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The biotransformation and excretion of darolutamide were investigated in a phase I study. Six healthy male volunteers received a single dose of 300 mg 14C-darolutamide as an oral solution in the fasted state. Plasma, urine, and feces samples were analyzed for mass balance evaluation by liquid scintillation counting (LSC). Metabolite profiling and identification were determined using liquid chromatography mass-spectrometry with off-line radioactivity detection using LSC. Complete mass balance was achieved, with mean radioactivity recovery of 95.9% within 168 hours (63.4% in urine, 32.4% in feces). The administered 1:1 ratio of (S,R)- and (S,S)-darolutamide changed to approximately 1:5, respectively, in plasma. Darolutamide and the oxidation product, keto-darolutamide, were the only components quantifiable by LSC in plasma, accounting for 87.4% of total radioactivity, with a 2.1-fold higher plasma exposure for keto-darolutamide. Aside from darolutamide, the most prominent metabolites in urine were O-glucoronide (M-7a/b) and N-glucuronide (M-15a/b), as well as pyrazole sulfates (M-29, M-24) and glucuronides (M-21, M-22) resulting from oxidative cleavage of the parent. The darolutamide diastereomers were mainly detected in feces. In vitro assays showed that darolutamide metabolism involves a complex interplay between oxidation and reduction, as well as glucuronidation. Interconversion of the diastereomers involves oxidation to keto-darolutamide, primarily mediated by CYP3A4, followed by reduction predominantly catalyzed by cytosolic reductase(s), with aldo-keto reductase 1C3 playing the major role. The latter reaction showed stereoselectivity with preferential formation of (S,S)-darolutamide. SIGNIFICANCE STATEMENT: The metabolism and excretion of darolutamide in humans revealed that oxidation (CYP3A4) and glucuronidation (UGT1A9, UGT1A1) were the main metabolic routes of elimination. Direct excretion also contributed to overall clearance. The two pharmacologically equipotent diastereomers of darolutamide interconvert primarily via oxidation to the active metabolite keto-darolutamide, followed by reduction predominantly by cytosolic reductase(s). The latter reaction showed stereoselectivity with preferential formation of (S,S)-darolutamide. Data indicate a low drug-drug interaction potential of darolutamide with inducers or inhibitors of metabolizing enzymes.

Here is reported the design and synthesis of a series of highly potent and selective α2C antagonists using benzodioxine methyl piperazine as a central scaffold by pharmacophoric analysis to improve the pharmacokinetics of suboptimal clinical candidate molecules.

Darolutamide is a second-generation androgen receptor inhibitor approved for the treatment of nonmetastatic castration-resistant prostate cancer at a dosage of 600 mg orally twice daily.We aimed to fully characterize the pharmacokinetic profile of darolutamide, its diastereomers, and its main active metabolite, keto-darolutamide.Single-dose and multiple-dose pharmacokinetics ofFollowing darolutamide oral tablet administration, peak plasma concentrations were reached 4-6 h after dosing. Darolutamide elimination was characterized by a half-life of 13 h. Steady state was reached after approximately 2 days of twice-daily dosing. Pharmacokinetics of the diastereomers and keto-darolutamide followed similar trends to the parent compound. Darolutamide absorption from the tablet was lower than from the oral solution; tablet absolute bioavailability was ~30% in the fasted state but improved to 60-75% when given with food. The unbound fraction of darolutamide in plasma was 7.8%. The administered 1:1 ratio of the diastereomers (S,R)-darolutamide and (S,S)-darolutamide changed to ~1:6 in plasma following multiple dosing. Similar exposure and diastereomer ratios after single and multiple dosing indicate time-independent (no autoinduction) linear pharmacokinetics. Darolutamide exposure increased in patients with moderate hepatic or severe renal impairment vs healthy subjects; dose adaptation at treatment initiation should be considered in these patients.Darolutamide 600 mg twice daily demonstrates predictable linear pharmacokinetics and sustainably high plasma concentrations, suggesting the potential for constant inhibition of the androgen receptor signaling pathway.NCT02418650, NCT02894385, NCT02671097.

In this manuscript, we report a series of benzodioxine methyl piperidine derivatives as highly potent and selective α2C antagonists by ligand design to improve the pharmacokinetics of a previous candidate molecule.

Background and Objectives Darolutamide is a novel androgen receptor (AR) antagonist approved for the treatment of nonmetastatic castration-resistant prostate cancer (nmCRPC). Accordingly, the drug–drug interaction (DDI) potential of darolutamide was investigated in both nonclinical and clinical studies. Methods In vitro studies were performed to determine the potential for darolutamide to be a substrate, inducer or inhibitor for cytochrome P450 (CYP) isoforms, other metabolizing enzymes and drug transporters. A phase I drug-interaction study in healthy volunteers evaluated the impact of co-administering rifampicin [CYP3A4 and P-glycoprotein (P-gp) inducer] and itraconazole [CYP3A4, P-gp and breast cancer resistance protein (BCRP) inhibitor] on the pharmacokinetics of darolutamide. Two further phase I studies assessed the impact of co-administering oral darolutamide on the pharmacokinetics of midazolam (sensitive CYP3A4 substrate) and dabigatran etexilate (P-gp substrate) and the impact on the pharmacokinetics of co-administered rosuvastatin [a substrate for BCRP, organic anion-transporting polypeptide (OATP)1B1, OATP1B3 and organic anion transporter (OAT)3]. Results In vitro, darolutamide was predominantly metabolized via oxidative biotransformation catalyzed by CYP3A4 and was identified as a substrate for P-gp and BCRP. The enzymatic activity of nine CYP isoforms was not inhibited or slightly inhibited in vitro with darolutamide, and a rank order and mechanistic static assessment indicated that risk of clinically relevant DDIs via CYP inhibition is very low. In vitro, darolutamide exhibited no relevant induction of CYP1A2 or CYP2B6 activity. Inhibition of BCRP-, P-gp-, OAT3-, MATE1-, MATE2-K-, OATP1B1- and OATP1B3-mediated transport was observed in vitro. Phase I data showed that darolutamide exposure increased 1.75-fold with co-administered itraconazole and decreased by 72% with rifampicin. Co-administration of darolutamide with CYP3A4/P-gp substrates showed no effect or only minor effects. Rosuvastatin exposure increased 5.2-fold with darolutamide because of BCRP and probably also OATPB1/OATPB3 inhibition. Conclusions Darolutamide has a low potential for clinically relevant DDIs with drugs that are substrates for CYP or P-gp; increased exposure of BCRP and probably OATP substrates was the main interaction of note. Electronic supplementary material The online version of this article (10.1007/s13318-019-00577-5) contains supplementary material, which is available to authorized users.

