Bromide Release Research Articles

Psiadia punctulata (DC.) Vatke induces cell apoptosis in highly metastatic MDA-MB-231 cells

Purpose: This study assessed the in vitro cell migration inhibitory and cell apoptotic effects of P. punctulata stem (PPS (and leaf hexane) PPL (extracts on breast cancer cell lines (MDA-MB-231 andMCF-7 cells).Methods: Cytotoxicity was quantified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LD) release test after 48 h exposure of MDA-MB-231 cells to 0 – 200 μg/mL of PPS and PPL hexane extract. Cell apoptosis was determined using Muse™ cell cytometry, while the phytoconstituents of PPS and PPL hexane extracts were identified by gas chromatography–mass spectrometry.Results: The half-maximal inhibitory concentration (IC50) for PPS and PPL hexane extracts against MDA-MB-231 cells was 44.33 and 52.16 μg/mL, respectively. T, whereas the IC50 of PPS and PPL hexane extracts was 102.22 and 59.53 μg/mL against MCF-7 cells, sequentially. Treatment with 100 and 200 μg/mL of PPS and PPL hexane extract increased late apoptosis in MDA-MB-231 cells to 16.005 ± 1.155 and 52.58 ± 3.02 %, respectively, for PPS hexane extract and 77.34 ± 0 % and 95.21 ± 1.61 %, respectively, for PPL hexane extract, when compared to control cells (3.81% ± 0.79%). PPL hexane extract decreased cell migration and filled ~15.5 % of the wound gap on MDA-MB-231 cells after 24 h, while PPS hexane extract decreased cell migration by ~35 and ~42.5 % at 24 and 48 h, respectively. PPS and PPL hexane extracts contained several phytocompounds. Stem and leaf extracts of P. punctulata showed significant (p < 0.05) cell apoptotic and migration inhibition activities.Conclusion: The extracts P. punctulata exhibit potent cytotoxic activity against the tested breast cancer cells. Further research is required to assess the acute and subacute toxicity of the extracts.

Aerobic degradation of decabrominated diphenyl ether through a novel bacterium isolated from municipal waste dumping site: Identification, degradation and metabolic pathway

In the present study, a novel bacterium capable of degrading BDE-209 aerobically was isolated from a municipal waste dumping site and identified as Bacillus tequilensis strain BDE-S1 through 16S rRNA gene sequencing. A correlation between BDE-209 and bromide concentration, COD, TOC, and cell biomass was established. 65% of 50 mg/L initial concentration of BDE-209 was degraded within eight days of incubation by BDE-S1 strain. Two hexa, two penta, one tetra-BDE congener, and benzamide were detected as metabolites. The bromide release, COD, TOC and cell biomass were found to be significantly correlated parameters with BDE-209 degradation. Based on the metabolite analysis, ortho and meta debromination, cleavage of diphenyl ether bond and ring-opening were suggested as possible degradation pathways. This is the first study demonstrating the use of indigenously isolated Bacillus tequilensis strain BDE-S1 for aerobic degradation of BDE-209, which could provide new comprehension for bioremediation of PBDEs from contaminated environments.

