Redox-mediator-free degradation of sulfathiazole and tetracycline using Phanerochaete chrysosporium.

The removal of two of the most commonly used antibiotics, tetracycline (TC) and sulfathiazole (STZ), using laccase-producing Phanerochaete chrysosporium was studied in liquid-phase batch experiments in the absence of any synthetic redox mediator. The removal of STZ and TC from single antibiotic spikes varied from 97.8% to 15.4% and 98.8% to 31%, respectively, with increasing initial doses of 10-250 mg L-1 within 14 days of incubation. The enzyme activity of P. chrysosporium was only minimally influenced by the concentrations of these antibiotics. The degradation of antibiotics initiated before an appreciable extracellular enzyme activity was noted in the fungal culture. The appearance of low-molecular weight molecular fragments from parent antibiotics in liquid chromatography-mass spectrometry confirmed the biodegradation process.

Uptake of the veterinary antibiotics chlortetracycline, enrofloxacin, and sulphathiazole from soil by radish.

Veterinary antibiotics are available for uptake by the plants through sources such as manure, irrigation, and atmospheric interaction. The present study was conducted to estimate the half-lives of three veterinary antibiotics, chlortetracycline (CTC), enrofloxacin (ENR), and sulphathiazole (STZ), in soil and experimentally explore their uptake from contaminated soil to radish roots and leaves. Samples were extracted using a modified citrate-buffered version of the quick, easy, cheap, effective, rugged, and safe "QuEChERS" method followed by liquid chromatography coupled with tandem mass spectrometric analysis (LC-MS/MS) in the positive ion mode. Good linearity was observed for the three tested antibiotics in soil and plants (roots and leaves) with high coefficients of determination (R2≥0.9922). The average recovery rates at two spiking levels with three replicates per level ranged between 77.1 and 114.8%, with a relative standard deviation (RSD)≤19.9% for all tested drugs. In a batch incubation experiment (in vitro study), the half-lives of CTC, ENR, and STZ ranged from 2.0-6.1, 2.2-4.5, and 1.1-2.2days, respectively. Under greenhouse conditions, the half-lives of the three target antibiotics in soil with and without radishes were 2.5-6.9 and 2.7-7.4; 4.7-16.7 and 10.3-14.6; and 4.4-4.9 and 2.5-2.8days, respectively. Trace amounts of the target antibiotics (CTC, ENR, and STZ) were taken up from soil via roots and entered the leaves of radishes. The concentration of CTC was lower than 2.73%, ENR was 0.08-3.90%, and <1.64% STZ was uptaken. In conclusion, the concentrations of the tested antibiotics decreased with time and consequently lower residues were observed in the radishes. The rapid degradation of the tested antibiotics in the present study might have only little impact on soil microorganisms, fauna, and plants.

Development and validation of a rapid test for anaerobic inhibition and toxicity.

Despite the importance of quantifying inhibitory capacity of compounds in anaerobic digestion, there is currently no well-defined method to assess it. Experimental methods in literature are frequently time-consuming and resource intensive. As a result, detailed inhibition testing rarely forms part of anaerobic digestion studies, despite the importance and utility of this information. This study develops and validates a simple and rapid inhibition test protocol, based on relative inhibition of acetoclastic methanogens. The inhibition potential of a compound is determined from the reduction in specific methanogenic activity as inhibitor concentration is increased. The method was successfully performed on two inoculums from different source environments and with both biostatic and biocidal inhibitors. Optimisation work indicated that: (i) sodium acetate is a preferred carbon source compared to acetic acid; (ii) an inoculum to acetate ratio of 5 g VS g(-1) acetate is preferred, and (iii) that the inoculum concentration should be normalised to 10 g L(-1) VS to reduce mass transfer problems and promote consistency. A key advantage over existing methods is that the sampling strategy has been optimised to three events over 1.5 days while effectively controlling the relative analytical error.

Fluorescence interaction and determination of sulfathiazole with trypsin.

The mechanism of interaction of trypsin with the sulfathiazole was studied through using fluorescence quenching and UV-visible absorption spectra at pH 7.4. The Stern-Volmer quenching constants, binding constants, number of binding sites and the corresponding thermodynamic parameters ΔH(o), ΔS(o) and ΔG(o) were calculated at different temperatures. The effect of common metal ions on the constants was also discussed. The results suggest that sulfathiazole can interact strongly trypsin and that there is the formation of trypsin-sulfathiazole complex and the interaction can be explained on the basis of hydrogen bonds and van der Waals forces. The binding distance (r) between the donor (trypsin) and acceptor (sulfathiazole) was 3.52 nm based on the Förster's non-radiative energy transfer theory. The detection and quantification limits of sulfathiazole were calculated as 2.52 and 8.40 µM in the presence of trypsin, respectively. The relative standard deviation (RSD) was 4.086% for determinations (n = 7) of a sulfathiazole solution with the concentration of 7.54 µM.