Background: Saini et al. recently investigated the pharmacokinetics of darolutamide and its diastereomers in vitro and in vivo in Balb/c mice, reporting higher levels of (S,S)-darolutamide than (S,R)-darolutamide following intravenous or oral dosing, and interconversion of (S,R)-darolutamide to (S,S)-darolutamide. Objective: To present our in vitro and in vivo studies of darolutamide pharmacokinetics in mice, which contrast with the findings of Saini et al. Methods: Nude male Balb/c mice were orally dosed for 7 days with 25, 50, or 100 mg/kg of darolutamide twice daily. Pharmacokinetic parameters in plasma and tissue samples were assessed by liquid chromatography-tandem mass spectrometry. Metabolism and interconversion of darolutamide and its diastereomers were investigated in cryopreserved Balb/c mouse hepatocytes. Protein binding was determined in plasma samples by equilibrium dialysis. Results : On day 7, Cmaxwas reached 30 min after the last dose. Rapid formation and greater exposure of keto-darolutamide versus darolutamide were observed. Plasma exposure of (S,R)-darolutamide was 3-5-fold higher than that of (S,S)-darolutamide. The fraction of unbound keto-darolutamide was almost 6-fold lower than for darolutamide. Conclusion: The darolutamide diastereomer ratio changes upon administration in mice and other species due to interconversion through keto-darolutamide. This is not considered clinically relevant since both diastereomers and keto- darolutamide are pharmacologically similar in vitro. Based on the high protein binding of keto-darolutamide, its contribution in vivo in humans is considered low.

1. Darolutamide is a novel selective androgen receptor antagonist consisting of two pharmacologically equipotent diastereoisomers. The absorption, distribution, metabolism and excretion properties of darolutamide in rats are reported.2. Non- or [

The release and absorption profile of an oral medication is influenced by the physicochemical properties of the drug and its formulation, as well as by the anatomy and physiology of the gastrointestinal (GI) tract. During drug development the bioavailability of a new drug is typically assessed in early clinical studies in a healthy adult population. However, many disease conditions are associated with an alteration of the anatomy and/or physiology of the GI tract. The same holds true for some subpopulations, such as paediatric or elderly patients, or populations with different ethnicity. The variation in GI tract conditions compared to healthy adults can directly affect the kinetics of drug absorption, and thus, safety and efficacy of an oral medication. This review provides an overview of GI tract properties in special populations compared to healthy adults and discusses how drug absorption is affected by these conditions. Particular focus is directed towards non-disease dependent conditions (age, sex, ethnicity, genetic factors, obesity, pregnancy), GI diseases (ulcerative colitis and Crohn's disease, celiac disease, cancer in the GI tract, Roux-en-Y gastric bypass, lactose intolerance, Helicobacter pylori infection, and infectious diseases of the GI tract), as well as systemic diseases that change the GI tract conditions (cystic fibrosis, diabetes, Parkinson's disease, HIV enteropathy, and critical illness). The current knowledge about GI conditions in special populations and their impact on drug absorption is still limited. Further research is required to improve confidence in pharmacokinetic predictions and dosing recommendations in the targeted patient population, and thus to ensure safe and effective drug therapies. ispartof: EUROPEAN JOURNAL OF PHARMACEUTICAL SCIENCES vol:147 ispartof: location:Netherlands status: published

Background Darolutamide, an androgen receptor antagonist with a distinct molecular structure, significantly prolonged metastasis-free survival versus placebo in the phase III ARAMIS study in men with nonmetastatic castration-resistant prostate cancer (nmCRPC). In this population, polypharmacy for age-related comorbidities is common and may increase drug–drug interaction (DDI) risks. Preclinical/phase I study data suggest darolutamide has a low DDI potential—other than breast cancer resistance protein/organic anion transporter protein substrates (e.g., statins), no clinically relevant effect on comedications is expected. Objective Our objective was to evaluate the effect of commonly administered drugs on the pharmacokinetics of darolutamide and the effect of comedications potentially affected by darolutamide on safety in patients with nmCRPC. Patients and Methods Comorbidities and comedication use in the 1509 ARAMIS participants treated with darolutamide 600 mg twice daily or placebo were assessed. A population pharmacokinetic analysis evaluated whether comedications affected the pharmacokinetics of darolutamide in a subset of 388 patients. A subgroup analysis of adverse events (AEs) in statin users versus nonusers was conducted. Results Most participants (median age 74 years) had at least one comorbidity (98.4% in both arms) and used at least one comedication (98.7% with darolutamide vs. 98.0% with placebo); these were similar across study arms. Despite frequent use of comedications with DDI potential, no significant effects on darolutamide pharmacokinetics were identified. Comedications included lipid-modifying agents (34.5%), β-blockers (29.7%), antithrombotics (42.8%), and systemic antibiotics (26.9%). AE incidence was similar across study arms in statin users and nonusers. Study limitations include the small sample size for sub-analyses. Conclusions These analyses suggest the pharmacokinetic profile of darolutamide is not affected by a number of commonly administered drugs in patients with nmCRPC. Although pharmacokinetic data have indicated that darolutamide has the potential to interact with rosuvastatin, used to assess DDI in these studies, this finding did not seem to translate into increased AEs due to statin use in the ARAMIS trial. Clinicaltrials.gov identifier: NCT02200614. Electronic supplementary material The online version of this article (10.1007/s11523-019-00674-0) contains supplementary material, which is available to authorized users., Plain Language Summary Background Darolutamide is a medicine used to treat men with prostate cancer that has not spread to other parts of the body (nonmetastatic). Often, these patients are taking other medicines for common age-related illnesses. Taking more than one medicine at the same time increases the chances of what is known as drug–drug interactions. Drug–drug interactions can decrease how well the medicines work or may sometimes increase side effects. Study Aim To test for possible drug–drug interactions in men with prostate cancer who take darolutamide alongside other medicines. Study Participants Men with nonmetastatic prostate cancer who were being treated with a medicine that lowers testosterone, a chemical in the body that causes prostate cancer tumors to grow. Participants took two darolutamide 300 mg tablets, or an inactive placebo, twice a day. What Did the Researchers Measure? The researchers documented the number of medicines taken by each participant and the number of other medical conditions that they had. Tests were done to find out whether other medicines affected the way that darolutamide works in the body and whether patients taking darolutamide alongside other medicines experienced more side effects. Results As would be expected, based on the typical age of patients with prostate cancer, more than 90% of participants in this study used medicines other than darolutamide to manage common age-related illnesses or medical conditions. Taking medicines alongside darolutamide did not impact how darolutamide worked in the body and did not increase the number of side effects experienced by patients. Darolutamide is known to interact with rosuvastatin, a cholesterol-lowering drug. However, in this study, there was no overall increase in side effects among darolutamide-treated patients who took this type of drug compared with in those who did not. Conclusion In this study of patients with nonmetastatic prostate cancer, limited drug–drug interactions were seen when taking darolutamide alongside other medicines given to these patients to manage age-related medical conditions. Electronic supplementary material The online version of this article (10.1007/s11523-019-00674-0) contains supplementary material, which is available to authorized users.

