István Jablonkai

Cloning, characterization and regulation of a family of phi class glutathione transferases from wheat

Six phi (F) class glutathione transferases (GSTs) were cloned from bread wheat (Triticum aestivum L.) treated with the herbicide safener fenchlorazole ethyl and named TaGSTF1–6. Recombinant TaGSTFs were assayed for glutathione conjugating activity towards xenobiotics including herbicides and for glutathione peroxidase (GPOX) activity. TaGSTF1, which resembled ZmGSTF1, the dominant GST in maize (Zea mays), was highly active in conjugating 1-chloro-2,4-dinitrobenezene (CDNB) but had low activities towards chloroacetanilide, diphenyl ether and aryloxphenoxypropionate herbicides. TaGSTF2, TaGSTF3 and TaGSTF4 all resembled the safener-inducible ZmGSTF2, with TaGSTF2 and TaGSTF3 being highly active GPOXs and rapidly detoxifying chloroacetanilides. TaGSTF5 resembled ZmGSTF3, having limited conjugating and GPOX activity. TaGSTF6 contained both ZmGSTF1- and ZmGSTF2-like sequences but was most similar to ZmGSTF1 in detoxifying activity. The expression of TaGSTFs in wheat seedlings was enhanced upon exposure to fenchlorazole ethyl, herbicides or other chemical inducing treatments. TaGSTFs were also enhanced by treatment with the natural products caffeic acid, 7,4-dihydroxyflavone and naringenin. The CDNB-conjugating activity of TaGSTF1, and to a lesser extent TaGSTF6, was highly sensitive to inhibition by flavonoids, particularly the chalcone isoliquiritigenin. The other TaGSTFs were much less sensitive to such inhibition. It was subsequently determined that isoliquiritigenin underwent glutathione conjugation, though this reversible reaction did not require the intervention of any TaGSTF. The potential importance of GSTFs and glutathione conjugation in flavonoid metabolism is discussed.

Specific interactions of chloroacetanilide herbicides with human ABC transporter proteins.

Chloroacetanilide herbicides are among the most commonly used herbicides in agriculture. Several studies have demonstrated a number of them to be carcinogenic. ATP binding cassette (ABC) transporters are efflux pumps expressed in cell membranes, which form an important wall of defense against xenobiotics from different sources. We tested the interaction of the herbicides acetochlor, alachlor, dimetachlor, metazachlor, metolachlor, propachlor and prynachlor with human multidrug resistance transporters MDR1, MRP1, MRP2 and BCRP. A number of metabolites were studied for interaction with MRP1, MRP2 and MRP3. Transporter interactions were studied by measuring ATPase activity, inhibition of fluorescent dye efflux and vesicular transport. Also inhibition of MDR1 was monitored by measuring digoxin transport on Caco-2 monolayers and paclitaxel toxicity on K562-MDR cells. Acetochlor, alachlor, metolachlor and metazachlor showed specific interactions with MDR1. Digoxin permeability and paclitaxel cytotoxicity studies revealed that these herbicides are potent inhibitors of MDR1 that can modulate drug absorption and cause chemosensitization of cells. MRP1 was demonstrated to transport an important intermediate of the acetochlor detoxification pathway. Several specific interactions were shown when studying the interaction of chloroacetanilides with human transporter proteins. This study suggests an important role for transporter proteins in hazard prediction of agrochemicals and demonstrates how transporter interactions can be easily detected using in vitro screening methods.