The influence of sulfathiazole on the macroalgae Ulva lactuca.

Sulfonamides (SA) are a class of antibiotics routinely found in environmental matrices and therefore their role as contaminants should be investigated in non-target organisms. With this purpose the present experimental work has evaluated the exposure of the chlorophycean Ulva lactuca L. to sulfathiazole (STZ), a SA drug commonly used in aquaculture, at two concentrations representing prophylactic (25 µg mL(-1)) and therapeutic (50 µg mL(-1)) administrations. Results showed that STZ exhibits high stability in seawater with only 18% degradation over the 5d assay at both dosages tested. Also, macroalgae demonstrated an efficient uptake capacity with constant internal concentrations after 24h regardless of the external solutions and thus should be considered as a bioindicator species in risk assessment. Both STZ concentrations induced a slight inhibition of the macroalgae growth after 96 h.

Identification and dynamic modeling of biomarkers for bacterial uptake and effect of sulfonamide antimicrobials.

The effects of sulfathiazole (STA) on Escherichia coli with glucose as a growth substrate was investigated to elucidate the effect-based reaction of sulfonamides in bacteria and to identify biomarkers for bacterial uptake and effect. The predominant metabolite was identified as pterine-sulfathiazole by LC-high resolution mass spectrometry. The formation of pterine-sulfathiazole per cell was constant and independent of the extracellular STA concentrations, as they exceeded the modeled half-saturation concentration K(M)(S) of 0.011 µmol L(-1). The concentration of the dihydrofolic acid precursor para-aminobenzoic acid (pABA) increased with growth and with concentrations of the competitor STA. This increase was counteracted for higher STA concentrations by growth inhibition as verified by model simulation of pABA dynamics. The EC value for the inhibition of pABA increase was 6.9 ± 0.7 µmol L(-1) STA, which is similar to that calculated from optical density dynamics indicating that pABA is a direct biomarker for the SA effect.

Reduction of antibiotics using microorganisms containing glutathione S-transferases under immobilized conditions.

The degradation of several antibiotics (tetracycline, sulfathiazole, ampicillin) was performed with immobilized bacterial cells containing the glutathione S-transferases (GSTs). Antibiotics in animal feed contaminated wastewater usually inhibit the growth of microorganisms that treat the wastewater, so a bio-friendly treatment method is required. Therefore, we have shown that the inhibitory effects of antibiotics on bacteria were reduced by microorganisms containing detoxifying enzyme GSTs by using a cell immobilizing method in a bioreactor. The initial concentrations of tetracycline, sulfathiazole and ampicillin were 100mg/L, 100mg/L and 50mg/L respectively, which are typical of the range detected in pig feed in Korea. In the results, we observed the removal efficiency of tetracycline to be almost 70% with Staphylococcus epidermidis in the bioreactor, suggesting that this method of antibiotic removal is worthy of further study.

Continuous degradation of a mixture of sulfonamides by Trametes versicolor and identification of metabolites from sulfapyridine and sulfathiazole.

In this study, we assessed the degradation of the sulfonamides sulfapyridine (SPY) and sulfathiazole (STZ) by the white-rot fungus Trametes versicolor. Complete degradation was accomplished in fungal cultures at initial pollutant concentrations of approximately 10 mg L(-1), although a longer period of time was needed to completely remove STZ in comparison to SPY. When cytochrome P450 inhibitors were added to the fungal cultures, STZ degradation was partially suppressed, while no additional effect was observed for SPY. Experiments with purified laccase and laccase mediators caused the removal of greater than 75% of each antibiotic. Ultra-performance liquid chromatography-quadupole time of flight mass spectrometry (UPLC-QqTOF-MS) analyses allowed the identification of a total of eight degradation intermediates of SPY in both the in vivo and the laccase experiments, being its desulfonated moiety the commonly detected product. For STZ, a total of five products were identified. A fluidized bed reactor with T. versicolor pellets degraded a mixture of sulfonamides (SPY, STZ and sulfamethazine, SMZ) by greater than 94% each at a hydraulic residence time of 72 h. Because wastewater contains many diverse pollutants, these results highlight the potential of T. versicolor as a bioremediation agent not only for the removal of antibiotics but also for the elimination of a wide range of contaminants.