Rapid reorganization and stabilization of the actin cytoskeleton in dendritic spines enables cellular processes underlying learning, such as long-term potentiation (LTP). Dendritic spines are enriched in exceptionally short and dynamic actin filaments, but the studies so far have not revealed the molecular mechanisms underlying the high actin dynamics in dendritic spines. Here, we show that actin in dendritic spines is dynamically phosphorylated at tyrosine-53 (Y53) in rat hippocampal and cortical neurons. Our findings show that actin phosphorylation increases the turnover rate of actin filaments and promotes the short-term dynamics of dendritic spines. During neuronal maturation, actin phosphorylation peaks at the first weeks of morphogenesis, when dendritic spines form, and the amount of Y53-phosphorylated actin decreases when spines mature and stabilize. Induction of LTP transiently increases the amount of phosphorylated actin and LTP induction is deficient in neurons expressing mutant actin that mimics phosphorylation. Actin phosphorylation provides a molecular mechanism to maintain the high actin dynamics in dendritic spines during neuronal development and to induce fast reorganization of the actin cytoskeleton in synaptic plasticity. In turn, dephosphorylation of actin is required for the stabilization of actin filaments that is necessary for proper dendritic spine maturation and LTP maintenance.SIGNIFICANCE STATEMENTDendritic spines are small protrusions from neuronal dendrites where the postsynaptic components of most excitatory synapses reside. Precise control of dendritic spine morphology and density is critical for normal brain function. Accordingly, aberrant spine morphology is linked to many neurological diseases. The actin cytoskeleton is a structural element underlying the proper morphology of dendritic spines. Therefore, defects in the regulation of the actin cytoskeleton in neurons have been implicated in neurological diseases. Here, we revealed a novel mechanism for regulating neuronal actin cytoskeleton that explains the specific organization and dynamics of actin in spines. The better we understand the regulation of the dendritic spine morphology, the better we understand what goes wrong in neurological diseases.

The majority of the postsynaptic terminals of excitatory synapses in the central nervous system exist on small bulbous structures on dendrites known as dendritic spines. The actin cytoskeleton is a structural element underlying the proper development and morphology of dendritic spines. Synaptic activity patterns rapidly change actin dynamics, leading to morphological changes in dendritic spines. In this mini-review, we will discuss recent findings on neuronal maturation and synaptic plasticity-induced changes in the dendritic spine actin cytoskeleton. We propose that actin dynamics in dendritic spines decrease through actin filament crosslinking during neuronal maturation. In long-term potentiation, we evaluate the model of fast breakdown of actin filaments through severing and rebuilding through polymerization and later stabilization through crosslinking. We will discuss the role of Ca(2+) in long-term depression, and suggest that actin filaments are dissolved through actin filament severing. © 2016 Wiley Periodicals, Inc.

Syftet med studien är att få reda på hur användningen av matematikens skriftliga språkande kan påverka elevernas lärande i räknesättet division. Informanterna i studien var fem elever i årskurs 4 som valdes bland de som samtyckte till deltagandet. I den praktiska delen av arbetet, konstruerade eleverna en textuppgift till matematiska uttrycket 24/6 - 3. Materialet bestod av intervjuer och det som eleverna producerade när de konstruerade uppgifter och deras svar. Intervjuerna analyserades med hjälp av en kvalitativ innehållsanalys. Resultatet visade att den största fördelen enligt eleverna var att matematikens språkande gör det roligare att arbeta med division. En annan viktig fördel var att elevernas förståelse i division ökade efter att de hade arbetat på detta sätt. De nackdelar som kom fram var att eleverna upplevde att det var arbetsamt och svårt att arbeta med metoden. Andra delen av resultatet visade att eleverna konstruerade uppgifter enbart med delningsdivision. De flesta elever gav siffran 24 kontexten av någonting ätbart. Största delen av eleverna menade att antalet 6 representerade människor. Eleverna definierade inte med tydlighet var antalet togs ifrån. Denna del av resultatet visade också att eleverna föredrar användning av symbolspråket och naturliga språket framför bildspråket i deras lösningar. Med stöd av det som resultatet visade kan språkandet anses vara ett bra sätt att arbeta med matematik i räknesättet division. The aim of the study is to see how the use of textual languaging of mathematics can affect pupils learning in an operation of division. Participants of the study consisted of five 4th grade pupils which were chosen among those who agreed to participate in the study. In the study, the pupils were supposed to construct a word problem for a mathematical expression 24/6 - 3. The research data consisted of interviews and of the material pupils produced when they constructed their word problems and their answers to them. The interviews were later analyzed with a qualitative content analysis. Results show that the most significant advantage according to the pupils were that languaging of mathematics makes it more fun to work with division. Another important result was that after the pupils had worked this way, they were able to understand the concept division a bit better. Disadvantages expressed by the participants were that they felt this kind of working method in mathematics may demand more work than what they are used to. The other part of the results show that the pupils constructed their word problems with division by parts. Almost all the pupils gave number 24 a context of something eatable. The most part of the pupils meant amount of 6 represent human beings, for example friends. Pupils didn’t clearly define where the amount of was taken away. This part of the results show also that the pupils preferred using symbolic language and natural language over pictorial language in their answers. Results indicates that languaging could be a recommended way to work with mathematics in division.

Background DARO is an androgen receptor antagonist with a distinct molecular structure, which has demonstrated prolonged metastasis-free survival vs placebo (PBO) in the phase 3 ARAMIS study in non-metastatic castration-resistant prostate cancer patients. These typically older patients often receive multiple comedications. We assessed DDIs with DARO both in vitro and in clinical trial development. Methods The effects of induction and inhibition of transporters and CYP enzymes on DARO, and the effect of DARO on other comedications were studied in vitro and in several phase 1 trials using rifampicin (CYP3A4, P-gp inducer), itraconazole (ICZ; CYP3A4, P-gp inhibitor), rosuvastatin (BCRP, OATP1B1, OATP1B3 and OAT3 substrate), midazolam (CYP3A4 substrate) and dabigatran etexilate (P-gp substrate). The impact of comedication on DARO was investigated in a population pharmacokinetic (popPK) and covariate analysis of a subset of ARAMIS patients. The adverse event (AE) profile for comedication use between PBO and DARO was also analysed. Results Concomitant rifampicin led to a 3.5-fold decrease in DARO area under the curve (AUC) vs DARO alone. ICZ resulted in 1.8-fold increase in DARO AUC vs DARO alone. No significant effect of comedications that are CYP or P-gp inhibitors was identified in the popPK analysis; proton pump inhibitors did not significantly affect the PK of DARO. Effects of DARO on CYP inhibition in vitro were negligible and coadministration of midazolam or dabigatran showed no clinically relevant effects. Rosuvastatin AUC increased by 5.2-fold with DARO vs rosuvastatin alone, attributed mainly to BCRP inhibition. The incidence of AEs in ARAMIS was low; comedication use (98.7% in DARO, 98.0% in PBO arms) and AE profile were similar between study arms. Conclusions DDIs with DARO and P-gp or CYP enzyme substrates, e.g. antithrombotics, calcium channel blockers or proton pump inhibitors, are not expected. Strong CYP3A4 inducers, e.g. rifampicin and carbamazepine, showed some interaction with DARO. Effects of CYP3A4 or P-gp inhibitors on DARO were not considered clinically relevant. DARO may increase the exposure of concomitant BCRP substrates, e.g. statins, although a safety and AE analysis from ARAMIS did not indicate any relevant impact of DARO. Clinical trial identification NCT03237416, NCT03048110, NCT02671097, NCT02200614. Editorial acknowledgement Medical writing support: Lucy Smithers, PhD, and editorial support: Beth King, both of Scion Medica, London, supported by Bayer according to Good Publication Practice guidelines. Legal entity responsible for the study Bayer. Funding Bayer and Orion Corporation. Disclosure C.R. Zurth: Full / Part-time employment: Bayer. K. Fizazi: Honoraria (self), Advisory / Consultancy: Merck, Sharp & Dohme; Advisory / Consultancy: Bayer; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Janssen; Advisory / Consultancy: AstraZeneca; Honoraria (self), Advisory / Consultancy: Sanofi; Honoraria (self), Advisory / Consultancy: Astellas; Advisory / Consultancy: Orion; Advisory / Consultancy: CureVac; Advisory / Consultancy: Clovis. R. Fricke: Full / Part-time employment: Bayer. H. Gieschen: Full / Part-time employment: Bayer. K. Graudenz: Full / Part-time employment: Bayer. M. Koskinen: Full / Part-time employment: Orion Corporation. B.A. Ploeger: Full / Part-time employment: Bayer. O. Prien: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bayer. M.R. Smith: Advisory / Consultancy: Bayer; Advisory / Consultancy: Amgen; Advisory / Consultancy: Astellas; Advisory / Consultancy: Clovis; Advisory / Consultancy: Gilead; Honoraria (self): Hinova; Honoraria (self), Advisory / Consultancy: Janssen; Honoraria (self), Advisory / Consultancy: Eli Lilly; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (self), Advisory / Consultancy: Pfizer. T. Tammela: Honoraria (self), Research grant / Funding (institution): Bayer; Advisory / Consultancy: Janssen; Advisory / Consultancy, Research grant / Funding (institution): Lidds AB; Advisory / Consultancy, Research grant / Funding (institution): Astellas. N.D. Shore: Advisory / Consultancy, Research grant / Funding (self): Ferring; Advisory / Consultancy, Speaker Bureau / Expert testimony: Bayer; Honoraria (self), Research grant / Funding (self): Amgen; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self): Janssen; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self): Dendreon; Advisory / Consultancy, Research grant / Funding (self): Tolmar; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self): Astellas.