Pan-Arctic surface ozone: modelling vs. measurements

Abstract. Within the framework of the International Arctic Systems for Observing the Atmosphere (IASOA), we report a modelling-based study on surface ozone across the Arctic. We use surface ozone from six sites – Summit (Greenland), Pallas (Finland), Barrow (USA), Alert (Canada), Tiksi (Russia), and Villum Research Station (VRS) at Station Nord (North Greenland, Danish realm) – and ozone-sonde data from three Canadian sites: Resolute, Eureka, and Alert. Two global chemistry models – a global chemistry transport model (parallelised-Tropospheric Offline Model of Chemistry and Transport, p-TOMCAT) and a global chemistry climate model (United Kingdom Chemistry and Aerosol, UKCA) – are used for model data comparisons. Remotely sensed data of BrO from the GOME-2 satellite instrument and ground-based multi-axis differential optical absorption spectroscopy (MAX-DOAS) at Eureka, Canada, are used for model validation. The observed climatology data show that spring surface ozone at coastal sites is heavily depleted, making ozone seasonality at Arctic coastal sites distinctly different from that at inland sites. Model simulations show that surface ozone can be greatly reduced by bromine chemistry. In April, bromine chemistry can cause a net ozone loss (monthly mean) of 10–20 ppbv, with almost half attributable to open-ocean-sourced bromine and the rest to sea-ice-sourced bromine. However, the open-ocean-sourced bromine, via sea spray bromide depletion, cannot by itself produce ozone depletion events (ODEs; defined as ozone volume mixing ratios, VMRs, < 10 ppbv). In contrast, sea-ice-sourced bromine, via sea salt aerosol (SSA) production from blowing snow, can produce ODEs even without bromine from sea spray, highlighting the importance of sea ice surface in polar boundary layer chemistry. Modelled total inorganic bromine (BrY) over the Arctic sea ice is sensitive to model configuration; e.g. under the same bromine loading, BrY in the Arctic spring boundary layer in the p-TOMCAT control run (i.e. with all bromine emissions) can be 2 times that in the UKCA control run. Despite the model differences, both model control runs can successfully reproduce large bromine explosion events (BEEs) and ODEs in polar spring. Model-integrated tropospheric-column BrO generally matches GOME-2 tropospheric columns within ∼ 50 % in UKCA and a factor of 2 in p-TOMCAT. The success of the models in reproducing both ODEs and BEEs in the Arctic indicates that the relevant parameterizations implemented in the models work reasonably well, which supports the proposed mechanism of SSA production and bromide release on sea ice. Given that sea ice is a large source of SSA and halogens, changes in sea ice type and extent in a warming climate will influence Arctic boundary layer chemistry, including the oxidation of atmospheric elemental mercury. Note that this work dose not necessary rule out other possibilities that may act as a source of reactive bromine from the sea ice zone.

Mechanistic insights into ethidium bromide removal by palygorskite from contaminated water

Ethidium bromide (EtBr)-containing wastewater can be hazardous to biodiversity when released into the soil and water bodies without treatment. EtBr can mutate living microbial cells and pose toxicity to even higher organisms. This work investigated the removal of EtBr from aqueous solutions by a naturally occurring palygorskite (PFl-1) clay mineral via systematic batch adsorption experiments under different physicochemical conditions. EtBr existed in an undissociated form at pH ~7, and was adsorbed on PFl-1 obeying the Freundlich isotherm model. The maximum EtBr adsorption capacity was 285 mmol/kg. The best fitted kinetic model for EtBr adsorption was the pseudo-second order model. The amounts of exchangeable cations desorbed from PFl-1 during EtBr adsorption was linearly correlated to the amounts of EtBr adsorbed, with a slope of 0.97, implying that a cation exchange-based adsorption mechanism was dominating. Additionally, dimerization of EtBr molecules via bromide release assisted an increased EtBr removal by PFl-1 at high adsorbate concentrations. Detailed x-ray diffraction, Fourier transform infrared, scanning electron imaging and energy dispersive x-ray analyses confirmed that EtBr adsorption occurred dominantly on the surface of palygorskite which mineralogically constituted 80% of the bulk PFl-1 adsorbent. A small portion of EtBr was also adsorbed by PFl-1 through intercalation onto the smectite impurity (10%) in PFl-1. This study suggested that PFl-1 could be an excellent natural material for removing EtBr from pharmaceutical and laboratory wastewater.