Catalysis and sulfa drug resistance in dihydropteroate synthase.

The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S(N)1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.

A comparison of spray drying and milling in the production of amorphous dispersions of sulfathiazole/polyvinylpyrrolidone and sulfadimidine/polyvinylpyrrolidone.

Formulations containing amorphous active pharmaceutical ingredients (APIs) present great potential to overcome problems of limited bioavailability of poorly soluble APIs. In this paper, we directly compare for the first time spray drying and milling as methods to produce amorphous dispersions for two binary systems (poorly soluble API)/excipient: sulfathiazole (STZ)/polyvinylpyrrolidone (PVP) and sulfadimidine (SDM)/PVP. The coprocessed mixtures were characterized by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and intrinsic dissolution tests. PXRD and DSC confirmed that homogeneous glassy solutions (mixture with a single glass transition) of STZ/PVP were obtained for 0.05 ≤ X(PVP) (PVP weight fraction) < 1 by spray drying and for 0.6 ≤ X(PVP) < 1 by milling (at 400 rpm), and homogeneous glassy solutions of SDM/PVP were obtained for 0 < X(PVP) < 1 by spray drying and for 0.7 ≤ X(PVP) < 1 by milling. For these amorphous composites, the value of T(g) for a particular API/PVP ratio did not depend on the processing technique used. Variation of T(g) versus concentration of PVP was monotonic for all the systems and matched values predicted by the Gordon-Taylor equation indicating that there are no strong interactions between the drugs and PVP. The fact that amorphous SDM can be obtained on spray drying but not amorphous STZ could not be anticipated from the thermodynamic driving force of crystallization, but may be due to the lower molecular mobility of amorphous SDM compared to amorphous STZ. The solubility of the crystalline APIs in PVP was determined and the activities of the two APIs were fitted to the Flory-Huggins model. Comparable values of the Flory-Huggins interaction parameter (χ) were determined for the two systems (χ = -1.8 for SDM, χ = -1.5 for STZ) indicating that the two APIs have similar miscibility with PVP. Zones of stability and instability of the amorphous dispersions as a function of composition and temperature were obtained from the Flory-Huggins theory and the Gordon-Taylor equation and were found to be comparable for the two APIs. Intrinsic dissolution studies in aqueous media revealed that dissolution rates increased in the following order: physical mix of unprocessed materials < physical mix of processed material < coprocessed materials. This last result showed that production of amorphous dispersions by co-milling can significantly enhance the dissolution of poorly soluble drugs to a similar magnitude as co-spray dried systems.

Evaluating the biodegradability of sulfamethazine, sulfamethoxazole, sulfathiazole, and trimethoprim at different stages of sewage treatment.

The aerobic biodegradability of four antimicrobials (sulfamethazine, sulfamethoxazole, sulfathiazole, and trimethoprim) was investigated in sewage collected at four treatment stages (primary treatment, activated sludge treatment, aerobic nitrification process, and after disinfection of treated sewage) of a municipal sewage treatment plant. The biodegradability tests were conducted in aerated batch reactors by spiking the sewage with 20 microg/L of each of the test substance. Concentration profiles of the assayed compounds were monitored during a 54-d period using liquid chromatography/electrospray ionization/mass spectrometry. Substantial differences in the degradation curves were observed between trimethoprim and the three sulfonamides. The behavior of the latter was characterized by a general biodegradability in the primary and secondary treatment. The highest degradation rates were obtained in the sewage from the activated sludge treatment, where no adaptation phase was observed. On the other hand, the onset of biodegradation in the sewage from the primary treatment was preceded by a lag phase ranging from 10 to 15 d. In contrast, trimethoprim displayed high resistance to microbial degradation in the sewage from the primary treatment and the activated sludge treatment. However, primary degradation of this compound was completed within only 3 d in the sewage from the nitrification process.

Spectroscopic features of radiolytic intermediates induced in gamma irradiated sulfatiazole: an ESR study.

Sulfonamides are used as active ingredients in different drugs to treat infections caused by bacteria. Sulfatiazole (STZ) is one of the commonly used sulfonamides as antibacterial agent in drugs, which constitute potential candidates for radiosterilization. However, the crucial point in this respect is to monitor the amount and characteristic features of the radiolytic intermediates produced after irradiation. Electron spin resonance (ESR) spectroscopy is extensively used for this purpose due to its high sensitivity toward intermediates exhibiting radicalic nature. Thus, the aim of the present work is to investigate the spectroscopic and kinetic features of the species having unpaired electrons induced in gamma irradiated STZ at room and different temperatures in the dose range of 5-50kGy using ESR spectroscopy. Spectra of irradiated STZ consisted of many resonance peaks in the studied dose and temperature ranges. Heights of the peaks measured with respect to the base line were used to monitor microwave, temperature, time-dependent features of the radical species contributing to the experimental ESR spectra. Four tentative species of different spectroscopic and structural features assigned as A, B, C and D were found well explaining the experimental ESR spectra of gamma irradiated STZ. Comparison between the principal IR bands of unirradiated and gamma irradiated samples showed no detectable changes and appearance of new bands.

