Species differences in the response of liver drug-metabolizing enzymes to (S)-4-O-tolylsulfanyl-2-(4-trifluormethyl-phenoxy)-butyric acid (EMD 392949) in vivo and in vitro.

Induction of drug-metabolizing enzymes (DMEs) is highly species-specific and can lead to drug-drug interaction and toxicities. In this series of studies we tested the species specificity of the antidiabetic drug development candidate and mixed peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist (S)-4-O-tolylsulfanyl-2-(4-trifluormethyl-phenoxy)-butyric acid (EMD 392949, EMD) with regard to the induction of gene expression and activities of DMEs, their regulators, and typical PPAR target genes. EMD clearly induced PPARalpha target genes in rats in vivo and in rat hepatocytes but lacked significant induction of DMEs, except for cytochrome P450 (P450) 4A. CYP2C and CYP3A were consistently induced in livers of EMD-treated monkeys. Interestingly, classic rodent peroxisomal proliferation markers were induced in monkeys after 17 weeks but not after a 4-week treatment, a fact also observed in human hepatocytes after 72 h but not 24 h of EMD treatment. In human hepatocyte cultures, EMD showed similar gene expression profiles and induction of P450 activities as in monkeys, indicating that the monkey is predictive for human P450 induction by EMD. In addition, EMD induced a similar gene expression pattern as the PPARalpha agonist fenofibrate in primary rat and human hepatocyte cultures. In conclusion, these data showed an excellent correlation of in vivo data on DME gene expression and activity levels with results generated in hepatocyte monolayer cultures, enabling a solid estimation of human P450 induction. This study also clearly highlighted major differences between primates and rodents in the regulation of major inducible P450s, with evidence of CYP3A and CYP2C inducibility by PPARalpha agonists in monkeys and humans.

CRF receptors in the rodent and human cardiovascular systems: Species differences

CRF has powerful receptor-mediated cardiovascular actions. To evaluate the precise distribution of CRF receptors, in vitro CRF receptor autoradiography with (125)I-[Tyr(0), Glu(1), Nle(17)]-sauvagine or [(125)I]-antisauvagine-30 was performed in the rodent and human cardiovascular system. An extremely high density of CRF(2) receptors was detected with both tracers in vessels of rodent lung, intestine, pancreas, mesenterium, kidney, urinary bladder, testis, heart, brain, and in heart muscle. In humans, CRF(2) receptors were detected with (125)I- antisauvagine-30 at low levels in vessels of kidneys, intestine, urinary bladder, testis, heart and in heart muscle, while only heart vessels were detected with (125)I-[Tyr(0), Glu(1), Nle(17)]-sauvagine. This is the first extensive morphological study reporting the extremely wide distribution of CRF(2) receptors in the rodent cardiovascular system and a more limited expression in man, suggesting a species-selective CRF receptor expression.

Inhibition of in vitro metabolism of testosterone in human, dog and horse liver microsomes to investigate species differences.

Testosterone hydroxylation was investigated in human, canine and equine liver microsomes and in human and canine single CYPs. The contribution of the CYP families 1, 2 and 3 was studied using chemical inhibitors. Testosterone metabolites were analyzed by HPLC. The metabolites androstenedione, 6β- and 11β-hydroxytestosterone were found in microsomes of all species, but the pattern of metabolites varied within species. Androstenedione was more prominent in the animal species, and an increase over time was seen in equines. Testosterone hydroxylation was predominantly catalyzed by the CYP3A subfamily in all three species. While CYP2C9 did not metabolise testosterone, the canine ortholog CYP2C21 produced androstenedione. Quercetin significantly inhibited 6β- and 11β-hydroxytestosterone in all species investigated, suggesting that CYP2C8 is involved in testosterone metabolism, whereas sulfaphenazole significantly inhibited the formation of 6β- and 11β-hydroxytestosterone in human microsomes, at 60min in equine microsomes, but not in canine microsomes. A contribution of CYP2B6 in testosterone metabolism was only found in human and equine microsomes. Inhibition of 17β-hydroxysteroid dehydrogenase 2 indicated its involvement in androstenedione formation in humans, increased androstenedione formation was found in equines and no involvement in canines. These findings provide improved understanding of differences in testosterone biotransformation in animal species.