Holistic approach for evaluation of landfill leachate pollution potential – From the waste to the aquifer

Leachate contamination from uncontrolled landfills is a long-lasting environmental hazard that threatens groundwater resources worldwide. We developed a holistic site-characterization approach that incorporates leachate data from the waste body, unsaturated zone, and groundwater with information on landfill geomorphology and climatic conditions. An advanced vadose-zone monitoring system was used to characterize the percolation patterns and chemical properties of the leachates in the waste body and underlying unsaturated zone; a set of observation wells was used to characterize the contaminants' distribution in the groundwater. Multivariate analysis of the chemical composition overcame multiparameter complexity, and pinpointed the dominant factors controlling contaminant migration dynamics. The landfill's mound morphology, constructed with steep slopes, led to runoff generation, limited water infiltration through the waste, and enhanced infiltration of contaminated water at the landfill margins. Aerated conditions in the unsaturated zone under the margins induced leachate degradation and oxidation processes. The chemical composition of leachates under the center of the landfill remained typically anaerobic (high DOC, NH4+, Fe2+) despite the low water penetration. The limited water-percolation rates through the waste body and substantial mixing of the leachates with the oxidizing aquifer water led to almost complete degradation of the organic matter and significant nitrogen attenuation in the groundwater. Bromide release from decomposing waste served as an effective tracer for leachate distribution in the subsurface. The holistic approach implemented in this study provides robust and valuable insights into the factors that control landfill contamination potential and can be implemented in other sites with different climatic and geomorphological features.

Joule heating of logs for phytosanitary purposes and timber processing pre-treatment

Joule heating has recently been investigated as a potential alternative to chemical fumigation for phytosanitary treatment of Pinus radiata logs exported from New Zealand. The research was driven by concern regarding the effects of methyl bromide release to atmosphere, following fumigation, as highlighted by the Montreal Protocol. The work has involved creating 1 dimensional and 3 dimensional computer models of the Joule heating process in P. radiata, building a laboratory-scale log test rig with suitable instrumentation and control systems, heating approximately 60 P. radiata logs and using the measured data to refine and validate the models. While engaged in this work it became clear that the process could also potentially be applied to heat veneer peeler logs. This paper describes the application of the 1 dimensional model and laboratory-scale test rig to a typical P. radiata export log and to a Eucalyptus nitens log prepared for veneer peeling. The results demonstrate that, despite the heterogeneities of the timber, the technique is capable of achieving the required temperature profiles for both applications and that the model is a sufficiently good representation of the process to provide a viable control method.

Antifungal effects of BiOBr nanosheets carrying surfactant cetyltrimethylammonium bromide.

BiOBr nanosheets are important photocatalytic nanomaterials. However, their biological effects remain to be explored. In this study, we investigated the antifungal effect of BiOBr nanosheets on Candida albicans. Strikingly, the nanosheets strongly inhibited the growth of C. albicans [IC50=(96±4.7) mg/L], hyphal development and biofilm formation. Compareed to the antifungal effect of the cationic surfactant cetyltrimethylammonium bromide, the inhibitory effect of the nanosheets on fungal pathogen was attributed to cetyltrimethylammonium bromide adsorbed by the nanosheets. Thermal gravity analysis and cetyltrimethylammonium bromide release experiment indicated that only 0.42% cetyltrimethylammonium bromide on BiOBr nanosheets was released. Taken together, this study uncovers the contribution of surfactant released from the nanosheets to their antifungal activity.

Resolution of the uncertainty in the kinetic mechanism for the trans-3-Chloroacrylic acid dehalogenase-catalyzed reaction

trans- and cis-3-Chloroacrylic acid dehalogenase (CaaD and cis-CaaD, respectively) catalyze the hydrolytic dehalogenation of their respective isomers and represent key steps in the bacterial conversion of 1,3-dichloropropene to acetaldehyde. In prior work, a kinetic mechanism for the CaaD-catalyzed reaction could not be unequivocally determined because (1) the order of product release could not be determined and (2) the fluorescence factor for the enzyme species, E*PQ (where P = bromide and Q = malonate semialdehyde, the two products of the reaction) could not be assigned. The ambiguities in the model have now been resolved by stopped-flow experiments following the reaction using an active site fluorescent probe, αY60W-CaaD and 3-bromopropiolate, previously shown to be a mechanism-based inhibitor of CaaD, coupled with the rate of bromide release in the course of CaaD inactivation. A global fit of the combined datasets provides a complete minimal model for the reaction of αY60W-CaaD and 3-bromoacrylate. In addition, the global fit produces kinetic constants for CaaD inactivation by 3-bromopropiolate and implicates the acyl bromide as the inactivating species. Finally, a comparison of the model with that for cis-CaaD shows that for both enzymes turnover is limited by product release and not chemistry.

