Chiral aspects of metabolism of antiinflammatory drug flobufen in human hepatocytes

The metabolism of the nonsteroidal antiinflammatory drug flobufen, 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid, was studied in primary cultures of human hepatocytes prepared by two-step collagenase perfusion of livers from four donors. Racemic flobufen or its individual enantiomers, R-(+)- and S-(-)-flobufen were used as substrates. Aliquots of culture medium were collected during 24-h incubation. The time-dependent disappearance of flobufen enantiomers and the formation of metabolites (stereoisomers of dihydroflobufen (DHF)) in hepatocytes were measured by chiral HPLC. The reduction of flobufen in human hepatocytes was stereoselective ((+)-R-flobufen was preferentially metabolized) and stereospecific ((2R;4S)-DHF and (2S;4S)-DHF stereoisomers were mostly formed). Although the structure of flobufen is different from the profens (2-arylpropionates), flobufen undergoes chiral inversion in human hepatocytes. The inversion of R-(+)-flobufen to S-(-)-flobufen predominates. The individual DHF stereoisomers were incubated in hepatocyte cultures and their biotransformation studied. The unidirectional chiral inversion of (2S;4S)-DHF to (2R;4S)-DHF and (2R;4R)-DHF to (2S;4R)-DHF was observed. Stereoselective oxidation of the DHFs to flobufen was also detected. Thus, flobufen metabolism in primary cultures of human hepatocytes is much more complicated (via chiral inversion and DHF re-oxidation) than was presumed from a preliminary achiral point of view.     

Heat shock proteins hsp32 and hsp70 as biomarkers of an early response? In vitro induction of heat shock proteins after exposure of cell culture to carcinogenic compounds and their real mixtures

Heat shock proteins (hsp) are a highly conserved group of proteins that are synthesized as a response to different forms of stress (heat, toxic chemicals, diseases, non-physiological pH changes). Because of their high sensitivity to changes in the environment, these proteins were suggested as possible early biomarkers of exposure in ecotoxicological studies. The purpose of the present study was to check the suitability of hsp 32 and hsp70 as biomarkers of in vitro exposure to environmentally relevant carcinogens: polycyclic aromatic hydrocarbons (PAHs), their nitro-derivates, aromatic amines, acrylonitrile (ACN) and the mixture of organic compounds adsorbed onto ambient airborne particles (extractable organic matter, EOM).The expression of hsp 32 and hsp70 was studied in human diploid lung fibroblasts (HEL cells) and human monocytic leukaemia cells (THP-1 cells) incubated in vitro with different concentrations of dibenzo[a,l]pyrene (DB[a,l]P), 1-nitropyrene, (NP), 4-aminobiphenyl (ABP), ACN and EOM for different periods of time. The incubation of cells with DB[a,l]P, NP, ABP and EOM did not result in increased levels of hsp 32 or hsp70, either in dose- or time-dependent manner. ACN induced the expression of hsp 32 as well as hsp70 in HEL and THP-1 cells, which probably reflects its ability to induce oxidative stress. We conclude that hsp 32 and hsp70 are not suitable biomarkers of an early exposure to PAHs, their nitro-derivates, aromatic amines or EOM under the conditions used.     

Life in Burrows Channelled the Morphological Evolution of the Skull in Rodents: the Case of African Mole-Rats (Bathyergidae,Rodentia)

African mole-rats are fossorial rodents that consist of five chisel-tooth digging genera (Heterocephalus, Heliophobius, Georychus, Fukomys, and Cryptomys) and one scratch digger (Bathyergus). They are characterized by striking physiological, morphological, and behavioral adaptations intimately related to their subterranean life. The influence of their mode of life in shaping the cranial morphology has yet to be evaluated in comparison to other Ctenohystrica, especially fossorial genera, which include the subterranean genera Spalacopus and Ctenomys. In our study, we seek to determine to what extent subterranean life affects the morpho-functional properties of the skull among fossorial ctenohystricans. 3D geometric morphometric analyses were performed on 277 skulls, encompassing 63 genera of Ctenohystrica, and complemented by biomechanical studies. African mole-rats and other subterranean Ctenohystrica, especially chisel-tooth diggers, have a short snout, a wide cranium with enlarged zygomatic arches, and a strongly hystricognathous mandible. Even if convergences are also manifest between most fossorial Ctenohystrica, subterranean rodents departed from the main ctenohystrican allometric trends in having a skull shape less size-dependent, but under stronger directional selection with intense digging activity as a major constraint. African mole-rats, notably chisel-tooth diggers, show important mechanical advantage for the temporalis muscles favoring higher forces at the bite point, while mechanical advantage of the superficial masseter muscles is lower compared to other Ctenohystrica. If subterranean species can be clearly discriminated based on their skull morphology, the intrinsic mosaic of anatomical characters of each genus (e.g., skull, teeth, and muscles) can be understood only in the light of their ecology and evolutionary history.     