MECHANISMS OF SPECIES DIFFERENCES IN BRAIN ACETYLCHOLINESTERASE SENSITIVITY TO ORGANOPHOSPHATE INHIBITION

In vitro incubation of acetylcholinesterase from brain tissue of several species with organophosphate compounds indicated that the concentrations required to inhibit 50% of acetylcholinesterase activity (IC(,50)) differed from species to species for the same compound (Murphy, et al., 1968; Andersen, et al., 1972, 1977 and 1978).^ The hypothesis that non-specific binding proteins (Lauwerys and Murphy, 1969a,b) exerts a protective effect on acetylcholinesterase, and thus cause the differences observed in IC(,50) studies was tested by a ('3)H-DFP binding experiment. It was found that differences in the amount of non-specific binding protein cannot explain the observed differences observed in IC(,50) studies.^ An alternative hypothesis, that acetylcholinesterase from different species have different affinities for binding and/or different rates of phosphorylation by organophosphate insecticides was tested by determining the apparent affinity constant (k(,a)) and apparent rate of phosphorylation (k(,p)). Kinetic studies indicated that acetylcholinesterases from different species have different sensitivities to inhibition by organophosphate insecticides, and the differences are due to different affinities for binding and/or different rates of phosphorylation by the same organophosphate compound.^ Studies of the spontaneous reactivation of acetylcholinesterase after inhibition by organophosphate insecticides also indicated that acetylcholinesterases from different species have different rates and extents of spontaneous reactivation. This further substantiates the hypothesis that acetylcholinesterases from different species have different kinetic characteristics with respect to organophosphate insecticides inhibition.^ Eleven paraoxon analogs were synthesized for a quantitative structure-activity relationship study. It was found that the electron-withdrawing power ((sigma)) and hydrophobicity ((PARAGR)) of the substituent are important in determining the anti-cholinesterase activity of paraoxon analogs. Thus, predictions of species differences in acetylcholinesterase sensitivities to paraoxon analogs can be made if the physicochemical parameters ((sigma) and (PARAGR)) of the substituents are known.^ In another approach, i.e. enzyme modeling, the sensitivity of rat brain acetylcholinesterase to organophosphate insecticides was used as the independent variable to predict the sensitivities of acetylcholinesterases from other species to the same compound. Regression equations were derived for each species based on nineteen organophosphate insecticides studied. It was found, that in addition to paraoxon analogs, this method is also applicable to other organophosphate compounds with wide variations in structure. Thus, the sensitivities of acetylcholinesterases from other species can also be predicted from the sensitivity of rat brain acetylcholinesterase. ^

Pollination Ecology Of Two Species Of Elleanthus (orchidaceae): Novel Mechanisms And Underlying Adaptations To Hummingbird Pollination.

Relationships among floral biology, floral micromorphology and pollinator behaviour in bird-pollinated orchids are important issues to understand the evolution of the huge flower diversity within Orchidaceae. We aimed to investigate floral mechanisms underlying the interaction with pollinators in two hummingbird-pollinated orchids occurring in the Atlantic forest. We assessed floral biology, nectar traits, nectary and column micromorphologies, breeding systems and pollinators. In both species, nectar is secreted by lip calli through spaces between the medial lamellar surfaces of epidermal cells. Such form of floral nectar secretion has not been previously described. Both species present functional protandry and are self-compatible yet pollinator-dependent. Fruit sets in hand-pollination experiments were more than twice those under natural conditions, evidencing pollen limitation. The absence of fruit set in interspecific crosses suggests the existence of post-pollination barriers between these synchronopatric species. In Elleanthus brasiliensis, fruits resulting from cross-pollination and natural conditions were heavier than those resulting from self-pollination, suggesting advantages to cross-pollination. Hummingbirds pollinated both species, which share at least one pollinator species. Species differences in floral morphologies led to distinct pollination mechanisms. In E. brasiliensis, attachment of pollinaria to the hummingbird bill occurs through a lever apparatus formed by an appendage in the column, another novelty to the knowledge of orchids. In E. crinipes, pollinaria attachment occurs by simple contact with the bill during insertion into the flower tube, which fits tightly around the bill. The novelties described here illustrate the overlooked richness in ecology and morphophysiology in Orchidaceae. This article is protected by copyright. All rights reserved.