Deposition kinetics, metabolism and urinary excretion of sulfathiazole following oral administration in sheep.

Deposition kinetics, metabolism and urinary excretion of sulfathiazole were investigated in German black head sheep following single oral administration (100 mg/kg). Kinetic evaluation of plasma levels was performed using a two-compartment best fit model. Sulfathiazole is significantly metabolized to N4-acetyl metabolite in the rumen fluid. The drug is very poorly absorbed since the minimum effective concentration in plasma was not attained at any time following oral administration. The prolonged elimination half-life in sheep may be due to a low rate of drug absorption from the rumen and gastro-intestinal tract. Sulfathiazole was mainly excreted in the urine as free drug and N4-acetyl metabolite.

In vitro metabolism of sulfathiazole in rumen fluid and its metabolism and disposition kinetics following intraruminal administration in sheep.

The antimicrobial agents may undergo a change in the complex stomach particularly in the rumen as a result of microbial fermentation in ruminants. Present investigation deals with the influence of ruminal fluid probably the role of ruminal microorganisms on the degradation of sulfathiazole in vitro and its metabolism and disposition following its single intraruminal administration (100 mg/kg) in adult german black head sheep. Sulfathiazole is metabolized to N4-acetyl sulfathiazole in the rumen fluid after its in vitro incubation at different concentrations (10-60 micrograms/ml) for varying time intervals (1-6 h) at a temperature of 38 +/- 0.5 degrees C. Likewise in vivo it is significantly metabolized to its N-acetyl metabolite in the rumen after an intraruminal administration. The levels of sulfathiazole are maintained above minimum effective therapeutic concentration (40 micrograms/ml) for more than 24 h in rumen fluid. The drug is poorly absorbed into the circulation after intraruminal administration since the levels in plasma could not reach up to minimum effective therapeutic concentration at any time. The biological half-life of sulfathiazole was found to be 16.7 h following single intraruminal administration. Results of this investigation suggest that oral or intraruminal application of sulfathiazole has only local effects in the rumen fluid. A systemic treatment is not possible after this path of application.

Comparison of the determination of four sulphonamides and their N4-acetyl metabolites in swine muscle tissue using liquid chromatography with ultraviolet and mass spectral detection.

A discharge-assisted LC-MS method has been developed and validated for the analysis of four sulphonamides (sulphathiazole, sulphadiazine, sulphamerazine and sulphadimidine) and their N4-acetyl metabolites in the muscle of swine treated with Polysulpha-Complex, which contains all four drugs. The clean-up procedure developed involved chloroform-acetone extraction followed by Sep-Pak silica solid-phase extraction. In parallel a LC-UV method was validated using the same clean-up procedure. Blank tissue was fortified at levels between 20 and 100 micrograms/kg. [13C]sulphadimidine was used as internal standard. The samples were analysed with thermospray LC-MS. The [M + H]+ ion was the major ion in all cases and was employed for single-ion monitoring. The limits of detection (LOD) were below 25 micrograms/kg and the limits of quantification (LOQ) for most sulphonamides were ca. 100 micrograms/kg. Incurred muscle tissues were measured by both LC methods and the concentrations of the sulphonamides were found to be similar. However, the LC-MS procedure is more suitable for confirmatory analysis due to its specificity.

Evidence for a large and flexible region of human serum albumin possessing high affinity binding sites for salicylate, warfarin, and other ligands.