The majority of the postsynaptic terminals of excitatory synapses in the central nervous system exist on small bulbous structures on dendrites known as dendritic spines. The actin cytoskeleton is a structural element underlying the proper development and morphology of dendritic spines. Synaptic activity patterns rapidly change actin dynamics, leading to morphological changes in dendritic spines. In this mini-review, we will discuss recent findings on neuronal maturation and synaptic plasticity-induced changes in the dendritic spine actin cytoskeleton. We propose that actin dynamics in dendritic spines decrease through actin filament crosslinking during neuronal maturation. In long-term potentiation, we evaluate the model of fast breakdown of actin filaments through severing and rebuilding through polymerization and later stabilization through crosslinking. We will discuss the role of Ca(2+) in long-term depression, and suggest that actin filaments are dissolved through actin filament severing. © 2016 Wiley Periodicals, Inc.

Objective To investigate the pharmacokinetics and excretion balance of [ 14 C]-OR-1896, a pharmacologically active metabolite of levosimendan, in six healthy male subjects. In addition, pharmacokinetic parameters of total radiocarbon and the deacetylated congener, OR-1855, were determined. Methods OR-1896 was administered as a single intravenous infusion of 200 μg of [ 14 C]-OR-1896 (specific activity 8.6 MBq/mg) over 10 min. The pharmacokinetic parameters were calculated by three-compartmental methods. Results During the 14-day collection of urine and faeces, excretion (±S.D.) averaged 94.2 ± 1.4% of the [ 14 C]-OR-1896 dose. Mean recovery of radiocarbon in urine was 86.8 ± 1.9% and in faeces 7.4 ± 1.5%. Mean terminal elimination half-life of OR-1896 ( t 1/2 ) was 70.0 ± 44.9 h. Maximum concentrations of OR-1855 were approximately 30% to that of OR-1896. Total clearance and the volume of distribution of OR-1896 were 2.0 ± 0.4 l/h and 175.6 ± 74.5 l, respectively. Renal clearances of OR-1896 and OR-1855 were 0.9 ± 0.4 l/h and (5.4 ± 2.3) × 10 −4 l/h, respectively. Conclusions This study provides data to demonstrate that nearly one half of OR-1896 is eliminated unchanged into urine and that the active metabolites metabolite of levosimendan remain in the body longer than levosimendan. The remaining half of OR-1896 dose is eliminated through other metabolic routes, partially through interconversion back to OR-1855 with further metabolism of OR-1855. Given the fact that the pharmacological activity and potency of OR-1896 is similar to levosimendan, these results emphasize the clinical significance of OR-1896 and its contribution to the long-lasting effects of levosimendan.

Nicotine biotransformation affects the smoking habits of addicted individuals and therefore their health risk. Using an improved analytical method, we have discovered that the human UDP-glucuronosyltransferase (UGT) 2B10, a liver enzyme previously unknown to conjugate nicotine or exhibit considerable activity toward any compound, plays a major role in nicotine inactivation by direct conjugation with glucuronic acid at the aromatic nitrogen atom. The K(m) value of recombinant UGT2B10 for nicotine (0.29 mM) was similar to that determined for human liver microsomes (0.33 mM), whereas the K(m) value of UGT1A4 for nicotine was almost 10-fold greater (2.4 mM). UGT2B10 was also more active than UGT1A4 in N-glucuronidation of cotinine (oxidative nicotine metabolite), whereas UGT2B7 exhibited only low nicotine glucuronidation activity and was essentially inactive toward cotinine. UGT1A9 did not glucuronidate nicotine or cotinine. Quantitative reverse transcription-polymerase chain reaction showed that UGT2B10 mRNA was exclusively expressed in human liver, whereas UGTs 1A4 and 2B7 were expressed at comparable, although somewhat lower, levels in liver and several other extrahepatic tissues, including kidney and intestine. These findings for UGT2B10 (but not for UGT1A4 and UGT2B7) were mirrored by human tissue activities because nicotine and cotinine glucuronidation rates in intestine microsomes were less than 0.1% that of human liver microsomes. These novel findings solve two seemingly separate questions: which UGT is primarily responsible for nicotine glucuronidation in human liver, and what conjugation reactions are catalyzed by UGT2B10.

This paper describes the Mobile Fair Diary (MFD), which is designed to allow a housing fair customer to make a personalized digital recording of his/her visit to a hectic fairground for later use. The MFD is a hybrid interface service comprising of an application for a camera equipped smart phone and a website accessed by a PC. The smart phone application is used for taking context-aware notes such as visual codes, photos, dictations and text. The notes are uploaded onto a website, where they can be viewed with a PC in a contextually ordered view for browsing, organizing and sharing. The MFD was empirically evaluated in a real-life environment of use with genuine end users by a large-scale field trial at a national housing fair. The results testify to the very successful design of the MFD with high usefulness.

An important issue for designing any conservation programme aimed at preserving genetic diversity is estimation of the scale at which genetic structuring occurs. Additional relevant factors include distinguishing whether or not population structuring is expected to be stable as predicted by the member-vagrant hypothesis, or alternatively, whether populations are more prone to local extinction-recolonization processes, as predicted by the metapopulation evolutionary model. In this study, the population genetic structure of Atlantic salmon from 11 locations within or nearby the Varzuga River tributary system was assessed using 17 microsatellites. Mantel tests and spatial autocorrelation analyses revealed a significant isolation-by-distance signal within the tributary system as well as a negative association between the level of genetic diversity and waterway distance from the river mouth, indicating that dispersal is less likely to occur to populations deep in the tributary system. Individual-level spatial autocorrelation analyses indicated that the majority of migration occurred between populations situated within 20 km of each other. The relatively high level of genetic structuring and significant isolation-by-distance signal observed in the Varzuga tributaries are concordant with the predictions of the member-vagrant evolutionary model. However, one subpopulation in particular revealed signs of instability which may be due to its location in the tidal zone, or due to the fact that it is more affected by human impacts. The results suggest that preservation of a number of spawning sites spaced throughout the tributary system is recommendable for ensuring sustainable fishing tourism in the river.