Evidence for inter- and intra-species biofilm formation variability among a small group of coagulase-negative staphylococci

Coagulase-negative staphylococci (CoNS) are common bacterial colonisers of the human skin. They are often involved in nosocomial infections due to biofilm formation in indwelling medical devices. While biofilm formation has been extensively studied in Staphylococcus epidermidis, little is known regarding other CoNS species. Here, biofilms from six different CoNS species were characterised in terms of biofilm composition and architecture. Interestingly, the ability to form a thick biofilm was not associated with any particular species, and high variability on biofilm accumulation was found within the same species. Cell viability assays also revealed different proportions of live and dead cells within biofilms formed by different species, although this parameter was particularly similar at the intra-species level. On the other hand, biofilm disruption assays demonstrated important inter- and intra-species differences regarding extracellular matrix composition. Lastly, confocal laser scanning microscopy (CLSM) experiments confirmed this variability, highlighting important differences and common features of CoNS biofilms. We hypothesised that the biofilm formation heterogeneity observed was rather associated with biofilm matrix composition than with cells themselves. Additionally, our results indicate that polysaccharides, DNA and proteins are fundamental pieces in the process of CoNS biofilm formation.

The pollutant diethylhexyl phthalate regulates hepatic energy metabolism via species-specific PPARalpha-dependent mechanisms.

Background: The modulation of energetic homeostasis by pollutants has recently emerged as a potential contributor to the onset of metabolic disorders. Diethylhexyl phthalate (DEHP) is a widely used industrial plasticizer to which humans are widely exposed. Phthalates can activate the three peroxisome proliferatoractivated receptor (PPAR) isotypes on cellular models and induce peroxisome proliferation in rodents.Objectives: In this study, we aimed to evaluate the systemic and metabolic consequences of DEHP exposure that have remained so far unexplored and to characterize the underlying molecular mechanisms of action.Methods: As a proof of concept and mechanism, genetically engineered mouse models of PPARs were exposed to high doses of DEHP, followed by metabolic and molecular analyses.Results: DEHP-treated mice were protected from diet-induced obesity via PPARalpha-dependent activation of hepatic fatty acid catabolism, whereas the activity of neither PPARbeta nor PPARgamma was affected. However, the lean phenotype observed in response to DEHP in wild-type mice was surprisingly abolished in PPARalpha-humanized mice. These species differences are associated with a different pattern of coregulator recruitment.Conclusion: These results demonstrate that DEHP exerts species-specific metabolic actions that rely to a large extent on PPARalpha signaling and highlight the metabolic importance of the species-specific activation of PPARalpha by xenobiotic compounds. Editor's SummaryDiethylhexyl phthalate (DEHP) is an industrial plasticizer used in cosmetics, medical devices, food packaging, and other applications. Evidence that DEHP metabolites can activate peroxisome proliferatoractivated receptors (PPARs) involved in fatty acid oxidation (PPARalpha and PPARbeta) and adiposite function and insulin resistance (PPARgamma) has raised concerns about potential effects of DEHP on metabolic homeostasis. In rodents, PPARalpha activation also induces hepatic peroxisome proliferation, but this response to PPARalpha activation is not observed in humans. Feige et al. (p. 234) evaluated systemic and metabolic consequences of high-dose oral DEHP in combination with a high-fat diet in wild-type mice and genetically engineered mouse PPAR models. The authors report that mice exposed to DEHP gained less weight than controls, without modifying their feeding behavior; they also exhibited lower triglyceride levels, smaller adipocytes, and improved glucose tolerance compared with controls. These effects, which were observed in mice fed both high-fat and standard diets, appeared to be mediated by PPARalpha-dependent activation of hepatic fatty acid catabolism without apparent involvement of PPARbeta or PPARgamma. However, mouse models that expressed human (versus mouse) PPARalpha tended to gain more weight on a high-fat diet than their DHEP-unexposed counterparts. The authors conclude that findings support species-specific metabolic effects of DEHP mediated by PPARalpha activation.

