Combined in silico and in vitro approaches to identify P-glycoprotein-inhibiting pesticides.

The P-glycoprotein (P-gp) efflux pump plays a major role in xenobiotic detoxification. The inhibition of its activity by environmental contaminants remains however rather little characterised. The present study was designed to develop a combination of different approaches to identify P-gp inhibitors among a large number of pesticides using in silico and in vitro models. First, the prediction performance of four web tools was evaluated alone or in combination using a set of recently marketed drugs. The best combination of web tools-AdmetSAR2.0/PgpRules/pkCSM-was next used to predict P-gp activity inhibition by 762 pesticides. Among the 187 pesticides predicted to be P-gp inhibitors, 11 were tested in vitro for their ability to inhibit the efflux of reference substrates (rhodamine 123 and Hoechst 33342) in P-gp overexpressing MCF7R cells and to inhibit the efflux of the reference substrate rhodamine 123 in the Caco-2 cell monolayer. In MCF7R cell assays, ivermectin B1a, emamectin B1 benzoate, spinosad, dimethomorph and tralkoxydim inhibited P-gp activity; ivermectin B1a, emamectin B1 benzoate and spinosad were determined to be stronger inhibitors (half-maximal inhibitory concentration [IC50 ] of 3 ± 1, 5 ± 1 and 7 ± 1 µM, respectively) than dimethomorph and tralkoxydim (IC50 of 102 ± 7 and 88 ± 7 µM, respectively). Ivermectin B1a, emamectin B1 benzoate, spinosad and dimethomorph also inhibited P-gp activity in Caco-2 cell monolayer assays, with dimethomorph being a weaker P-gp inhibitor. These combined approaches could be used to identify P-gp inhibitors among food contaminants, but need to be optimised and adapted for high-throughput screening.

Comparative in silico prediction of P-glycoprotein-mediated transport for 2010-2020 US FDA-approved drugs using six Web-tools.

P-glycoprotein (P-gp) is an efflux pump implicated in pharmacokinetics and drug-drug interactions. The identification of its substrates is consequently an important issue, notably for drugs under development. For such a purpose, various in silico methods have been developed, but their relevance remains to be fully established. The present study was designed to get insight about this point, through determining the performance values of six freely accessible Web-tools (ADMETlab, AdmetSAR2.0, PgpRules, pkCSM, SwissADME and vNN-ADMET), computationally predicting P-gp-mediated transport. Using an external test set of 231 marketed drugs, approved over the 2010-2020 period by the US Food and Drug Administration and fully in vitro characterized for their P-gp substrate status, various performance parameters (including sensitivity, specificity, accuracy, Matthews correlation coefficient and area under the receiver operating characteristics curve) were determined. They were found to rather poorly meet criteria commonly required for acceptable prediction, whatever the Web-tools were used alone or in combination. Predictions of being P-gp substrate or non-substrate by these online in silico methods may therefore be considered with caution.

Differential interactions of carbamate pesticides with drug transporters.

Pesticides are now recognised to interact with drug transporters, but only few data are available on this issue for carbamate pesticides, a widely used class of agrochemicals, to which humans are highly exposed. The present study was therefore designed to determine whether four representative carbamate pesticides, i.e. the insecticides aminocarb and carbofuran, the herbicide chlorpropham and the fungicide propamocarb, may impair activities of main drug transporters implicated in pharmacokinetics. The interactions of carbamates with solute carrier and ATP-binding cassette transporters were investigated using cultured transporter-overexpressing cells, reference substrates and spectrofluorimetry-, liquid chomatography/tandem mass spectrometry- or radioactivity-based methods. Aminocarb and carbofuran exerted no or minimal effects on transporter activities, whereas chlorpropham inhibited BCRP and OAT3 activities and propamocarb decreased those of OCT1 and OCT2, but cis-stimulated that of MATE2-K. Such alterations of transporters however required chlorpropham/propamocarb concentrations in the 5-50 µM range, likely not relevant to environmental exposure. Trans-stimulation assays and propamocarb accumulation experiments additionally suggested that propamocarb is not a substrate for OCT1, OCT2 and MATE2-K. These data indicate that some carbamate pesticides can interact in vitro with some drug transporters, but only when used at concentrations higher than those expected to occur in environmentally exposed humans.