This study describes urinary excretion of two nucleobase adducts derived from styrene 7,8-oxide (SO), i.e., 7-(2-hydroxy-1-phenylethyl)guanine (N7alphaG) and 7-(2-hydroxy-2-phenylethyl)guanine (N7betaG), as well as a formation of N7-SO-guanine adducts in lungs and liver of two month old male NMRI mice exposed to styrene by inhalation in a 3-week subacute study. Strikingly higher excretion of both isomeric nucleobase adducts in the first day of exposure was recorded, while the daily excretion of nucleobase adducts in following time intervals reached the steady-state level at 4.32+1.14 and 6.91+1.17 pmol/animal for lower and higher styrene exposure, respectively. beta-SO-guanine DNA adducts in lungs increased with exposure in a linear way (F=13.7 for linearity and 0.17 for non-linearity, respectively), reaching at the 21st day the level of 23.0 adducts/10(8) normal nucleotides, i.e., 0.74 fmol/microg DNA of 7-alkylguanine DNA adducts for the concentration of 1500 mg/m3, while no 7-SO-guanine DNA adducts were detected in the liver after 21 days of inhalation exposure to both of styrene concentrations. A comparison of 7-alkylguanines excreted in urine with 7-SO-guanines in lungs (after correction for depurination and for missing alpha-isomers) revealed that persisting 7-SO-guanine DNA adducts in lungs account for about 0.5% of the total alkylation at N7 of guanine. The total styrene-specific 7-guanine alkylation accounts for about 1.0x10(-5)% of the total styrene uptake, while N1-adenine alkylation contributes to this percentage only negligibly.

This report reviews styrene biotransformation, including minor metabolic routes, and relates metabolism to the genotoxic effects and possible styrene-related carcinogenicity. Styrene is shown to require metabolic activation in order to become notably genotoxic and styrene 7,8-oxide is shown to contribute quantitatively by far the most (in humans more than 95%) to the genotoxicity of styrene, while minor ring oxidation products are also shown to contribute to local toxicities, especially in the respiratory system. Individual susceptibility depending on metabolism polymorphisms and individual DNA repair capacity as well as the dependence of the nonlinearity of the dose-response relationships in the species in question and the consequences for risk evaluation are analyzd.

1-SO-adenine DNA adducts, DNA single-strand breaks (SBs), chromosomal aberrations (CAs), mutant frequency (MF) at the HPRT gene, and immune parameters (hematological and of humoral immunity) were studied in styrene-exposed human subjects and controls. Results were correlated with genetic polymorphisms in DNA repair genes (XPD, exon 23, XPG, exon 15, XPC, exon 15, XRCC1, exon 10, XRCC3, exon 7) and cell cycle gene cyclin D1. Results for biomarkers of genotoxicity after stratification for the different DNA repair genetic polymorphisms showed that the polymorphism in exon 23 of the XPD gene modulates levels of chromosomal and DNA damage, HPRT MF, and moderately affects DNA adduct levels. The highest levels of biomarkers were associated with the wild-type homozygous AA genotype. The exposed individuals with the wild-type GG genotype for XRCC1 gene exhibited the lowest CA frequencies, compared to those with an A allele (P < 0.05). Cyclin D1 polymorphism seems to modulate the number of leukocytes and lymphocytes in the analyzed subjects. The number of eosinophiles was positively associated with XPD variant C allele and negatively with XRCC1 variant A allele (P < 0.05) and XPC variant C allele (P < 0.05). Immunoglobulin IgA was positively associated with an XRCC3 variant T allele (P < 0.01) and negatively with XPC variant C allele (P < 0.05). Both C3- and C4-complement components were lower in individuals with XRCC3 CT (P < 0.05) and TT genotypes (P < 0.01). Adhesion molecules sL-selectin and sICAM-1 were associated with XPC genotype (P < 0.05). Individual susceptibility may be reflected in genotoxic and immunotoxic responses to environmental and occupational exposures to xenobiotics.

We evaluated our data on the occupational exposure to styrene in lamination workers. The battery of parameters included markers of external and internal exposure and biomarkers of biological effects and susceptibility. DNA repair capacities have been determined in both exposed and control groups. Styrene workplace concentration significantly correlated with styrene concentration in blood, exhaled air and urinary mandelic acid. Haemoglobin and O 6 -styrene oxide (SO)-guanine DNA adducts were significantly higher in exposed subjects as compared to controls and correlated with exposure parameters. In styrene-exposed workers 1-SO-adenine DNA adducts were detected (2.6 per 10 9 dNp), while in controls these adducts were below the detection limit. 1-SO-adenine adduct levels were affected by both acute and cumulative exposure ( P =0.001, F =86.0 and P =0.017, F =59.0, respectively) and associated with cytochrome P450 2E1 (CYP2E1) polymorphisms ( R 2 =0.442). Mutant frequencies (MF) at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus appeared to accumulate with exposure over time and were associated with glutathione S -transferase P1 (GSTP1) polymorphism. DNA repair capacity increased with the exposure, except for the group exposed to the highest styrene concentration. In this particular group, increased DNA repair capacity to remove oxidative DNA damage was found.

Theories on the hydrological history of Lake Baikal, the world’s oldest and deepest body of freshwater, and its surrounding great rivers, are currently based solely on geological evidence and are conflicting. Baikal is inhabited by numerous zoogeographical enigmas but their high level of endemism has hindered phylogeographic inferences. We provide a biological perspective of the region’s palaeo-hydrological development based on the demographic and genealogical history of the widespread Thymallus spp. (grayling). Phylogenetic reconstruction reveals that old lineages of grayling (pre-Pleistocene) currently inhabit the Enisey, Lena and Amur River basins. For Lake Baikal however, we conclude that a mid-Pleistocene colonization (110 000—450 000 years ago) of an unoccupied niche has occurred. Population genetic inferences support an Enisey—Angara river route of colonization into Baikal, corresponding to the cataclysmic palaeo-hydrological event that led to the formation of the lake’s only contemporary outlet, and a subsequent range expansion several thousand kilometres into the uppermost reaches of the Selenga River basin. The evolutionary history of Lake Baikal grayling is congruent with the controversial hypothesis of repeated glaciation. However, considering the extraordinary levels of endemism and proposed Miocene or Oligocene coalescence of other faunal lineages, a less profound but equally consequential cycle of environmental perturbations may have taken place. Bi-parentally inherited microsatellite DNA loci supported the phylogenetic relationships of Thymallus spp. and the geographical expansion of Baikal grayling strikingly well. A Markov Chain Monte Carlo modelling approach suggested severe contemporary population decline during the last century, possibly reflecting the influence of an uncontrolled fishery on this treasured ecosystem. These complementary pictures of the demographic history of grayling underscore the breadth of historical inquiry that can be entertained through the modelling of sufficient molecular data, and may significantly alter the zoogeographical and limnological perspectives of Baikal’s history.

