DNA adducts, strand breaks and micronuclei in mice exposed to styrene by inhalation

Genotoxic and clastogenic effects of styrene were studied in mice. Male NMRI mice were exposed by inhalation to styrene in concentrations of 750 and 1500 mg/m3 for 21, 7, 3 and 1 days (6 h/day, 7 days/week). Followed parameters included styrene in blood, specific styrene oxide (SO) induced DNA adducts, DNA strand breaks and micronuclei. The formation of SO induced 7-SO-guanines and 1-SO-adenines in DNA was analysed from lung tissues by two versions of the 32P-postlabeling technique. In lungs after 21 days of exposure to 1500 mg/m3 the level of 7-SO-guanine was 23.0+/-11.9 adducts/10(8) normal nucleotides, while 1-SO-adenine was detected at the levels of 0.6+/-0.2 adducts/10(8) normal nucleotides. Both 7-SO-guanines and 1-SO-adenines strongly correlated with exposure parameters, particularly with styrene concentration in blood (r=0.875, P=0.0002 and r=0.793, P=0.002, respectively). DNA breaks were measured in peripheral lymphocytes, bone marrow cells and liver cells using comet assay. To discern oxidative damage and abasic sites, endonuclease III was used. In bone marrow of exposed mice slight increase of strand breaks can be detected after 7 days of inhalation. A significant increase was revealed in the endonuclease III-sensitive sites after 21 days of inhalation in bone marrow. In the liver cells inhalation exposure to both concentrations of styrene did not virtually affect either levels of DNA single-strand breaks or endonuclease III-sensitive sites. The inhalation of 1500 mg/m3 of styrene induced significant increase of micronuclei after 7 days of exposure (10.4+/-2.5/1000 cells, i.e. twice higher micronuclei frequency than in controls). After 21 days of inhalation no significant difference between the control group and the two exposed groups was observed. Whether the decrease of micronuclei after 21 days of inhalation was due to the inhibition of cell proliferation caused by styrene or due to the natural elimination of chromatide fragments, remains to be clarified. An interesting link has been found between DNA single-strand breaks in bone marrow and frequencies of micronuclei (r=0.721, P=0.028).     

The one that did not get away: individual assignment using microsatellite data detects a case of fishing competition fraud

Assignment of an individual to the population from which it most probably originated based on its multilocus genotype has been widely applied in recent years. In this study, individual assignment based on microsatellite data was used to identify a case of fishing competition fraud. Despite the fact that the true population of origin was most probably not among the reference populations, recent modifications of the assignment tests were used in confidently excluding (p < 0.0001) the possibility of a 5.5 kg salmon (Salmo salar) originating from the fishing competition location, Lake Saimaa (south-east Finland). In fact, the probability of the suspect salmon originating from one of the regions that supply most of Finland's fish markets was found to be over 600 times higher than it originating from Lake Saimaa. When presented with this evidence, the offender confessed to purchasing the salmon at a local fish shop and criminal charges were laid. This study emphasizes the potential practical application of the individual assignment procedure, in particular the usefulness of confidently excluding populations as the origin of an individual. A similar strategy could be also used, for example in suspected cases of illegal poaching, in order to assign or exclude individuals from originating from a claimed population.     

Production and purification of recombinant human α2C2 adrenergic receptor using Saccharomyces cerevisiae

The objective is to generate milligram quantities of recombinant human ₂C2 adrenergic receptor for X-ray crystallographic studies. It has been cloned in Saccharomyces cerevisiae, and the production level is at best about 13 pmol/mg of membrane protein, as estimated by radio-ligand binding assay. The receptor is solubilized with sucrose monolaurate followed by immunoaffinity purification and reconstitution into phospholipid vesicles. The efficiency of solubilization and immuno-purification are 60% and 91%, respectively.     

Numerical response of small mustelids to vole abundance: delayed or not?