Prediction of plant species distributions across six millennia.

The usefulness of species distribution models (SDMs) in predicting impacts of climate change on biodiversity is difficult to assess because changes in species ranges may take decades or centuries to occur. One alternative way to evaluate the predictive ability of SDMs across time is to compare their predictions with data on past species distributions. We use data on plant distributions, fossil pollen and current and mid-Holocene climate to test the ability of SDMs to predict past climate-change impacts. We find that species showing little change in the estimated position of their realized niche, with resulting good model performance, tend to be dominant competitors for light. Different mechanisms appear to be responsible for among-species differences in model performance. Confidence in predictions of the impacts of climate change could be improved by selecting species with characteristics that suggest little change is expected in the relationships between species occurrence and climate patterns.

Comparing multiple criteria for species identification in two recently diverged seabirds

Correct species identification is a crucial issue in systematics with key implications for prioritising conservation effort. However, it can be particularly challenging in recently diverged species due to their strong similarity and relatedness. In such cases, species identification requires multiple and integrative approaches. In this study we used multiple criteria, namely plumage colouration, biometric measurements, geometric morphometrics, stable isotopes analysis (SIA) and genetics (mtDNA), to identify the species of 107 bycatch birds from two closely related seabird species, the Balearic (Puffinus mauretanicus) and Yelkouan (P. yelkouan) shearwaters. Biometric measurements, stable isotopes and genetic data produced two stable clusters of bycatch birds matching the two study species, as indicated by reference birds of known origin. Geometric morphometrics was excluded as a species identification criterion since the two clusters were not stable. The combination of plumage colouration, linear biometrics, stable isotope and genetic criteria was crucial to infer the species of 103 of the bycatch specimens. In the present study, particularly SIA emerged as a powerful criterion for species identification, but temporal stability of the isotopic values is critical for this purpose. Indeed, we found some variability in stable isotope values over the years within each species, but species differences explained most of the variance in the isotopic data. Yet this result pinpoints the importance of examining sources of variability in the isotopic data in a case-by-case basis prior to the cross-application of the SIA approach to other species. Our findings illustrate how the integration of several methodological approaches can help to correctly identify individuals from recently diverged species, as each criterion measures different biological phenomena and species divergence is not expressed simultaneously in all biological traits.

Seedling growth of fifteen Brazilian tropical tree species differing in successional status

Growth of seedlings of fifteen tropical tree species representative, at the adult stage, of different successional positions, was studied under field conditions. Seedlings were grown in three treatments: full sun (FS), artificial shade imposed by neutral screens (AS) and natural shade imposed by a closed canopy in a Forest Reserve in Southeast Brazil (NS). Most of the studied species survived in both shade treatments, although their growth was severely affected. Decreases in height, internode numbers, dry weight, leaf area, root:shoot ratio (R:S) and increases in leaf mass ratio (LMR), leaf area ratio (LAR) and specific leaf area (SLA) were common responses to shade. Relative growth rates (RGRs) and net assimilation rates (NARs) were consistently lower in the shaded treatments than in full sun. RGR was significantly correlated with NAR in the FS and NS treatments, whereas it was correlated with LAR in the AS treatment. Natural shade had more severe effects than artificial shade on leaf area reduction and RGR. Between-species differences in R:S, LMR, SLA and LAR were not related to the successional status of species. However, there was a tendency for early-successional species to have higher RGRs than late successional ones, regardless of the light environment. Late-successional species also showed less pronounced responses to shade than early ones. The characteristics presented by the late-successional species may be associated with shade tolerance, enabling their persistence under dense canopies.