Novel Insights on the Toxicity of Phycotoxins on the Gut through the Targeting of Enteric Glial Cells.

In vitro and in vivo studies have shown that phycotoxins can impact intestinal epithelial cells and can cross the intestinal barrier to some extent. Therefore, phycotoxins can reach cells underlying the epithelium, such as enteric glial cells (EGCs), which are involved in gut homeostasis, motility, and barrier integrity. This study compared the toxicological effects of pectenotoxin-2 (PTX2), yessotoxin (YTX), okadaic acid (OA), azaspiracid-1 (AZA1), 13-desmethyl-spirolide C (SPX), and palytoxin (PlTX) on the rat EGC cell line CRL2690. Cell viability, morphology, oxidative stress, inflammation, cell cycle, and specific glial markers were evaluated using RT-qPCR and high content analysis (HCA) approaches. PTX2, YTX, OA, AZA1, and PlTX induced neurite alterations, oxidative stress, cell cycle disturbance, and increase of specific EGC markers. An inflammatory response for YTX, OA, and AZA1 was suggested by the nuclear translocation of NF-κB. Caspase-3-dependent apoptosis and induction of DNA double strand breaks (γH2AX) were also observed with PTX2, YTX, OA, and AZA1. These findings suggest that PTX2, YTX, OA, AZA1, and PlTX may affect intestinal barrier integrity through alterations of the human enteric glial system. Our results provide novel insight into the toxicological effects of phycotoxins on the gut.

Identifying the spatial pattern and the drivers of the decline in the eastern English Channel chlorophyll-a surface concentration over the last two decades.

It has been established from previous studies that chlorophyll-a surface concentration has been declining in the eastern English Channel. This decline has been attributed to a decrease in nutrient concentrations in the rivers. However, the decrease in river discharge could also be a cause. In our study, rivers outflows and in-situ data have been compared to time series of satellite-derived chlorophyll-a concentrations. Dynamic Linear Model has been used to extract the dynamic and seasonally adjusted trends of several environmental variables. The results showed that, for the 1998-2019 period, chlorophyll-a levels stayed significantly lower than average and satellite images revealed a coast to offshore gradient. Chlorophyll-a concentration of coastal stations appeared to be related to the declining fluxes of phosphate while offshore stations were more related to nitrate-nitrite. Therefore, we can exclude that the climate variability, through river flows alone, has a dominant effect on the decline of chlorophyll-a concentration.

Comparative cytotoxicity, oxidative stress, and cytokine secretion induced by two cyanotoxin variants, microcystin LR and RR, in human intestinal Caco-2 cells.

While MC-LR and MC-RR share significant structural similarity, MC-RR is less cytotoxic than MC-LR. In the current study, we have compared the effects of MC-LR and MC-RR in Caco-2 cells by evaluating cytotoxicity, oxidative stress (reactive oxygen species production), and the cellular proinflammatory response (IL-6 and IL-8 production). Following treatment with 100 µM microcystins (MC), cytotoxicity was two-fold greater with MC-LR as compared to MC-RR after 24 h exposure. Whereas the reactive oxygen species production and IL-6 secretion were similar following a 24-h treatment with either MC, 100 µM MC-LR induced a five-fold greater IL-8 secretion when compared to MC-RR. Our study has demonstrated that, although both MC-LR and MC-RR induced some cytotoxicity in human intestinal cells, a major difference in IL-8 production was observed between the two variants.

Polyhexamethylene biguanide promotes adaptive cross-resistance to gentamicin in Escherichia coli biofilms.