One of the most studied problems in population ecology has been to understand the relative roles of top–down and bottom–up forces in regulating animal populations. This has also been a key issue in studies of vole population dyna mics. Vole populations exhibit a wide variation of dynamics, from seasonal fluctuations to multiannual variations or cyclicity. One of the hypotheses to explain cyclic population dynamics is predation by the specialist predators. A common counterargument against the predation hypothesis has been the lack of conclusive observations of the time delay in the predators’ numerical response. We studied the interaction between voles and their specialist small mustelid predators, the stoat Mustela erminea and the least weasel Mustela n. nivalis, by modelling their interaction to data sets that cover large areas of Finland. Vole abundance was monitored with biannual trappings and their predators with snow‐tracking. Results show a high dependence of the predators on the voles, and this connection is generally tighter in weasels than in stoats. Weasel abundance is affected most strongly by the vole abundance in previous spring, 8.5– 10 months earlier, while in stoats the effect of autumn abundance of voles, 2.5–6 months earlier, was the strongest. These results, together with the observation that the weasels’ effects on voles are stronger after a time lag of 6–9.5 than 2–4.5 months, indicate the existence of a time lag in weasels’ numerical response. A time lag in the predators’ numerical response is a necessary condition for the predators to drive population cycles in its prey, and therefore our results support the specialist predation hypothesis.     

32P-postlabelling analysis of 1,3-butadiene-induced DNA adducts in vivo and in vitro

Butadiene monoepoxide (BMO), epoxybutanediol (EBD) and diepoxybutane (DEB) are reactive metabolites of 1,3-butadiene (BD), an important industrial chemical classified as a probable human carcinogen. The covalent interactions of these metabolites with DNA lead to the formation of DNA adducts which may induce mutations or other types of DNA damage, resulting in tumour formation. In the present study, two pairs of diastereomeric N-1-BMO-adenine adducts were identified in the reaction of BMO with 2´-deoxyadenosine-5´-monophosphate (5´-dAMP). The major products formed by reacting EBD with 2´-deoxyguanosine-5´-monophosphate (5´-dGMP) were characterized as diastereomeric N-7-(2´,3´,4´-trihydroxybut-1´-yl)-5´-dGMP by UV and electrospray mass spectrometry. The formation of N-7-BMO-guanine adducts (1´-carbon, 60; 2´carbon, 54/10⁴ nucleotides) in BMO-treated DNA was about four times higher than that of N-1-BMO-adenine adducts (1´-carbon, 20; 2´-carbon, 8.7/10⁴ nucleotides). However, the recovery of N-1-BMO-adenine adducts in DNA (45 ± 5%) was two times higher than that of N-7-guanine adducts (20 ± 4%) by 32P-postlabelling analysis. Using the 32P-postlabelling/ HPLC assay, N-1-BMO-adenine, N-7-BMO-guanine and N-7-EBDguanine adducts were detected in BMO- or DEB-treated DNA and in liver DNA of rats exposed to BD by inhalation. The amount of N-7-EBD-guanine adducts (11/10⁸ nucleotides) in rat liver was about three-fold higher than N-7-BMO-guanine adducts (4.0/10⁸ nucleotides). The novel finding of N-1-BMO-adenine adducts formed in vivo may contribute to the understanding of the mechanisms of BD carcinogenic action.     

Postprandial metabolism of docosapentaenoic acid (DPA, 22:5n−3) and eicosapentaenoic acid (EPA, 20:5n−3) in humans

The study of the metabolism of docosapentaenoic acid (DPA, 22:5n?3) in humans has been limited by the unavailability of pure DPA and the fact that DPA is found in combination with eicosapentaenoic acid (EPA, 20:5n?3) and docosahexaenoic acid (DHA, 22:6n?3) in natural products. In this double blind cross over study, pure DPA and EPA were incorporated in meals served to healthy female volunteers. Mass spectrometric methods were used to study the chylomicron lipidomics. Plasma chylomicronemia was significantly reduced after the meal containing DPA compared with the meal containing EPA or olive oil only. Both EPA and DPA were incorporated into chylomicron TAGs, while there was less incorporation into chylomicron phospholipids. Lipidomic analysis of the chylomicron TAGs revealed the dynamic nature of chylomicron TAGs. The main TAG species that EPA and DPA were incorporated into were EPA/18:1/18:1, DPA/18:1/16:0 and DPA/18:1/18:1. There was very limited conversion of DPA and EPA to DHA and there were no increases in EPA levels during the 5 h postprandial period after the DPA meal. In conclusion, EPA and DPA showed different metabolic fates, and DPA hindered the digestion, ingestion or incorporation into chylomicrons of the olive oil present in the meal.     