Vocal repertoire of the social Mashona mole-rat (Fukomys darlingi) and how it compares with other mole-rats

Vocalizations play a major role in communication of mammals with subterranean lifestyles since other senses are dramatically restricted in the environment of below ground burrows. In our study, we recorded vocalizations of 10 adult males and 10 adult females of a social bathyergid, the Mashona mole-rat (Fukomys darlingi) in different behavioural contexts. Vocalizations were divided into four categories according to behavioural contexts. Similar to other subterranean mammals, the range of Mashona mole-rat calls is shifted towards lower frequencies. We described 10 types of true vocal signals and 2 mechanical sounds. The vocal repertoire of the Mashona mole-rat is less rich compared to other social mole-rats, corresponding with its low mean family size. Interestingly, this species has a higher diversity in contact and distress calls, while using a relatively low number of aggressive signals.     

Poikilothermic traits in Mashona mole-rat (Fukomys darlingi). Reality or myth?

The African mole-rats (Bathyergidae, Rodentia) is a mammalian family well known for a variety of ecophysiological adaptations for strictly belowground life. The smallest bathyergid, the hairless naked mole-rat from arid areas in Eastern Africa, is even famous as the only truly poikilothermic mammal. Another bathyergid, the Mashona mole-rat (Fukomys darlingi) from Zimbabwe, is supposed to have strong poikilothermic traits, because it is not able to maintain a stable body temperature at ambient temperatures below 20 °C. This is surprising because, compared to the naked mole-rat, this species, together with all congenerics, is larger, haired, and living in more seasonal environment. In addition, other Fukomys mole-rats show typical mammalian pattern in resting metabolic rates. In our study, we measured resting metabolic rate and body temperature of Mashona mole-rats from Malawi across a gradient of ambient temperatures to test its poikilothermic traits. We found that the adult mass specific resting metabolic rate was 0.76±0.20 ml O₂ g⁻¹ h⁻¹ and body temperature 34.8±1.1 °C in the thermoneutral zone (27–34 °C). Body temperature was stable (33.0±0.5 °C) at ambient temperatures from 10 to 25 °C. We thus cannot confirm poikilothermic traits in this species, at least for its Malawian population. Factors potentially explaining the observed discrepancy in Mashona mole-rat energetics are discussed.     

Repeatability of nest predation in passerines depends on predator species and time scale

It has been proposed that some specific locations of bird's nests have higher intrinsic chances of being depredated than other locations. This predicts that fates of consecutive nesting attempts at the same site should be repeatable. We used 20 pairs of old thrush nests to simulate repeated nesting attempts at the same sites, both within and between breeding seasons (n=40  sites×2  trials×2  years=160). Each nest was monitored by a camera to record multiple predation events and to identify predators. Predation by all predator species was repeatable during a 15-day trial. Predation by principal predators (jay Garrulus glandarius , marten Martes martes / foina ) and total predation (all species combined) was not correlated within pairs of simultaneously exposed nests or within samples of nests from particular study plot, and not repeatable for individual nests between-trials or between-years. These findings suggest short-term effect of predator memory causing revisitation of previously depredated nests during a current nesting trial (all predators); do not support an effect of nest site features on multiple nest discoveries and/or an effect of nest location on repeated random encounters with the same nest (principal predators). Long-term repeatability and correlation within pairs of simultaneously exposed nests was detectable only in occasional predators (great spotted woodpecker Dendrocopos major , possibly also squirrel Sciurus vulgaris ), which suggests effect of nest location combined with site fidelity and individual foraging specialization of these predators. We conclude that repeatability of nest predation depends on the time scale considered and the local predator community. We caution against spurious findings of repeatable nest predation resulting simply from statistical properties of correlation in binary data (nest fates).     

Synthesis and antimycobacterial properties of N-substituted 6-amino-5-cyanopyrazine-2-carboxamides

A series of fifteen new compounds related to pyrazinamide (PZA) were synthesized, characterized with analytical data and screened for antimycobacterial, antifungal and antibacterial activity. The series consists of 6-chloro-5-cyanopyrazine-2-carboxamide and N-substituted 6-amino-5-cyanopyrazine-2-carboxamides, derived from the previous by nucleophilic substitution with various non-aromatic amines (alkylamines, cycloalkylamines, heterocyclic amines). Some of the compounds exerted antimycobacterial activity against Mycobacterium tuberculosis equal to pyrazinamide (12.5-25 μg/mL). More importantly, 6-chloro-5-cyanopyrazine-2-carboxamide and 5-cyano-6-(heptylamino)pyrazine-2-carboxamide were active against Mycobacterium kansasii and Mycobacterium avium, which are unsusceptible to PZA. Basic structure-activity relationships are presented. Only weak antifungal and no antibacterial activity was detected.