Phenols and anilines degradation by permanganate in the absence/presence of carbon nanotubes: Oxidation and dehalogenation

Abstract Synergistic enhancement of phenols and anilines removal was investigated in permanganate oxidation in the presence of multi-walled carbon nanotubes (CNTs). Then the attendant dehalogenation was explored for bromophenols and bromoanilines from the perspective of dehalogenation number (DN). Permanganate decay and total manganese concentration decrease in the presence of CNTs indicates considerable formation of manganese dioxide (MnO 2 ) on carbon surface. The CNTs-enhanced phenol degradation is more favorable under lower pH, confirming that the generation of MnO 2 induced by the reaction between permanganate and CNTs enhances the phenol oxidation by permanganate. The first order rate constants of respective bromophenols and bromoanilines suggest the synergistic enhancement of CNTs and permanganate in removing these micropollutants. Bromide release experiments clearly demonstrate that the subsequent dehalogenation accompanying the oxidation of bromophenols and bromoanilines is significant for permanganate oxidation in the absence/presence of CNTs. Dehalogenation by permanganate follows the order of 4-bromophenol > 2-bromophenol > 3-bromophenol and 4-bromoaniline > 2-bromoaniline > 3-bromoaniline, respectively. Higher DN values in the presence of CNTs indicate a reduced dehalogenation. In real water application, permanganate in the presence of CNTs could effectively remove 4-bromoaniline with a maximum removal efficiency of ∼95% and a bromide release of 2 μM.

Neuroprotective effects of pramipexole against tunicamycin-induced cell death in PC12 cells

1. Pramipexole (PPX), a dopamine D2 and D3 receptor agonist, exerts neuroprotective effects via both dopamine receptor-mediated and non-dopaminergic mechanisms. In the present study, we demonstrate that PPX reduces the toxicity of tunicamycin, a typical endoplasmic reticulum (ER) stressor, in PC12h cells, a subline of PC12 cells. 2. The PC12h cells were treated with 300 micromol / L PPX in the presence of 0.5 micromol / L tunicamycin for 24 h. The neuroprotective effects of PPX against tunicamycin-induced cell death were evaluated using 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays, Hoechst 33258 staining and western blot analysis. 3. Tunicamycin (0.2, 0.3 and 0.5 microg / mL) dose-dependently decreased MTT activity and increased LDH release from PC12h cells. Treatment with 300 micromol / L PPX rescued the tunicamycin-induced decrease in cell viability. 4. Spiperone (10 micromol / L), a dopamine D2 and D4 receptor antagonist, had no effect on PPX neuroprotection against tunicamycin in these cells. Marker proteins of ER stress and apoptosis are known to be upregulated by tunicamycin, but we detected no significant effects of PPX on these factors. 5. In conclusion, we speculate that a combination of several mechanisms may be involved in PPX-induced neuroprotection.