The value of oviposition timing,queen presence and kinship in a social insect

Reproductive cooperation confers benefits, but simultaneously creates conflicts among cooperators. Queens in multi-queen colonies of ants share a nest and its resources, but reproductive competition among queens often results in unequal reproduction. Two mutually non-exclusive factors may produce such inequality in reproduction: worker intervention or queen traits. Workers may intervene by favouring some queens over others, owing to either kinship or queen signals. Queens may differ in their intrinsic fecundity at the onset of oviposition or in their timing of the onset of oviposition, leading to their unequal representation in the brood. Here, we test the role of queen kin value (relatedness) to workers, timing of the onset of oviposition and signals of presence by queens in determining the maternity of offspring. We show that queens of the ant Formica fusca gained a significantly higher proportion of sexuals in the brood when ovipositing early, and that the presence of a caged queen resulted in a significant increase in both her share of sexual brood and her overall reproductive share. Moreover, the lower the kin value of the queen, the more the workers invested in their own reproduction by producing males. Our results show that both kinship and breeding phenology influence the outcome of reproductive conflicts, and the balance of direct and indirect fitness benefits in the multi-queen colonies of F. fusca.     

CD73 Is a Major Regulator of Adenosinergic Signalling in Mouse Brain

CD73 (ecto-5’-nucleotidase) is a cell surface enzyme that regulates purinergic signalling by desphosphorylating extracellular AMP to adenosine. 5′-nucleotidases are known to be expressed in brain, but the expression of CD73 and its putative physiological functions at this location remain elusive. Here we found, using immunohistochemistry of wild-type and CD73 deficient mice, that CD73 is prominently expressed in the basal ganglia core comprised of striatum (caudate nucleus and putamen) and globus pallidus. Furthermore, meninges and the olfactory tubercle were found to specifically express CD73. Analysis of wild type (wt) and CD73 deficient mice revealed that CD73 confers the majority of 5’-nucleotidase activity in several areas of the brain. In a battery of behavioural tests and in IntelliCage studies, the CD73 deficient mice demonstrated significantly enhanced exploratory locomotor activity, which probably reflects the prominent expression of CD73 in striatum and globus pallidus that are known to control locomotion. Furthermore, the CD73 deficient mice displayed altered social behaviour. Overall, our data provide a novel mechanistic insight into adenosinergic signalling in brain, which is implicated in the regulation of normal and pathological behaviour.     

The Impact of Bdnf Gene Deficiency to the Memory Impairment and Brain Pathology of APPswe/PS1dE9 Mouse Model of Alzheimer’s Disease

Brain-derived neurotrophic factor (BDNF) importantly regulates learning and memory and supports the survival of injured neurons. Reduced BDNF levels have been detected in the brains of Alzheimer’s disease (AD) patients but the exact role of BDNF in the pathophysiology of the disorder remains obscure. We have recently shown that reduced signaling of BDNF receptor TrkB aggravates memory impairment in APPswe/PS1dE9 (APdE9) mice, a model of AD. The present study examined the influence of Bdnf gene deficiency (heterozygous knockout) on spatial learning, spontaneous exploratory activity and motor coordination/balance in middle-aged male and female APdE9 mice. We also studied brain BDNF protein levels in APdE9 mice in different ages showing progressive amyloid pathology. Both APdE9 and Bdnf mutations impaired spatial learning in males and showed a similar trend in females. Importantly, the effect was additive, so that double mutant mice performed the worst. However, APdE9 and Bdnf mutations influenced spontaneous locomotion in contrasting ways, such that locomotor hyperactivity observed in APdE9 mice was normalized by Bdnf deficiency. Obesity associated with Bdnf deficiency did not account for the reduced hyperactivity in double mutant mice. Bdnf deficiency did not alter amyloid plaque formation in APdE9 mice. Before plaque formation (3 months), BDNF protein levels where either reduced (female) or unaltered (male) in the APdE9 mouse cortex. Unexpectedly, this was followed by an age-dependent increase in mature BDNF protein. Bdnf mRNA and phospho-TrkB levels remained unaltered in the cortical tissue samples of middle-aged APdE9 mice. Immunohistological studies revealed increased BDNF immunoreactivity around amyloid plaques indicating that the plaques may sequester BDNF protein and prevent it from activating TrkB. If similar BDNF accumulation happens in human AD brains, it would suggest that functional BDNF levels in the AD brains are even lower than reported, which could partially contribute to learning and memory problems of AD patients.