Polyamidoamine dendrimer impairs mitochondrial oxidation in brain tissue

BackgroundThe potential nanocarrier polyamidoamine (PAMAM) generation 5 (G5-NH2) dendrimer has been shown to evoke lasting neuronal depolarization and cell death in a concentration-dependent manner. In this study we explored the early progression of G5-NH2 action in brain tissue on neuronal and astroglial cells.ResultsIn order to describe early mechanisms of G5-NH2 dendrimer action in brain tissue we assessed G5-NH2 trafficking, free intracellular Ca2+ and mitochondrial membrane potential (ΨMITO) changes in the rat hippocampal slice by microfluorimetry. With the help of fluorescent dye conjugated G5-NH2, we observed predominant appearance of the dendrimer in the plasma membrane of pyramidal neurons and glial cells within 30 min. Under this condition, G5-NH2 evoked robust intracellular Ca2+ enhancements and ΨMITO depolarization both in pyramidal neurons and astroglial cells. Intracellular Ca2+ enhancements clearly preceded ΨMITO depolarization in astroglial cells. Comparing activation dynamics, neurons and glia showed prevalence of lasting and transient ΨMITO depolarization, respectively. Transient as opposed to lasting ΨMITO changes to short-term G5-NH2 application suggested better survival of astroglia, as observed in the CA3 stratum radiatum area. We also showed that direct effect of G5-NH2 on astroglial ΨMITO was significantly enhanced by neuron-astroglia interaction, subsequent to G5-NH2 evoked neuronal activation.ConclusionThese findings indicate that the interaction of the PAMAM dendrimer with the plasma membrane leads to robust activation of neurons and astroglial cells, leading to mitochondrial depolarization. Distinguishable dynamics of mitochondrial depolarization in neurons and astroglia suggest that the enhanced mitochondrial depolarization followed by impaired oxidative metabolism of neurons may be the primary basis of neurotoxicity.

Microbial and photolytic degradation of the herbicide acetochlor.

Abstract Time-course of the microbial degradation of the herbicide acetochlor was studied in commercial black mold and products of degradation were followed using gas chromatography-mass spectrometry (GC-MS). After 1-year-exposure in soil only 9% of the parent molecule was found and two metabolites were identified. The estimated half-life (t1/2) of the acetochlor was approximately 90 days. In photolytic degradation studies irradiation of acetochlor resulted in 50% conversion of the parent molecule yielding at least six products, of which five photoproducts accounted for 43 % were tentatively identified using GC-MS and their structures were confirmed by chemical synthesis. Products formed by microbial degradation were less phytotoxic to maize, oat and ryegrass seedlings than parent acetochlor.

Synthesis and cytotoxic activity of novel 5-substituted-1-(β-L- arabinofuranosyl) pyrimidine nucleosides

A series of new 5-halogeno-1-(ß-L-arabinofuranosyl)uracils and their cytosine analogues were synthesized by halogenation of ara-L-uridine and ara-L-cytidine, respectively. The 5-(2-thienyl) and 5-halogenothienyl derivatives of both series were also prepared in excellent yields by Stille coupling followed by halogenation. All of these syntheses were based on benzoyl-protected derivatives. In vitro cytotoxicity experiments carried out using L1210 mouse leukemia cells showed that 5-(2-thienyl)-ara-L-uridine was the most potent compound of the new compounds; the majority of the analogues were not effective up to 200 μM concentrations.

Comment on "Dual fluorescence of ellipticine: excited state proton transfer from solvent versus solvent mediated intramolecular proton transfer".