Antimicrobial resistance is a critical public health issue that requires a thorough understanding of the factors that influence the selection and spread of antibiotic-resistant bacteria. Biocides, which are widely used in cleaning and disinfection procedures in a variety of settings, may contribute to this resistance by inducing similar defense mechanisms in bacteria against both biocides and antibiotics. However, the strategies used by bacteria to adapt and develop cross-resistance remain poorly understood, particularly within biofilms -a widespread bacterial habitat that significantly influences bacterial tolerance and adaptive strategies. Using a combination of adaptive laboratory evolution experiments, genomic and RT-qPCR analyses, and biofilm structural characterization using confocal microscopy, we investigated in this study how Escherichia coli biofilms adapted after 28 days of exposure to three biocidal active substances and the effects on cross-resistance to antibiotics. Interestingly, polyhexamethylene biguanide (PHMB) exposure led to an increase of gentamicin resistance (GenR) phenotypes in biofilms formed by most of the seven E. coli strains tested. Nevertheless, most variants that emerged under biocidal conditions did not retain the GenR phenotype after removal of antimicrobial stress, suggesting a transient adaptation (adaptive resistance). The whole genome sequencing of variants with stable GenR phenotypes revealed recurrent mutations in genes associated with cellular respiration, including cytochrome oxidase (cydA, cyoC) and ATP synthase (atpG). RT-qPCR analysis revealed an induction of gene expression associated with biofilm matrix production (especially curli synthesis), stress responses, active and passive transport and cell respiration during PHMB exposure, providing insight into potential physiological responses associated with adaptive crossresistance. In addition, confocal laser scanning microscopy (CLSM) observations demonstrated a global effect of PHMB on biofilm architectures and compositions formed by most E. coli strains, with the appearance of dense cellular clusters after a 24h-exposure. In conclusion, our results showed that the PHMB exposure stimulated the emergence of an adaptive cross-resistance to gentamicin in biofilms, likely induced through the activation of physiological responses and biofilm structural modulations altering gradients and microenvironmental conditions in the biological edifice.

Transcriptomic comparison of cyanotoxin variants in a human intestinal model revealed major differences in oxidative stress response: effects of MC-RR and MC-LR on Caco-2 cells.

Microcystins (MCs) are cyclic hepatotoxins produced by various species of cyanobacteria. Their structure includes two variable amino acids (AA) giving rise to more than 90 MC variants, however most of the studies to date have focused on the most toxic variant: microcystin LR (MC-LR). Ingestion is the major route of human exposure to MCs and several in vivo studies have demonstrated macroscopic effects on the gastro-intestinal tract. However, little information exists concerning the pathways affected by MC variants on intestinal cells. In the current study, we have investigated the effects of MC-RR and MC-LR on the human intestinal cell line Caco-2 using a non-selective method and compared their response at the pangenomic scale. The cells were incubated for 4h or 24h with a range of non-toxic concentrations of MC-RR or MC-LR. Minimal effects were observed after short term exposures (4h) to either MC variant. In contrast, dose dependent modulations of gene transcription levels were observed with MC-RR and MC-LR after 24h. The transcriptomic profiles induced by MC-RR were quite similar to those induced by MC-LR, suggestive of a largely common mechanism of toxicity. However, changes in total gene expression were more pronounced following exposure to MC-LR compared to MC-RR, as revealed by functional annotation. MC-LR affected two principal pathways, the oxidative stress response and cell cycle regulation, which did not elicit significant alteration following MC-RR exposure. This work is the first comparative description of the effects of MC-LR and MC-RR in a human intestinal cell model at the pangenomic scale. It has allowed us to propose differences in the mechanism of toxicity for MC-RR and MC-LR. These results illustrate that taking into account the toxicity of MC variants remains a key point for risk assessment.

Role of enteric glial cells in the toxicity of phycotoxins: Investigation with a tri-culture intestinal cell model.