Oxidation State of BZ Reaction Mixtures

The unstirred, ferroin (Fe(phen)(3)2+)-catalyzed Belousov-Zhabotinsky (BZ) reaction1-4 is the prototype oscillatory chemical system. After an induction period of several minutes, one sees "spontaneous" formation of "pacemaker" sites, which oscillate between a blue, oxidized state (high [Fe(phen)3(3+)]) and a red, reduced state (low [Fe(phen)(3)3+]). The reaction medium appears red (reduced) during the induction phase, and the pacemaker sites generate target patterns of concentric, outwardly moving waves of oxidation (blue). Auto-oscillatory behavior is also seen in the Oregonator model of Field, Korös, and Noyes (FKN), a robust, reduced model which captures qualitative BZ kinetics in the auto-oscillatory regime. However, the Oregonator model predicts a blue (oxidized) induction phase. Here, we show that including reaction R8 of the FKN mechanism, not incorporated in the original Oregonator, accounts for bromide release during the induction phase, thus producing the observed red oxidation state.

Halogens in the coastal snow pack near Barrow, Alaska: Evidence for active bromine air‐snow chemistry during springtime

We measured halide concentrations of snow and frost flowers in the vicinity of Barrow, Alaska. We find that the ratio of bromide to sodium in frost flowers is slightly enhanced (≈10%) as compared to sea water. In contrast, the ratio of bromide to sodium in some snow samples is more than an order of magnitude enhanced, and in other samples is more than an order of magnitude depleted. We interpret the bromide depleted snow as having been processed by heterogeneous chemistry and providing reactive halogen compounds to the atmosphere. The eventual end product of reactive bromine chemistry is HBr that is then deposited over a wide region, enhancing bromide in inland snow samples. Although frost flowers or open leads are likely to be the original source of halides that become reactive halogen gases, we find that the bromide release often occurs subsequent to production of aerosol from marine sources.

Kinetic mechanism and enantioselectivity of halohydrin dehalogenase from Agrobacterium radiobacter.

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the reversible intramolecular nucleophilic displacement of a halogen by a hydroxyl group in vicinal haloalcohols, producing the corresponding epoxides. The enzyme displays high enantioselectivity toward some aromatic halohydrins. To understand the kinetic mechanism and enantioselectivity of the enzyme, steady-state and pre-steady-state kinetic analysis was performed with p-nitro-2-bromo-1-phenylethanol (PNSHH) as a model substrate. Steady-state kinetic analyses indicated that the k(cat) of the enzyme with the (R)-enantiomer (22 s(-1)) is 3-fold higher than with the (S)-enantiomer and that the K(m) for the (R)-enantiomer (0.009 mM) is about 45-fold lower than that for the (S)-enantiomer, resulting in a high enantiopreference for the (R)-enantiomer. Product inhibition studies revealed that HheC follows an ordered Uni Bi mechanism for both enantiomers, with halide as the first product to be released. To identify the rate-limiting step in the catalytic cycle, pre-steady-state experiments were performed using stopped-flow and rapid-quench methods. The results revealed the existence of a pre-steady-state burst phase during conversion of (R)-PNSHH, whereas no such burst was observed with the (S)-enantiomer. This indicates that a product release step is rate-limiting for the (R)-enantiomer but not for the (S)-enantiomer. This was further examined by doing single-turnover experiments, which revealed that during conversion of the (R)-enantiomer the rate of bromide release is 21 s(-1). Furthermore, multiple turnover analyses showed that the binding of (R)-PNSHH is a rapid equilibrium step and that the rate of formation of product ternary complex is 380 s(-1). Taken together, these findings enabled the formulation of an ordered Uni Bi kinetic mechanism for the conversion of (R)-PNSHH by HheC in which all of the rate constants are obtained. The high enantiopreference for the (R)-enantiomer can be explained by weak substrate binding of the (S)-enantiomer and a lower rate of reaction at the active site.

Phenotyping of human glutathione S-transferase hGSTT1-1: a comparison of two ex vivo routine procedures.