Mobilization of seed reserves during germination of four tropical species of the Amazon Rainforest

Seed reserve mobilization during germination of the Amazonian species Myciaria dubia (camu-camu), Eugenia stipitata(araca-boi), Dipteryx odorata (cumaru) and Hymenaea courbaril (jatoba) was evaluated. Seeds were placed in germination chambers at 30 degrees C with it 12 h photoperiod. Analysis of primary metabolites (carbohydrates, lipids and proteins) and fatty acid composition were carried out in quiescent seeds and at four germination stages after radicle protrusion. Germination was high in all species but there were statistically significant differences between species. Differences were also observed with regard to the duration of the germination period. The seeds showed variation in the content and composition of the analyzed compounds. indicating that the mobilization rates of these compounds may affect germination velocity.

Production and carbon allocation in monocultures and mixed-species plantations of Eucalyptus grandis and Acacia mangium in Brazil

Introducing nitrogen-fixing tree species in fast-growing eucalypt plantations has the potential to improve soil nitrogen availability compared with eucalypt monocultures. Whether or not the changes in soil nutrient status and stand structure will lead to mixtures that out-yield monocultures depends on the balance between positive interactions and the negative effects of interspecific competition, and on their effect on carbon (C) uptake and partitioning. We used a C budget approach to quantify growth, C uptake and C partitioning in monocultures of Eucalyptus grandis (W. Hill ex Maiden) and Acacia mangium (Willd.) (treatments E100 and A100, respectively), and in a mixture at the same stocking density with the two species at a proportion of 1 : 1 (treatment MS). Allometric relationships established over the whole rotation, and measurements of soil CO2 efflux and aboveground litterfall for ages 4-6 years after planting were used to estimate aboveground net primary production (ANPP), total belowground carbon flux (TBCF) and gross primary production (GPP). We tested the hypotheses that (i) species differences for wood production between E. grandis and A. mangium monocultures were partly explained by different C partitioning strategies, and (ii) the observed lower wood production in the mixture compared with eucalypt monoculture was mostly explained by a lower partitioning aboveground. At the end of the rotation, total aboveground biomass was lowest in A100 (10.5 kg DM m(-2)), intermediate in MS (12.2 kg DM m(-2)) and highest in E100 (13.9 kg DM m(-2)). The results did not support our first hypothesis of contrasting C partitioning strategies between E. grandis and A. mangium monocultures: the 21% lower growth (delta B-w) in A100 compared with E100 was almost entirely explained by a 23% lower GPP, with little or no species difference in ratios such as TBCF/GPP, ANPP/TBCF, delta B-w/ANPP and delta B-w/GPP. In contrast, the 28% lower delta B-w in MS than in E100 was explained both by a 15% lower GPP and by a 15% lower fraction of GPP allocated to wood growth, thus partially supporting our second hypothesis: mixing the two species led to shifts in C allocations from above- to belowground, and from growth to litter production, for both species.