The relations between the single high affinity binding sites for azapropazone, phenylbutazone, chlorpropamide, sulfathiazole, and iophenoxate and the binding regions of human serum albumin represented by the marker ligands diazepam, phenol red, salicylate, and warfarin were examined by a series of competition experiments. Binding was determined by equilibrium dialysis at pH 7.0. In order to ensure an accurate analysis of the competition experiment, the number of moles of ligand bound per mole of protein was usually 0.4 or less to minimize ligand binding to weaker sites. Furthermore, binding of both ligands was determined in all experiments (except for iophenozate). None of the test ligands competed with diazepam for a common high affinity binding site, but, surprisingly, they were all able to displace two or three of the other marker ligands according to a competitive scheme. These findings show, first, the existence of a particular serum albumin region for high affinity binding of diazepam. Secondly, they imply that it is not necessary to assume the existence of new drug binding regions beyond those existing for phenol red, salicylate, and warfarin. On the contrary, the relatively many examples of competitive binding indicate that the binding regions represented by the last-mentioned three marker ligands are placed quite close to each other in the albumin molecule in a common region, which is suggested to be located at subdomains 1C and 2A-B. The region must be relatively large, because in some cases independent high affinity binding of pairs of ligands is observed. It is probably also rather flexible, inasmuch as no clear relation could be found between the chemical structure of the test ligands and the two or three marker ligands with which they compete. Correlations between primary association constants and partition coefficients for both marker ligands and test ligands, in the unionized forms, between n-hexane or 1-octanol and aqueous media showed that hydrophobic forces are important for the binding processes. However, the data also showed that other attractive forces must be operative as well.

The effect of nitrite on 14C-sulphathiazole (4-amino-N-2-thiazolyl[U-14C]benzenesulphonamide) metabolism in the rat.

1. Rats given a meal containing 613 p.p.m. of 14C-sulphathiazole (4-amino-N-2-thiazolyl[14C]benzenesulphonamide) excreted less 14C-activity in urine and more 14C-activity in faeces as nitrite in the meal was increased (0, 10, 100 or 1000 p.p.m.). As nitrite in the meal was increased from 0 to 1000 p.p.m. the total 14C-residues in the gastrointestinal tract six hours after dosing increased, but decreased in other tissues. 2. High nitrite in the meal resulted in increased methanol insoluble 14C-activity in the gastrointestinal tract but had little or no effect on the methanol-insoluble activity in liver and blood. 3. Conversion of 14C-sulphathiazole to 14C-desaminosulphathiazole (N-2-thiazolyl[U-14C]benzenesulphonamide) in the rat was greatly increased by nitrite in the meal.

Studies on drug-milk freeze-dried formulations. I: Bioavailability of sulfamethizole and dicumarol formulations.

In this study, solid dispersion formulations of dicumarol (3,3'-methylenebis[4-hydroxycoumarin]) and sulfamethizole (N'-(5-methyl-1,3, 4-thiadiazol-2-yl)sulfanilamide) in defatted milk were prepared by freeze-drying. X-ray crystallographic data showed that both drugs were dispersed in the formulations in an amorphous state. Bioequivalency comparisons between freeze-dried formulations, after regeneration with water, and control capsules containing the pure drug substances were studied in four male volunteers. Determination of the plasma dicumarol levels indicated superiority of the dicumarol-milk formulation. Statistically significant differences were found between area under the curve, maximum plasma concentration, and apparent elimination rates. Analysis of the urine sulfamethizole data revealed that the two formulations exhibit statistically equivalent rates and extents of excretion of unchanged sulfamethizole. The binding of both drugs to casein and their solubility in the presence of casein were measured in vitro. The presence of casein caused an increase in the solubility of dicumarol, while it had no effect on the solubility of sulfamethizole. Normal protein binding cannot be responsible for the effects noted. Extrapolation of the in vitro data to the in vivo situation was attempted. Drug-milk freeze-dried formulations are promising for the enhancement of the bioavailability of sparingly water soluble drugs.

Urine flow dependence of renal clearance and interrelation of renal reabsorption and physicochemical properties of drugs.

A physiologically based model is presented for the analysis of the reabsorption kinetics of drugs in the renal tubules. The two reabsorption parameters, AR(I) X Pe and sigma, expressing the permeability of a drug through lipoidal membranes and the reflection coefficient, respectively, are obtained on the basis of this model using the results of clearance experiments in rats. These two reabsorption parameters are compared among four drugs, sulfanilamide, sulfamethizole, theophylline, and ethanol. Urine flow dependence of the percentage of reabsorbed drugs is reasonably expressed by the model. Moreover, the percentage of reabsorbed drugs which are highly reflected at the membranes is affected by the change of the glomerular filtration rate. In contrast, the percentage of reabsorbed drugs, such as ethanol, having a small reflection coefficient is not sensitive to the glomerular filtration rate. A good linear relationship is obtained between AR(I) X Pe and the n-octanol/water partition coefficient. The reflection coefficient is related to the molecular weight of the compound. The reflection coefficient of ethanol is small because of its low molecular weight, whereas the reflection coefficient is almost unity when the molecular weight is higher than 170. Accordingly, this physiological model approach can be acceptable to the reabsorption kinetics of drugs in the renal tubules.