The human glutathione S-transferase hGSTT1-1 is characterized by a polymorphism displaying three phenotypes: the "non-conjugator" (NC) phenotype expresses no or only a residual activity due to a homozygous deletion of the hGSTT1 gene, a medium hGSTT1-1 activity can be demonstrated for the "low conjugator" (LC) phenotype as the heterozygous bearer of one hGSTT1 allele, and a high hGSTT1-1 activity is detected for the "high conjugator" (HC) phenotype as the homozygous bearer of two hGSTT1 alleles. We have developed a routine ex vivo photometric phenotyping procedure based on the determination of bromide release rates from the hGSTT1-1-catalyzed glutathione conjugation of the substrate methyl bromide in EDTA blood samples under standard conditions (1,000 ppm methyl bromide, 10 min incubation). The bromide release rates were standardized to the hemoglobin (Hb) value. Twenty-six individuals were phenotyped following the new procedure. Four individuals were classified as NCs (24-33 pmol Br(-)/mg Hb per min), 21 individuals were regarded as LCs (107-206 pmol Br(-)/mg Hb per min) and one person of the study group was designated HC (236 pmol Br(-)/mg Hb per min). The results were validated by qualitative hGSTT1 genotyping and demonstrated a 100% match for conjugators and non-conjugators. A second HPLC phenotyping routine procedure based on the formation of S-methylglutathione from methyl chloride in erythrocyte lysate incubations (Müller et al. 2001, Arch Toxicol 74:760-767) was established and validated by genotyping. The phenotyping results obtained with both methods were correlated, resulting in a good correlation with R(2)=0.64 (y=0.8997x +51.535). Three distinct phenotype clusters for NCs, LCs and HCs, consistent with the proposed genetics, were demonstrated. Assay-dependent storage experiments revealed an excellent stability of the hGSTT1-1 activity. In conclusion, the evaluated methods provide powerful tools for determination of hGSTT1-1 activity as a clinical parameter.

알기론 정(브롬화 시메트로피움 50 mg)에 대한 알피트 정의 생물학적 동등성

Cimetropium bromide, a quaternary ammonium compound which is chemically related to scopolamine, exhibits its antispasmodic activity by competing with acetylcholine for the muscarinic receptors of the smooth muscle of gastrointestinal tract. The drug has been used for the treatment of various disorders involving spasms of the musculature of the gastrointestinal, biliary and genitourinary tracts. The purpose of the present study was to evaluate the bioequivalence of two cimetropium bromide tablets, (Boehringer Ingelheim Korea Ltd.) and (Hana Pharmaceutical Co., Ltd.), according to the prior and revised guidelines of Korea Food and Drug Administration (KFDA). The cimetropium bromide release from the two cimetropium bromide tablets in vitro was tested using KP VII Apparatus II method with various different kinds of dissolution media (pH 1.2, 4.0, 6.8 buffer solution and water). Twenty normal male volunteers, years in age and kg in body weight, were divided into two groups and a randomized cross-over study was employed. After three tablets containing 50 mg of cimetropium bromide per tablet were orally administered, blood was taken at predetermined time intervals and the concentrations of cimetropium bromide in serum were determined using HPLC method with UV detector. The dissolution profiles of two cimetropium bromide tablets were very similar at all dissolution media. Besides, the pharmacokinetic parameters such as were calculated and ANOVA test was utilized for the statistical analysis of the parameters using non-transformed and logarithmically transformed . The results showed that the differences in between two tablets based on the were 2.19%, -5.97% and 3.49%, respectively. Minimum detectable differences were less than 20% (e.g., 13.71 %, 19.05% and 15.11% for , respectively). The powers , were 97.79%, 83.22% and 95.60%, respectively. The 90% confidence intervals were within (e.g., , respectively). There were no sequence effect between two tablets in logarithmically transformed . The 90% confidence intervals using logarithmically transformed data were within the acceptance range of log(0.8) to log(1.25) (e.g., , respectively). Two parameters met the criteria of prior and revised KFDA guideline for bioequivalence, indicating that tablet is bioequivalent to tablet.