B et al. have recently reported in this journal the fluorescent behavior of ellipticine in various solvents. They confirmed our previous findings that dual fluorescence is emitted in methanol and ethylene glycol, whereas protonated ellipticine is produced in the ground state in hexafluoro2-propanol. However, our result on the ellipticine absorption spectrum in methanol is incorrectly represented in ref 1. We did not claim that ellipticine protonation occurs in the ground state in neat methanol. We emphasized that “Despite the negligible ground-state protonation of E (ellipticine) and ME (6-methylellipticine) in methanol, two fluorescence bands were detected, suggesting that the basicity increase upon light absorption facilitates the protonation in the excited state.” Moreover, Figure 1a in ref 2 demonstrates that no band appears above 430 nm in the absorption spectrum of ellipticine in methanol. This is in accordance with the spectrum presented by Banerjee et al. It is obvious that the absorption maxima in methanol given in Table 1 in ref 2 correspond to the two lowest-energy absorption maxima of the spectra shown in Figure 3a therein. The text and Figure 3a in ref 2 clearly show that no absorption is detected above 430 nm in neat methanol and the longwavelength absorption band emerges only upon addition of trifluoroacetic acid (TFA). In ref 1, the long-wavelength fluorescence in methanol was attributed to a tautomer, which is formed by solvent-mediated excited-state intramolecular proton transfer from the pyrrole nitrogen to the pyridine nitrogen. The long-distance process was proposed to occur via a hydrogen-bonded methanol chain connecting the two nitrogen atoms of ellipticine. This hypothesis is inconsistent with the most important result reported in ref 2. Namely, we found that the photophysical behavior of ellipticine and its 6-methyl derivative (Scheme 1) are analogous, and both compounds emit dual fluorescence in methanol and ethylene glycol. This is convincing evidence against the reaction mechanism proposed by Banerjee et al. The N-methyl substitution of the pyrrole ring removes the sole dissociable hydrogen from the molecule, precluding thereby the possibility of the photoinduced intramolecular proton transfer to the pyridyl nitrogen. Therefore, we ascribed the dual fluorescence to excited-state protonation by the solvent. The long-wavelength fluorescence band in methanol cannot be assigned to a tautomer because (i) it matches the fluorescence spectrum of the protonated ellipticine (Figure 1) and (ii) the fluorescence decay time of the long-wavelength emission corresponds to that of the protonated ellipticine (τf = 8.5 ns) formed in the presence of 11 μMTFA. Figure 1A displays the resolution of the fluorescence spectrum of ellipticine in methanol. As we have shown, the long-wavelength band gradually vanishes upon addition of tetrabutylammonium hydroxide (Bu4NOH). In the presence of 0.12 M Bu4NOH, only short-wavelength fluorescence is observed. The subtraction of this emission from the fluorescence spectrum of ellipticine in neat methanol provides the spectrum of the long-wavelength fluorescence, which matches the spectrum of the protonated ellipticine in the presence of 11 μM TFA (Figure 1B). The difference of the two spectra is displayed as a function of the wavelength in the lowest panel of Figure 1. The negligible deviation is strong evidence that the long-wavelength emission is due to the protonated ellipticine instead of the tautomer proposed in ref 1. It is very unlikely that both the spectrum and the decay time of the fluorescence are identical for the ellipticine tautomer and protonated ellipticine. Therefore, the long-wavelength emission cannot be assigned to the tautomer. All papers on the fluorescence of β-carbolines, the alkaloids structurally related to ellipticine, agree that the fluorescence lifetimes of the tautomer and the protonated form are markedly different. Moreover, it is generally accepted that the fluorescence band of the tautomer is significantly red-shifted compared to the band of the protonated species for all β-carbolines. The faster rise of the long-wavelength emission in ethylene glycol compared to that in methanol corroborates that photoinduced intermolecular proton transfer occurs from the solvent to the singlet excited ellipticine. Because ethylene glycol is a stronger acid than methanol, more rapid excited-state protonation is expected and indeed found in the former solvent. For the relative acidity of ethylene glycol compared to that of methanol, about 6-fold and 10-fold larger values were reported in water and isopropanol, respectively. Due to the significantly larger acidity, the long-wavelength absorption band emerges above 420 nm in ethylene glycol, indicating the partial protonation of ellipticine in the ground state. Such an effect is not observed in the less acidic methanol. Scheme 1

A transgenic rat hepatocyte - Kupffer cell co-culture model for evaluation of direct and macrophage-related effect of poly(amidoamine) dendrimers.

Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in the development of drug induced liver injury (DILI). Furthermore, elevated intracellular Ca2+ level of hepatocytes is considered as a common marker of DILI. Here we applied an in vitro model based on hepatocyte mono- and hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing the GCaMP2 fluorescent Ca2+ sensor protein to investigate the effects of polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5 Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity assay and by Ca2+ imaging, while hepatocyte functions were studied by CYP2B1/2 inducibility, and bilirubin and taurocholate transport. G5 was significantly more cytotoxic than G4.5 for hepatocytes and induced Ca2+ oscillation and sustained Ca2+ signals at 1μM and10 μM, respectively both in hepatocytes and KCs. Dendrimer-induced Ca2+ signals in hepatocytes were attenuated by macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the inducibility of CYP2B1/2, which was restored by depleting the KCs with gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In conclusion, H/KC provides a good model for the prediction of hepatotoxic potential of drugs, especially of nanomaterials known to be trapped by macrophages, activation of which presumably contributes to DILI.