The relative importance of natural selection and random drift in phenotypic evolution has been discussed since the introduction of the first population genetic models. The empirical evidence used to evaluate the evolutionary theories of Fisher and Wright remains obscure because formal tests for neutral divergence or sensitive attempts to separate the effects of selection and drift are scarce, subject to error, and have not been interpreted in the light of well-known population demography. We combined quantitative genetic and microsatellite DNA analyses to investigate the determinants of contemporary life-history evolution in isolated populations of grayling (Thymallus thymallus, Salmonidae) that originated from a common source 80-120 years ago. Here we show that natural selection was the dominant diversifying agent in the evolution of the quantitative traits. However, the populations were founded by a small number of individuals, exhibit very low microsatellite-based effective sizes and show genetic imprints of severe 'bottlenecks'; which are conditions often suggested to constrain selection and favour drift. This study demonstrates a very clear case of fisherian evolution in small natural populations across a contemporary timescale.

Objective. Based on our previous studies we analysed DNA and haemoglobin adducts, single-strand breaks in DNA, chromosomal aberrations and HPRT mutant frequencies in styrene-exposed workers, in relation to employment time, with respect to markers of individual susceptibility. In our study the risk assessment of genotoxic styrene in occupationally exposed humans is reviewed in the light of adaptation and/or population selection. Various styrene-induced DNA adducts (representing important integral measures of DNA damage) are discussed as potential powerful biomarkers of styrene exposure, with respect to DNA repair. Methods. The complexity of followed markers required multiple methodological approaches, including modified 32P-post-labelling assay, PCR-based methods for genotyping, cytogenetic analysis, T-cell cloning assay, comet assay and tests for DNA repair capacity, and statistical tests. Results. Haemoglobin and DNA adducts, single-strand breaks in DNA, chromosomal aberrations and mutant frequencies at the HPRT gene were assessed with respect to the duration of exposure. No apparent accumulation of these biomarkers was found to be dependent on the years of employment, suggesting that adaptive processes play a role in chronically exposed workers. Statistically significant differences between the exposed and control individuals were found for the deduced epoxide hydrolase activity (P=0.041) and the frequency of heterozygous Ala/Val genotype in GSTP1, exon 5 (P=0.025). Significantly increased DNA repair capacity was found in styrene-exposed laminators compared with control individuals. Conclusions. In the light of present knowledge well-designed population studies on styrene genotoxicity are needed. Such studies should cover important areas of individual susceptibility (metabolising and repair enzymes, distributions of genetic polymorphisms) and possible role of adaptation, as well as the crucial role of DNA repair. Additionally, genotoxic effects of in-vivo formed 3,4-styrene oxide should be also addressed.

During the last decade, numerous studies have demonstrated that the actin cytoskeleton plays a pivotal role in the control of dendritic spine shape. Synaptic stimulation rapidly changes the actin dynamics and many actin regulators have been shown to play roles in neuron functionality. Accordingly, defects in the regulation of the actin cytoskeleton in neurons have been implicated in memory disorders. Due to the small size of spines, it is difficult to detect changes in the actin structures in dendritic spines by conventional light microscopy imaging. Instead, to know how tightly actin filaments are bundled together, and how fast the filaments turnover, we need to use advanced microscopy techniques, such as fluorescence recovery after photobleaching (FRAP), photoactivatable green fluorescent protein (PAGFP) fluorescence decay and fluorescence anisotropy. Fluorescence anisotropy, which measures the Förster resonance energy transfer (FRET) between two GFP fluorophores, has been proposed as a method to measure the level of actin polymerization. Here, we propose a novel idea that fluorescence anisotropy could be more suitable to study the level of actin filament bundling instead of actin polymerization. We validate the method in U2OS cell line where the actin structures can be clearly distinguished and apply the method to analyze how actin filament organization in dendritic spines changes during neuronal maturation. In addition to fluorescence anisotropy validation, we take a critical look at the properties and limitations of FRAP and PAGFP fluorescence decay methods and offer our proposals for the analysis methods for these approaches. These three methods complement each other, each providing additional information about actin dynamics and organization in dendritic spines.

The possible carcinogenicity of styrene is believed to be related to the DNA-binding properties of styrene 7,8-oxide (SO). In order to compare the intrinsic reactivity of the different nucleophilic sites in DNA towards SO and to evaluate the candidates for human biomonitoring we have determined the second-order rate constants and stabilities of several SO-adducts in double-stranded DNA. These include alpha- and beta-isomers of N7-substituted and alphaN(2)-substituted guanines, alpha- and betaN3-substituted and alphaN(6)-substituted adenines as well as betaN3- and alphaN(4)-substituted cytosines. The highest rate constants were found for the spontaneously depurinating N7-guanines being ca. 3-15-fold higher than those for the stable adducts. When the relative proportions of different alkylation products were determined in course of time, after a single addition of SO, the labile N7-guanines and N3-adenines were the major products at early time points. After 144 h of incubation at 37 degrees C, alphaN(6)-SO-adenine and alphaN(2)-SO-guanine as well as betaN3-SO-uracil were the major adducts. Regarding human biomonitoring, the N7-substituted guanines should be one of the main targets because of the high reactivity of the N7-atom of guanine. However, in the case of chronic styrene exposures the chemically more stable DNA adducts may become important.

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).

In order to provide guidelines for conservationand management of the severely declining LakeSaimaa (eastern Finland) European grayling(Thymallus thymallus, Salmonidae), weinvestigated the microgeographic geneticstructure of three populations from the watersystem using 17 microsatellite loci andmitochondrial DNA polymerase chainreaction-restriction fragment lengthpolymorphism analysis. Microsatellites revealedlow levels of intrapopulation genetic diversityand substantial divergence between populationssampled from spawning sites separated by aslittle as 55 kilometers. Mitochondrial analysisindicated the occurrence of two compositehaplotypes within Lake Saimaa. The nucleotidesubstitution estimates between the haplotypessuggested their separation to markedly predatethe late Pleistocene period. The populationsexhibited marked frequency differences for thetwo mitochondrial haplotypes, reinforcing theview of limited interpopulation gene flowwithin Lake Saimaa. An individual basedmicrosatellite Neighbor-Joining dendrogramdemonstrated clustering of all the specimensaccording to their sampling origin. Individualassignment tests revealed 100% assignmentsuccess of individuals into their population oforigin and 100% exclusion (p

Drug metabolism can result in the formation of highly reactive metabolites that are known to play a role in toxicity resulting in a significant proportion of attrition during drug development and clinical use. Thus, the earlier such reactivity was detected, the better. This review summarizes our multi-year project, together with pertinent literature, to examine a battery of in vitro tests capable of detecting the formation of reactive metabolites. Principal prerequisites for such tests were delineated: chemicals known/not known to cause tissue injury and produce reactive metabolites, activation system (mainly human-derived), small- and large-molecular targets (small-molecular trappers, peptides, proteins), analytical techniques (mass spectrometry), and cellular toxicity biomarkers. The current status of in vitro tools to detect reactive intermediates is the following: 1. Small-molecular trapping agents such glutathione or cyanide detect the production of reactive species with high sensitivity by proper MS technique. However, it seems that also putative “negatives” give rise to corresponding adducts. 2. Results from peptide and dG (DNA targeting) trapper studies are generally in line with those of small-molecular trappers, although also important differences exist. These two trapping platforms do not overlap. 3. It is anticipated that the in vitro adduct studies could be fully interpreted only in conjunction with toxicity biomarker (such as the Nrf2 pathway) information from whole cells or tissues. However, while there are tools to characterize the chemical liability and there are correlation between individual/integrated endpoints and toxicity, there are still severe gaps in understanding the mechanisms behind the link between reactive metabolites and adverse effects.