Temporal trends in bromide release following rewetting of a naturally drained gully mire

Abstract. Recently, there has been interest in the occurrence of bromide (Br‐) in natural waters since it has been demonstrated that Br‐, in association with humic substances in raw waters, is readily incorporated into haloacetic acids in the form of organically bound bromine (Br) during water chlorination. We report results of the effects of experimentally rewetting a naturally drained gully mire on the hydrochemistry of Br‐, iron (Fe) and dissolved organic carbon (DOC) in the peat water. Results obtained over a three year period showed that rewetting substantially increased the concentrations of these solutes in the pore water, with peak values of 1 mg dm‐3 (Br‐), > 60 mg dm‐3 (Fe) and > 300 mg dm‐3 (DOC) detected in some samples after rewetting, compared with typical values < 0.05 mg dm‐3 (Br‐), < 1 mg dm‐3 (Fe) and < 15 mg dm‐3 (DOC) under the drained conditions. Bromide, Fe and DOC release were highly seasonal, with the largest concentrations observed in late‐summer to autumn. However, whereas seasonal peak concentrations of Fe and DOC have since remained at these higher levels, seasonal peak concentrations of Br‐ were progressively attenuated over time, suggesting the latter phenomenon is a flush effect, with no longer‐term consequences for water quality.

Kinetic analysis and X-ray structure of haloalkane dehalogenase with a modified halide-binding site.

Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. Trp175 forms a halogen/halide-binding site in the active-site cavity together with Trp125. To get more insight in the role of Trp175 in DhlA, we mutated residue 175 and explored the kinetics and X-ray structure of the Trp175Tyr enzyme. The mutagenesis study indicated that an aromatic residue at position 175 is important for the catalytic performance of DhlA. Pre-steady-state kinetic analysis of Trp175Tyr-DhlA showed that the observed 6-fold increase of the Km for 1,2-dibromoethane (DBE) results from reduced rates of both DBE binding and cleavage of the carbon-bromine bond. Furthermore, the enzyme isomerization preceding bromide release became 4-fold faster in the mutant enzyme. As a result, the rate of hydrolysis of the alkyl-enzyme intermediate became the main determinant of the kcat for DBE, which was 2-fold higher than the wild-type kcat. The X-ray structure of the mutant enzyme at pH 6 showed that the backbone structure of the enzyme remains intact and that the tyrosine side chain lies in the same plane as Trp175 in the wild-type enzyme. The Clalpha-stabilizing aromatic rings of Tyr175 and Trp125 are 0.7 A further apart and due to the smaller size of the mutated residue, the volume of the cavity has increased by one-fifth. X-ray structures of mutant and wild-type enzyme at pH 5 demonstrated that the Tyr175 side chain rotated away upon binding of an acetic acid molecule, leaving one of its oxygen atoms hydrogen bonded to the indole nitrogen of Trp125 only. These structural changes indicate a weakened interaction between residue 175 and the halogen atom or halide ion in the active site and help to explain the kinetic changes induced by the Trp175Tyr mutation.

Photoinduced electron-transfer reactions of homonaphthoquinones with amine and arene donors

Photoreactions of monoaryl- and diaryl-homonaphthoquinones bearing methyl, chloro and bromo substituents have been studied in the presence of amine and arene donors. The products of these photolyses are dependent on the substituents and the identity of the added donors. Irradiation of bromo-substituted diarylhomoquinones 1a, b with amines such as triethylamine and diethylamine in various aprotic solvents resulted in the formation of (±)- and meso-3,3′-bis(diphenylmethylene)-2,2′,3,3′-tetrahydro-2,2′-bi-1,4-naphthoquinones 2a, bvia the dimerization of intermediary allyl radicals arising from bromide release accompanied by ring-opening of the cyclopropane ring. Besides dimer 2, a 1:1 amine adduct 4 was also obtained with N,N-dimethylaniline as donor. However, methyl- and chloro-substituted homoquinones remained intact due to the poor leaving ability of these substituents. A similar reaction of bromo-substituted methylphenylhomoquinones 1e gave naphthofuran derivative 6via 2-(α-phenylvinyl)-1,4-naphthoquinone III. In contrast, photoreactions of 1b in the presence of arene donors, naphthalene and methoxy-substituted benzenes, gave no dimeric product but instead gave photoisomerized 2-bromo-3-diphenylmethylene-2,3-dihydronaphthoquinone 3 and its photocyclized xanthylium salt 7. This reaction does not occur in nonpolar solvents. In the case of xanthene as donor, the dimer 2b was again formed in addition to the 3 and 7. These reaction features were interpreted in terms of a difference between proton donating ability of the donor cation radicals.