Lipophilic phycotoxins are secondary metabolites produced by phytoplankton. They can accumulate in edible filtering-shellfish and cause human intoxications, particularly gastrointestinal symptoms. Up to now, the in vitro intestinal effects of these toxins have been mainly investigated on simple monolayers of intestinal cells such as the enterocyte-like Caco-2 cell line. Recently, the combination of Caco-2 cells with mucus secreting HT29-MTX cell line has been also used to mimic the complexity of the human intestinal epithelium. Besides, enteric glial cells (EGC) from the enteric nervous system identified in the gut mucosa have been largely shown to be involved in gut functions. Therefore, using a novel model integrating Caco-2 and HT29-MTX cells co-cultured on inserts with EGC seeded in the basolateral compartment, we examined the toxicological effects of two phycotoxins, pectenotoxin-2 (PTX2) and okadaic acid (OA). Cell viability, morphology, barrier integrity, inflammation, barrier crossing, and the response of some specific glial markers were evaluated using a broad set of methodologies. The toxicity of PTX2 was depicted by a slight decrease of viability and integrity as well as a slight increase of inflammation of the Caco-2/HT29-MTX co-cultures. PTX2 induced some modifications of EGC morphology. OA induced IL-8 release and decreased viability and integrity of Caco-2/HT29-MTX cell monolayers. EGC viability was slightly affected by OA. The presence of EGC reinforced barrier integrity and reduced the inflammatory response of the epithelial barrier following OA exposure. The release of GDNF and BDNF gliomediators by EGC could be implicated in the protection observed.

Permeability of the Cyanotoxin Microcystin-RR across a Caco-2 Cells Monolayer.

Microcystins (MCs) are toxins produced by several cyanobacterial species found worldwide. While MCs have a common structure, the variation of two amino acids in their structure affects their toxicity. As toxicodynamics are very similar between the MC variants, their differential toxicity could rather be explained by toxicokinetic parameters. Microcystin-RR (MC-RR) is the second most abundant congener and induces toxicity through oral exposure. As intestinal permeability is a key parameter of oral toxicokinetics, the apparent permeability of MC-RR across a differentiated intestinal Caco-2 cell monolayer was investigated. We observed a rapid and large decrease of MC-RR levels in the donor compartment. However, irrespective of the loaded concentration and exposure time, the permeabilities were very low from apical to basolateral compartments (from 4 to 15 × 10-8 cm·s-1) and from basolateral to apical compartments (from 2 to 37 × 10-8 cm·s-1). Our results suggested that MC-RR would be poorly absorbed orally. As similar low permeability was reported for the most abundant congener microcystin-LR, and this variant presented a greater acute oral toxicity than MC-RR, we concluded that the intestinal permeability was probably not involved in the differential toxicity between them, in contrast to the hepatic uptake and metabolism.

Co-culture model of Caco-2/HT29-MTX cells: A promising tool for investigation of phycotoxins toxicity on the intestinal barrier.

Most lipophilic phycotoxins have been involved in human intoxications but some of these toxins have never been proven to induce human gastro-intestinal symptoms, although intestinal damage in rodents has been documented. For investigating the in vitro toxicological profile of lipophilic phycotoxins on intestine, the epithelial Caco-2 cell line has been the most commonly used model. Nevertheless, considering the complexity of the intestinal epithelium, in vitro co-cultures integrating enterocyte-like and mucus-secreting cell types are expected to provide more relevant data. In this study, the toxic effects (viability, inflammation, cellular monolayer integrity, modulation of cell type proportion and production of mucus) of four lipophilic phycotoxins (PTX2, YTX, AZA1 and OA) were evaluated in Caco-2/HT29-MTX co-cultured cells. The four toxins induced a reduction of viability from 20% to 50% and affected the monolayer integrity. Our results showed that the HT29-MTX cells population were more sensitive to OA and PTX2 than Caco-2 cells. Among the four phycotoxins, OA induced inflammation (28-fold increase of IL-8 release) and also a slight increase of both mucus production (up-regulation of mucins mRNA expression) and mucus secretion (mucus area and density). For PTX2 we observed an increase of IL-8 release but weaker than OA. Intestinal cell models integrating several cell types can contribute to improve hazard characterization and to describe more accurately the modes of action of phycotoxins.

Differences in Toxic Response Induced by Three Variants of the Diarrheic Shellfish Poisoning Phycotoxins in Human Intestinal Epithelial Caco-2 Cells.

