for the AhR-mediated activity and whether regular exposure to these compounds can cause effects comparable to, e.g. dioxins. The present study aimed at developing a method for identifying unknown Ah-receptor agonists in citrus products based on bioassay directed analysis, using marmalade as a first target. Following extraction with hexane and purification on an aluminium oxide-column, the extract was fractionated by HPLC using a C-18 semi-preparative column. Fractions were extracted, solvent-exchanged into dimethylsulfoxide and subsequently tested with DR CALUX. Extracts were shown to contain primarily coumarins, furocoumarins (FCs) and polymethoxyflavones (PMFs). Identification of fractions most active in the bioassay via LC/MS revealed that bergapten (an FC) is the most important Ah-receptor agonist in marmalade. The approach and method developed resulted in the successful identification of the bioactive component. However, potential pitfalls of the procedure will be discussed.