Enantioselective CE analysis of hepatic ketamine metabolism in different species in vitro

Ketamine, an injectable anesthetic and analgesic consisting of a racemic mixture of S-and R-ketamine, is routinely used in veterinary and human medicine. Nevertheless, metabolism and pharmacokinetics of ketamine have not been characterized sufficiently in most animal species. An enantioselective CE assay for ketamine and its metabolites in microsomal preparations is described. Racemic ketamine was incubated with pooled microsomes from humans, horses and dogs over a 3 h time interval with frequent sample collection. CE data revealed that ketamine is metabolized enantioselectively to norketamine (NK), dehydronorketamine and three hydroxylated NK metabolites in all three species. The metabolic patterns formed differ in production rates of the metabolites and in stereoselectivity of the hydroxylated NK metabolites. In vitro pharmacokinetics of ketamine N-demethylation were established by incubating ten different concentrations of racemic ketamine and the single enantiomers of ketamine for 8 min and data modeling was based on Michaelis-Menten kinetics. These data revealed a reduced intrinsic clearance of the S-enantiomer in the racemic mixture compared with the single S-enantiomer in human microsomes, no difference in equine microsomes and the opposite effect in canine microsomes. The findings indicate species differences with possible relevance for the use of single S-ketamine versus racemic ketamine in the clinic.

Inducible nitric oxide synthase and nitrotyrosine in listeric encephalitis: a cross-species study in ruminants

Listeria monocytogenes (LM) is a Gram-positive facultative intracellular bacterium that causes fatal meningoencephalitis in humans and ruminants. A current paradigm predicts that intracellular bacteria are controlled by nitric oxide (NO) whose synthesis is catalyzed by inducible nitric oxide synthase (iNOS). The ability of macrophages (Mphi) to express iNOS shows extreme interspecies variability. Here the expression of iNOS and synthesis of NO was studied in listeric encephalitis of cattle, sheep, and goats. iNOS was expressed by a subset of Mphi in cerebral microabscesses in all three species. The level of iNOS expression and the density of cells per lesion expressing iNOS was highest in cattle, intermediate in sheep, and lowest in goats. The accumulation of nitrotyrosine (NT), an indicator of local NO synthesis, was observed in lesions of cattle but not in those of small ruminants. The density of iNOS-expressing cells in lesions was inversely correlated with the number of bacteria. No species differences were observed in regard to reactive oxygen intermediate (ROI) production by stimulated granulocytes, using the flow cytometric dihydrorhodamine-123 (DHR) method indicating ROI generation. Thus, the marked species differences in iNOS expression, NT accumulation, and LM content in lesions of ruminants with listeric encephalitis are explained by different amounts of ROI produced. It suggests that variations in the ability of Mphi to synthesize NO are of pathophysiological significance in listeriosis.

The selection of a model microalgal species as biomaterial for a novel aquatic phytotoxicity assay

A phytotoxicity assay based on the ToxY-PAM dual-channel yield analyser has been developed and successfully incorporated into field assessments for the detection of phytotoxicants in water. As a means of further exploring the scope of the assay application and of selecting a model biomaterial to complement the instrument design, nine algal species were exposed to four chemical substances deemed of priority for water quality monitoring purposes (chlorpyrifos, copper, diuron and nonylphenol ethoxylate). Inter-species differences in sensitivity to the four toxicants varied by a factor of 1.9-100. Measurements of photosystem-II quantum yield using these nine single-celled microalgae as biomaterial corroborated previous studies which have shown that the ToxY-PAM dual-channel yield analyser is a highly sensitive method for the detection of PS-II impacting herbicides. Besides Phaeodactylum tricornutum, the previously applied biomaterial, three other species consistently performed well (Nitzschia closterium, Chlorella vulgaris and Dunaliella tertiolecta) and will be used in further test optimisation experiments. In addition to sensitivity, response time was evaluated and revealed a high degree of variation between species and toxicants. While most species displayed relatively weak and slow responses to copper, C. vulgaris demonstrated an IC10 of 51 μ g L-1, with maximum response measured within 25 minutes and inhibition being accompanied by a large decrease in fluorescence yield. The potential for this C vulgaris-based bioassay to be used for the detection of copper is discussed. There was no evidence that the standard ToxY-PAM protocol, using these unicellular algae species, could be used for the detection of chlorpyrifos or nonylphenol ethoxylate at environmentally relevant levels. © 2005 Elsevier B.V. All rights reserved.