The Janus Facet of Nanomaterials

Application of nanoscale materials (NMs) displays a rapidly increasing trend in electronics, optics, chemical catalysis, biotechnology, and medicine due to versatile nature of NMs and easily adjustable physical, physicochemical, and chemical properties. However, the increasing abundance of NMs also poses significant new and emerging health and environmental risks. Despite growing efforts, understanding toxicity of NMs does not seem to cope with the demand, because NMs usually act entirely different from those of conventional small molecule drugs. Currently, large-scale application of available safety assessment protocols, as well as their furthering through case-by-case practice, is advisable. We define a standard work-scheme for nanotoxicity evaluation of NMs, comprising thorough characterization of structural, physical, physicochemical, and chemical traits, followed by measuring biodistribution in live tissue and blood combined with investigation of organ-specific effects especially regarding the function of the brain and the liver. We propose a range of biochemical, cellular, and immunological processes to be explored in order to provide information on the early effects of NMs on some basic physiological functions and chemical defense mechanisms. Together, these contributions give an overview with important implications for the understanding of many aspects of nanotoxicity.

Basis for Differential Chemical Selectivity of MG-191 Safener against Acetochlor and EPTC Injury to Maize

Abstract The influence of MG-191 safener on the uptake, translocation and metabolism of [14C]acetochlor and [14C]EPTC was studied. The amounts of absorbed radioactivity by maize seedlings at 3, 6, 24, and 72 h after applications of [14C]labeled herbicides and [14C]MG-191 were different. Plants treated with [14C]acetochlor took up 30-to 50-fold more radiolabel within 72 h than [14C]EPTC-or [14C]MG-191-treated plants. Addition of MG-191 caused only minor changes in the rate of herbicide absorption. EPTC and MG-191 and/or their metabolites moved quickly acropetally and partitioned equally between root and shoot tissues up to 72 h. The amount of acetochlor and/or its labeled metabolites translocated to shoot tissues was less than 10%. MG-191 practically had no influence on herbicide translocation rates. With all chemicals the amounts of water-soluble and unextractable fractions increased while the ratio of hexane-extractable metabolites decreased with time. TLC analyses of both water-and hexane-soluble metabolites confirmed the fast metabolism of acetochlor. The acetochlor metabolism took place via GSH conjugation and more polar, non-conjugated metabolites compared to parent molecule were detected in hexane-soluble fraction. MG-191 enhanced acetochlor metabolism by decreasing the portion of non-metabolized acetochlor. EPTC metabolism resulted in water-soluble metabolites having similar chromatographic properties to those of acetochlor. However, there was no safener effect on non-metabolized EPTC content of plants. It appears that MG-191 protects maize against EPTC by enhancing the early rate of conjugation with GSH after initial oxidative metabolism.

Multiple inclusion complex formation of protonated ellipticine with cucurbit[8]uril: thermodynamics and fluorescence properties

Abstract The encapsulation of protonated ellipticine (EH+) in the cavity of cucurbit[8]uril (CB8) was studied in water at pH 4 with spectrophotometric, fluorescence spectroscopic and isothermal calorimetric measurements. The formation of three types of inclusion complexes was observed depending on the host and guest concentrations. Not only one, but also two EH+ was capable of encapsulation in CB8 in 37 μM EH+ solution and the thermodynamics of the binding steps were revealed. The produced very stable complexes showed markedly different absorption and fluorescence properties. When large excess of CB8 was employed in dilute (0.49 μM) EH+ solution, sequential binding of two CB8 occurred to the monomer alkaloid bringing about a substantial alteration in the fluorescence decay kinetics. The driving force of the 1:2 guest:host complex formation was much lower than that of 1:1 encapsulation.