In stark contrast to other species within the Salmonidae family, phylogeographic information on European grayling, Thymallus thymallus, is virtually nonexistent. In this paper, we utilized mitochondrial DNA polymerase chain reaction-restriction fragment length polymorphism (mtDNA PCR-RFLP) and sequence variation to infer the postglacial dispersal routes of T. thymallus into and within northern Europe, and to locate geographically, potential evolutionarily distinct populations. Mitochondrial analyses revealed a total of 27 T. thymallus haplotypes which clustered into three distinct lineages. Average pairwise interlineage divergence was four and nine times higher than average intralineage divergence for RFLP and sequence data, respectively. Two European grayling individuals from the easternmost sample in Russia exhibited haplotypes more genetically diverged from any T. thymallus haplotype than T. arcticus haplotype, and suggested that hybridization/introgression zone of these two sister species may extend much further west than previously thought. Geographic division of the lineages was generally very clear with northern Europe comprising of two genetically differentiated areas: (i) Finland, Estonia and north-western Russia; and (ii) central Germany, Poland and western Fennoscandia. Average interpopulation divergence in North European T. thymallus was 10 times higher than that observed in a recent mtDNA study of North American T. arcticus. We conclude that (i) North European T. thymallus populations have survived dramatic Pleistocene temperature oscillations and originate from ancient eastern and central European refugia; (ii) genetic divergence of population groups within northern Europe is substantial and geographically distinct; and (iii) the remainder of Europe harbours additional differentiated assemblages that likely descend from a Danubian refugium. These findings should provide useful information for developing appropriate conservation strategies for European grayling and exemplify a case with a clear need for multinational co-operation for managing and conserving biodiversity.

The reaction between 2'-deoxycytidine and styrene 7,8-oxide (SO) resulted in alkylation at the 3-position and at the O(2)-position through the alpha- and beta-carbons of the epoxide but at the N(4)-position only through the alpha-carbon. The 3-alkylated adducts were found to deaminate to the corresponding 2'-deoxyuridine adducts (37 degrees C, pH 7.4) with half-lives of 6 min and 2.4 h for the alpha- and beta-isomers, respectively. The N(4)-alkylated products were stable at neutral pH. The O(2)-alkylated products were unstable being prone to depyrimidation and to isomerisation between alpha- and beta-isomers. In SO-treated double-stranded DNA, enzymatic hydrolysis allowed the identification of the beta3-deoxyuridine and alphaN(4)-deoxycytidine adducts (1.9 and 0.5% of total alkylation, respectively), in addition to the previously identified DNA-adducts. The 3-substituted uracil may have implications for the mutagenicity of SO.

FC1271a is a novel triphenylethylene compound with a tissue-selective profile of estrogen agonistic and weak antagonistic effects. It specifically binds to the estrogen receptor alpha and beta with affinity closely similar to that of toremifene and tamoxifen. To study the in vivo effects of the compound, 4-month-old rats were sham operated (sham) or ovariectomized (OVX) and treated daily for 4 weeks with various doses of FC1271a or vehicle (orally). FC1271a was able to oppose OVX-induced bone loss by maintaining the trabecular bone volume of the distal femur. Accordingly, the OVX-induced loss of bone strength was prevented at doses of 1 and 10 mg/kg. FC1271a also prevented the OVX-induced increase in serum cholesterol in a dose-dependent manner. No significant changes in uterine wet weight or morphology were observed in the OVX-rats treated with 0.1 or 1 mg/kg FC1271a, but at a dose of 10 mg/kg it had a slightly estrogenic effect. In immature rats the effect of FC1271a on uterine wet weight was less stimulatory than that of toremifene or tamoxifen, but more stimulatory than that of raloxifene or droloxifene. The appearance of the dimethylbenzanthracene (DMBA)-induced mammary tumors was inhibited by treatment of DMBA-treated rats with FC1271a in a dose-dependent manner. In human MCF-7 breast cancer cell tumors raised in nude mice in the presence of estrogen, the growth and expression of pS2 marker gene could not be maintained after estrogen withdrawal by treatment with FC1271a. No formation of DNA adducts was observed in the liver of the FC1271a-treated rats. In conclusion, the bone-sparing, antitumor, and cholesterol-lowering effects of FC1271a combined with a low uterotropic activity and lack of liver toxicity indicate that FC1271a could be an important alternative in planning antiosteoporosis therapy for estrogen deficiency.

Styrene 7,8-oxide (SO), a major metabolite of styrene, is classified as a probable human carcinogen. In the present work, salmon testis DNA was reacted with SO and the alkylation products were analysed after sequential depurination in neutral or acidic conditions followed by HPLC separation and UV-detection. A novel finding was that the N-3 position of adenine was the next most reactive alkylation site in double-stranded DNA, comprising 4% of the total alkylation, as compared to alkylation at the N-7 position of guanine, 93% of the total alkylation. Both alpha- and beta-products of SO were formed at these two sites. Other modified sites were N2-guanine (1.5%, alpha-isomer), 1-adenine (0.4%, both isomers) and N6-adenine (0.7%, both isomers) as well as 1-hypoxanthine (0.1%, alpha-isomer), formed by deamination of the corresponding 1-adenine adduct. The results indicated that in double-stranded DNA N-7 of guanine and N-3 of adenine account for 97% of alkylation by SO. However, these abundant adducts are not stable, the half-life of depurination in DNA for 3-substituted adenines being approximately 10 and approximately 20 h, for alpha- and beta-isomers, respectively, and 51 h for both isomers of 7-substituted guanines.

Styrene 7,8-oxide (SO), a reactive epoxide that has been classified as a probable human carcinogen, was allowed to react under alkaline conditions with 2′-deoxyguanosine and thymidine monophosphates as well as with the dinucleotide dGpdT. The reaction products were separated by high-performance liquid chromatography and were characterised by UV and electrospray mass spectroscopy, the latter showing the ability to differentiate the isomerism of the hydroxyphenylethyl moiety of the adduct. The main alkylation products of the deoxyguanosine monophosphates at high pH were those reacted at the 1- (through the β-carbon of the epoxide), N2- (α-carbon) and 7-positions (β- and α-carbons) of guanine followed by alkylation of the phosphate group. The formation of a novel diastereomeric pair of N 2-guanine adducts connected to the β-carbon of SO was identified and characterised. Furthermore, two different geometrical isomers of 1-guanine were detected. For thymidine nucleotide, base alkylation under neutral conditions was almost negligible, but at pH 10.5, alkylation at the 3-position was very prominent. The same base-adducts and the pH-effects were observed in the case of dGpdT alkylation. However, no phosphate alkylation was detected in the case of the dinucleotide.

The structures and protonation energies of aminoguanidine and aminoguanidinium ion were obtained from quantum chemical calculations by using the density functional method B3-LYP and the standard basis set 6 -31 G(d ). The energy differences between the various forms of the two possible tautomers, the endiamine and the imidamide form, were investigated. The endiamine was found to be more stable than the most stable form of the imidamide by 5 kcal/mol in the gas phase. The proton affinity of am inoguanidine was found to be 241.4 kcal/mol (B3 -LYP/6 -31 G(d )) in the gas phase. The liquidation pKa2 for amininoguanidine and the guanidinium ion was determined to be 11.5 ± 0.1. The reference values for guanidine free base and the guanidinium ion are 235.7 kcal/mol and 13.6, respectively. The difference in the order of basicity in the gas phase and in aqueous solution between aminoguanidine and guanidine is discussed in terms of symmetry, resonance, delocalisation, rehybridisation of nitrogen atoms, and electrostatic repulsion.