Amylase release from streptolysin O-permeabilized pancreatic acini

Intracellular mediators of exocytosis were investigated using isolated mouse pancreatic acini permeabilized with the bacterial toxin streptolysin O (SLO). Permeabilization was demonstrated by fluorescent staining with ethidium bromide and fluorescein diacetate and release of cytoplasmic lactate dehydrogenase. When SLO-permeabilized acini were incubated at 37 degrees C in Ca2(+)-EGTA buffers containing MgATP, amylase secretion was Ca2+ dependent with an EC50 of 0.40 microM Ca2+ and a maximally effective Ca2+ concentration of 1 microM. Maximal amylase secretion was 330% of that in Ca2(+)-free buffer (basal). The nonhydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 30 microM) increased the maximal secretion to 451% of basal in the presence of 1 microM Ca2+ and decreased the EC50 to 0.14 microM Ca2+. Removal of ATP plus addition of antimycin A and 2-deoxy-D-glucose inhibited Ca2(+)-dependent, GTP gamma S-enhanced amylase secretion by 56%. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 microM) also enhanced maximal secretion to 450% of basal and decreased the EC50 to 0.18 microM Ca2+. Enhancement of amylase secretion by submaximal concentrations of GTP gamma S or TPA was inhibited by the protein kinase C inhibitor staurosporine. These results suggest that Ca2+ stimulation of amylase secretion is potentiated by activation of protein kinase C. However, the enhancement of secretion by GTP gamma S and TPA was additive at their maximally effective concentrations, suggesting that another G protein(s) maybe involved in the terminal steps of exocytosis.

Species differences in testicular necrosis and DNA damage, distribution and metabolism of 1,2-dibromo-3-chloropropane (DBCP)

The human testicular toxicant 1,2-dibromo-3-chloropropane (DBCP) was studied for the same end-point in 4 different species of laboratory animals. Marked necrosis and atrophy of the seminiferous epithelium were observed in rats and guinea pigs 10 days after a single i.p. administration of DBCP (170–340 μmol/kg), whereas significantly less damage was observed in hamsters and mice. The testicular concentrations of DBCP measured at various time-points after the i.p. injection of DBCP indicated that factors in addition to tissue concentration were of importance for the observed species differences in sensitivity towards DBCP-induced testicular damage. Also, there did not seem to be any direct correlation between DBCP-induced in vivo testicular toxicity and in vitro GSH-dependent dehalogenation, inasmuch as the rate of bromide release from DBCP with hamster testicular cytosol was as fast as that with rat cytosol. Testicular DNA damage, as determined by alkaline elution 60 min after in vivo administration of 170 μmol/kg DBCP, was observed only in rats and guinea pigs. Thus, induction of DNA damage correlates with the relative susceptibilities of the species towards DBCP-induced testicular necrosis. To further study species differences in testicular activation of DBCP to DNA-damaging intermediate(s), cells isolated from the testes of the 4 species were incubated with DBCP. Testicular cells from rats and guinea pigs were the only preparations developing substantial DNA damage after 60 min incubation with low concentrations of DBCP (5–50 μM). The findings indicate that rats are sensitive towards DBCP-induced testicular necrosis because rat testicular cells easily activate DBCP to a DNA-damaging intermediate(s). The relative high testicular DBCP concentration as well as the ability to activate DBCP may explain the sensitivity of guinea pigs towards DBCP-induced testicular toxicity.