Diarrheic shellfish poisoning (DSP) is caused by the consumption of shellfish contaminated with a group of phycotoxins that includes okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2). These toxins are inhibitors of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), but show distinct levels of toxicity. Aside from a difference in protein phosphatases (PP) inhibition potency that would explain these differences in toxicity, others mechanisms of action are thought to be involved. Therefore, we investigated and compared which mechanisms are involved in the toxicity of these three analogues. As the intestine is one of the target organs, we studied the transcriptomic profiles of human intestinal epithelial Caco-2 cells exposed to OA, DTX-1, and DTX-2. The pathways specifically affected by each toxin treatment were further confirmed through the expression of key genes and markers of toxicity. Our results did not identify any distinct biological mechanism for OA and DTX-2. However, only DTX-1 induced up-regulation of the MAPK transduction signalling pathway, and down-regulation of gene products involved in the regulation of DNA repair. As a consequence, based on transcriptomic results, we demonstrated that the higher toxicity of DTX-1 compared to OA and DTX-2 was consistent with certain specific pathways involved in intestinal cell response.

Identification of key pathways involved in the toxic response of the cyanobacterial toxin cylindrospermopsin in human hepatic HepaRG cells.

The hepatotoxin cylindrospermopsin (CYN) has been involved in cases of poisoning in humans following ingestion. As its liver toxicity process is complex, we studied the transcriptomic profile of HepaRG cells exposed to CYN. The affected pathways were confirmed through the expression of key genes and the investigation of toxicity markers. In addition, CYP450 activities and cell redox homeostasis were investigated following acute and repeated exposure. CYN induced the down-regulation of genes involved in xenobiotic metabolism and cell cycle progression. There was cell cycle disturbance characterised by an accumulation of G1/S and G2/M cells and an increase in phospho-H3-positive cells. This was linked to the induction of DNA damage demonstrated by an increase in γH2AX-positive cells as well as an accumulation of sub-G1 cells indicating apoptosis but not involving caspase-3. While glutathione (GSH) content sharply decreased following acute exposure to CYN, it increased following repeated exposure, reflecting an adaptive response of cell redox homeostasis. However, our data also suggested that CYN induced the down-regulation of phase I and II metabolism gene products, and CYP450 activities were affected following both acute and repeated exposure to CYN. Our study indicated that repeated exposure of liver cells to low concentrations of CYN may affect their detoxification capacities.

Metabolism of the lipophilic phycotoxin 13-Desmethylspirolide C using human and rat in vitro liver models.

13-Desmethylspirolide C (13-SPX-C) is a phycotoxin produced by dinoflagellates which can accumulate in shellfish. 13-SPX-C induces neurotoxic effects in rodents through blockade of nicotinic acetylcholine receptors. As no human intoxication has been to date attributed to the consumption of 13-SPX-C-contaminated seafood, this toxin is not regulated according to the Codex Alimentarius. Nevertheless, shellfish consumers can be exposed to 13-SPX-C via shellfish consumption. In order to follow the fate of the toxin after ingestion and to verify whether metabolic detoxification could explain the lack of human intoxications, we assessed the metabolism of 13-SPX-C using several in vitro liver systems. First, both phase I and II reactions occurring with rat and human liver S9 fractions were screened. Our results indicated that 13-SPX-C was almost completely metabolized with both rat and human liver S9. Using a receptor binding assay towards nicotinic acetylcholine receptors we demonstrated that the resulting metabolites showed less affinity towards nicotinic acetylcholine receptors than 13-SPX-C. Finally, we showed that 13-SPX-C induced a pronounced increase of gene expression of the drug-metabolizing enzyme cytochrome P450 (CYP) CYP1A2. The role of this CYP in 13-SPX-C metabolism was clarified using an innovative in vitro tool, CYP1A2-Silensomes™. In summary, this study highlights that liver first-pass metabolism can contribute to the detoxification of 13-SPX-C.

Metabolism of the Marine Phycotoxin PTX-2 and Its Effects on Hepatic Xenobiotic Metabolism: Activation of Nuclear Receptors and Modulation of the Phase I Cytochrome P450.