DNA isolated from livers of rats receiving tamoxifen was analysed by the 32P-postlabelling method. The postlabelled DNA hydrolysis mixture was analysed both by reversed-phase HPLC with 32P on-line detection and by TLC on polyethyleneimine plates followed by autoradiography. Using the HPLC method, five well separated adduct peaks could be detected, while by the TLC method, two groups of adduct spots were observed. The detection limit of the TLC assay was lower (0.5 adducts/10(10) nucleotides) than that of the HPLC assay (3 adducts/10(10) nucleotides). Thus, the TLC assay is more sensitive but also more laborious. The advantages of the HPLC assay were, in addition to better resolution, the ease of quantification and operation.

The crystal and molecular structure of aminoguanidine hemioxalate, a salt in which aminoguanidine exists in the monocation form, was determined by single crystal X-ray diffraction. The salt crystallizes in the monoclinic space group P2(1)/n with unit cell dimensions ofa=4.95,b=10.46,c=10.40 A, β=92.57°, andZ=4. The structure contains one oxalate ion for every two CN4H 7 + ions, the latter being practically planar. The structure of the monocation is largely similar to those of aminoguanidine dications except that the monocation is devoid of one of the protons attached to the terminal hydrazine nitrogen. This result is of interest considering the synthesis of amidinohydrazones, indicating that the concentration of the active nucleophile is nearly maximal even when aminoguanidine exists in the monocation form. Therefore, the synthesis of amidinohydrazones should be performed in thepH range in which aminoguanidine exists mainly in the monocation form,i.e. at apH higher than 2. There is, however, no need to elevate thepH to values at which a considerable proportion of aminoguanidine exists as the free base.

Precise control of the formation and development of dendritic spines is critical for synaptic plasticity. Consequently, abnormal spine development is linked to various neurological disorders. The actin cytoskeleton is a structural element generating specific changes in dendritic spine morphology. Although mechanisms underlying dendritic filopodia elongation and spine head growth are relatively well understood, it is still not known how spine heads are enlarged and stabilized during dendritic spine maturation. By using rat hippocampal neurons, we demonstrate that the size of the stable actin pool increases during the neuronal maturation process. Simultaneously, the treadmilling rate of the dynamic actin pool increases. We further show that myosin IIb controls dendritic spine actin cytoskeleton by regulating these two different pools of F-actin via distinct mechanisms. The findings indicate that myosin IIb stabilizes the stable F-actin pool through actin cross-linking. Simultaneously, activation of myosin IIb contractility increases the treadmilling rate of the dynamic pool of actin. Collectively, these data show that myosin IIb has a major role in the regulation of actin filament stability in dendritic spines, and elucidate the complex mechanism through which myosin IIb functions in this process. These new insights into the mechanisms underlying dendritic spine maturation further the model of dendritic spine morphogenesis.

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, 54/10(4) nucleotides) in BMO-treated DNA was about four times higher than that of N-1-BMO-adenine adducts (1´-carbon, 20; 2´-carbon, 8.7/10(4) 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 (11/10(8) nucleotides) in rat liver was about three-fold higher than N-7-BMO-guanine adducts (4.0/10(8) 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.

Styrene oxide (SO), a reactive metabolite of styrene, modifies DNA at several nucleophilic sites. In the present work we have determined the SO-DNA adducts in vitro and in vivo by two different versions of (32)P-postlabelling/HPLC assays. When anionexchange cartridges were used for adduct enrichment the β-isomer of 7-substituted guanines was detected in in vitro SO-treated DNA as well as in mice lungs exposed to styrene at 750 and 1500 mg m(-3) for 21 days (6 h day(-1), 7 days week(-1)). In the lungs, the adduct levels were 6.5 and 23 per 10(8) nucleotides for the two doses, respectively. When the nuclease P1 resistant adducts were studied by the (32)P-postlabelling/HPLC assay involving nuclease P1/prostatic acid phosphatase hydrolysis, the main adducts in in vitro-treated DNA were the α-isomer of N(2)-substituted guanine, β-isomers of 1-substituted adenine and 3-substituted uracil. β1-SO-adenine adduct was detected in the mice lung tissues after conversion of the 1-substituted adduct to the βN(6)-SO-adenine adduct by the Dimroth rearrangement. The 1-adenine adduct levels for the two doses were found to be 0.17 and 0.51 per 10(8) nucleotides. The current results show the potential of using the 7-guanine and 1-adenine adducts as biomarkers in biomonitoring of styrene exposure.

The crystal and molecular structure of aminoguanidine monohydrochloride, CN4H7 +.CL- in which aminonoguanidine exists in the monocation form, was determined by single-crystal X-ray diffraction. The structure of the monocation is largely similar to that of aminoguanidine dication as present in previously studied divalent salts. The monocation was found to exist in the form of the tautom er that allows strong resonance in the guanyl group. As compared to the dication, the terminal hydrazine nitrogen atom bears one hydrogen atom less. The monocation is planar, the only atoms deviating from the plane being the hydrogens attached to the terminal hydrazine nitrogen. Quantum chemical calculations on the endiamine tautomer of aminoguanidine free base as well as on aminoguanidine monocation and dication were also perform ed by using the HF and MP2 ab initio methods and also the B3- LYP and B-LYP methods based on density functional theory. On the basis of the calculations, a predicted structure of the endiamine tautom er of aminoguanidine free base is presented

A reversed-phase (C18) HPLC method with diode-array detection was developed for the separation and determination of methylglyoxal bis(amidinohydrazone) (mitoguazone) and seven closely related aliphatic analogs thereof, namely the bis(amidinohydrazones) of glyoxal, dimethylglyoxal, ethylmethylglyoxal, methylpropylglyoxal, butylmethylglyoxal, diethylglyoxal and dipropylglyoxal. The mobile phase consisted of a non-linear binary gradient of methanol and 0.03 M aqueous sodium acetate buffer (pH 4.3). Good separation of the eight congeners was achieved. On increasing the methanol content of the eluent, the bis(amidinohydrazones) eluted in the order of increasing number of carbon atoms in the side-chains. The method was also applied to the quantitative analysis of the compounds in aqueous solution and, combined with ultrafiltration, for the separation of the eight congeners in spiked human blood serum. A separate simplified method for the quantitative determination of each of the compounds in spiked human blood serum samples was also developed. The methods developed made for the first time possible the simultaneous HPLC analysis of more than one bis(amidinohydrazones). The results obtained indicate that the bis(amidinohydrazones) studied obviously have a distinct tendency to form ion associates with acetate ions and probably also other carboxylate ions in aqueous solution. This aspect may be of biochemical significance, especially concerning the intracellular binding of the compounds. Each one of the compounds studied invariably gave rise to one peak only, this result supporting the theory that the conventional synthesis of each of the compounds gives rise to one geometrical isomer only. This result is completely in agreement with the results of previous proton and carbon NMR spectroscopic as well as X-ray diffraction studies.