PTX-2 is a marine biotoxin frequently found in shellfish that can lead to food intoxication in humans. Information regarding PTX-2 metabolism is scarce, and little is known of its effect on xenobiotic-metabolizing enzymes (XME) or its molecular pathways. The aim of this study was consequently to examine PTX-2 Phase I metabolism using rat and human liver S9 fractions, and also to assess the capability of PTX-2: (i) to modulate the gene expression of a panel of Phase I (CYP450) and II (UGT, SULT, NAT, and GST) enzymes, as well as the Phase III or 0 (ABC and SLCO) transporters in the human hepatic HepaRG cell line using qPCR; (ii) to induce specific CYP450 in HepaRG cells measured by immunolabeling detection and the measurement of the cells' activities; and (iii) to activate nuclear receptors and induce CYP promoter activities in HEK-T and HepG2 transfected cell lines using transactivation and reporter gene assay, respectively. Our results indicate that PTX-2 hydroxylation occurred with both rat and human S9 fractions. Whereas PTX-2 mostly upregulated the gene expression of CYP1A1 and 1A2, no induction of these two CYP activities was observed. Lastly, PTX-2 did not act as an agonist of CAR or PXR. Due to its effects on some key XME, more attention should be paid to possible drug-drug interactions with phycotoxins, especially as shellfish can accumulate several phycotoxins as well as other kinds of contaminants.

Low in vitro permeability of the cyanotoxin microcystin-LR across a Caco-2 monolayer: with identification of the limiting factors using modelling.

Microcystins (MCs) are toxins produced by several cyanobacteria species found worldwide. MC-LR is the most frequent. Here, we used the human Caco-2 cell line grown on semi-permeable filter supports as an in vitro model for determining MC-LR intestinal bidirectional transport. In this study, there was very low and time-dependent apparent permeability of MC-LR. To identify the limiting factors involved in the low permeability of MC-LR, a mathematical model was constructed to get physiologically relevant and informative parameters. The apical-to-basolateral transport was characterised by a rapid and substantial decrease in apical MC-LR concentrations (24-40% of the initial amount). In the basolateral compartment, the concentrations increased slowly after a lag time, but represented only a small fraction of the loaded concentrations (0.3-1.3%) after 24 h. This weak permeability was mainly due to a low clearance of efflux (from the cellular to the basolateral compartment) and effective secretion (from the cellular to the apical compartment). During the basolateral-to-apical transport, we observed a slow decrease in basolateral concentrations and a rapid increase in apical concentrations. In conclusion, modelling has the potential to highlight the key mechanisms involved in the complex kinetics of toxin transport.

Modulation of chromatin remodelling induced by the freshwater cyanotoxin cylindrospermopsin in human intestinal caco-2 cells.

Cylindrospermopsin (CYN) is a cyanotoxin that has been recognised as an emerging potential public health risk. Although CYN toxicity has been demonstrated, the mechanisms involved have not been fully characterised. To identify some key pathways related to this toxicity, we studied the transcriptomic profile of human intestinal Caco-2 cells exposed to a sub-toxic concentration of CYN (1.6 µM for 24hrs) using a non-targeted approach. CYN was shown to modulate different biological functions which were related to growth arrest (with down-regulation of cdkn1a and uhrf1 genes), and DNA recombination and repair (with up-regulation of aptx and pms2 genes). Our main results reported an increased expression of some histone-modifying enzymes (histone acetyl and methyltransferases MYST1, KAT5 and EHMT2) involved in chromatin remodelling, which is essential for initiating transcription. We also detected greater levels of acetylated histone H2A (Lys5) and dimethylated histone H3 (Lys4), two products of these enzymes. In conclusion, CYN overexpressed proteins involved in DNA damage repair and transcription, including modifications of nucleosomal histones. Our results highlighted some new cell processes induced by CYN.

Defatted bovine colostrum-supplemented diet around weaning improves exocrine pancreatic secretion by means of volume, digestive enzymes, and antibacterial activity.

OBJECTIVES: Exocrine pancreatic secretion contributes to limit pathogenic bacteria-associated diarrhea. Bovine colostrum, used in the treatment of diarrhea, reduces symptoms originating from gut pathogenic bacteria overgrowth. We hypothesized that bovine colostrum may stimulate the exocrine pancreatic secretion. METHODS: Eighteen piglets fitted with 2 permanent catheters (for pancreatic juice collection and reintroduction) were allocated to 1 of the following 2 dietary treatments for 5 days: a control diet or a diet supplemented with defatted bovine colostrum. Pancreatic juice was collected daily, and digestive enzyme activities and antibacterial activity were determined. RESULTS: The prandial pancreatic juice outflow, the basal and prandial lipase output, and the basal secretion of the antibacterial activity were, respectively, 60% (P = 0.08), 154% (P = 0.08), 92% (P = 0.06), and 72% (P < 0.05) higher in piglets fed a diet supplemented with defatted bovine colostrum. CONCLUSIONS: With defatted bovine colostrum, the increased antibacterial activity secretion against Escherichia coli may limit pathogenic bacteria overgrowth of the gut and reduce diarrheal episodes. The role of secretin in the increased pancreatic juice flow and lipase secretion was considered.

Effects of a bovine colostrum-supplemented diet on some gut parameters in weaned piglets.

The present study investigated the effects of a bovine colostrum-supplemented diet on gut post-weaning adaptation and health in piglets. Thirty-six 21-d-old piglets were allocated to one of the three following dietary treatments: sow-reared (SR), weaned on a control starter diet (WCtrl) or on a starter diet supplemented with bovine colostrum (WCol) until slaughter at 28 d or 35 d of age. Gastric pH and intestinal bacteriological, structural and functional parameters were determined. Compared to WCtrl, the gastric pH was lower (P < 0.05) and the duodenal lactobacilli:coliform ratio was higher (P = 0.05) in WCol piglets. The relative small intestine weight was 18% (P < 0.05) higher in WCol piglets than in SR piglets. Duodenal villous height was lower (P < 0.01) in WCtrl than in SR piglets, whereas the value for WCol piglets was intermediate. The weaning-increased crypt cell proliferation was not affected by bovine colostrum supplementation. The mucosal ribosomal capacity was higher (P < 0.05) in W than in SR piglets. In conclusion, a diet supplemented with colostrum induced, although not always significantly, variations of gut parameters, suggesting that globally, colostrum may limit weaning-induced gut structural and microbial alterations. The observed effects occurred early and were maintained throughout the post-weaning adaptive phase.

Differences in regulation of the first two M-phases in Xenopus laevis embryo cell-free extracts.

The first embryonic M-phase is special, being the time when paternal and maternal chromosomes mix together for the first time. Reports from a variety of species suggest that the regulation of first M-phase has many particularities; however, no systematic comparative study of the biochemical aspects of first and the following M-phases has been previously undertaken. Here, we ask whether the regulation of the first embryonic M-phase is modified, using Xenopus cell-free extracts. We developed new types of extract specific for the first and the second M-phase obtained either from parthenogenetic or from in vitro fertilized embryos. Analyses of these extracts confirmed that the amplitude of histone H1 kinase activity reflecting CDK1/cyclin B (or MPF for M-phase Promoting Factor) activity is higher and persists longer than during the second M-phase, and that levels of cyclins B1 and B2 are correspondingly higher during the first than the second embryonic M-phase. Inhibition of protein synthesis shortly before M-phase entry reduced mitotic histone H1 kinase amplitude, shortened the period of mitotic phosphorylation of chosen marker proteins, and reduced cyclin B1 and B2 levels, suggesting a role of B-type cyclins in regulating the duration of mitotic events. Moreover, addition of exogenous cyclin B to the extract prior the second mitosis brought forward the activation of mitotic histone H1 kinase but prolonged the duration of this activity. We also confirmed that the inhibitory phosphorylation of CDK1 on tyrosine 15 oscillates between the first two embryonic M-phases, but is clearly more pronounced before the first than the second mitosis, while the MAP kinase ERK2 tended to show greater activation during the first embryonic M-phase but with a similar duration of activation. We conclude that discrete differences exist between the first two M-phases in Xenopus embryo and that higher CDK1/cyclin B activity and B-type cyclin levels could account for the different characteristics of these M-phases.