Peter Scherpenisse

Using polyacrylate-coated SPME fibers to quantify sorption of polar and ionic organic contaminants to dissolved organic carbon.

A passive sampling method using polyacrylate-coated solid-phase microextraction (SPME) fibers was applied to determine sorption of polar and ionic organic contaminants to dissolved organic carbon (DOC). The tested contaminants included pharmaceuticals, industrial chemicals, hormones, and pesticides and represented neutral, anionic, and cationic structures. Prior to the passive sampler application, sorption of the chemicals to the fibers was characterized. This was needed in order to accurately translate concentrations measured in fibers to freely dissolved aqueous concentrations during the sorption tests with DOC. Sorption isotherms of neutral compounds to the fiber were linear, whereas isotherms of basic chemicals covered a nonlinear and a linear range. Sorption of acidic and basic compounds to the fiber was pH-dependent and was dominated by sorption of the neutral sorbate species. Fiber- and DOC-water partition coefficients of neutral compounds were both linearly related to octanol-water partition coefficients (log Kow). The results of this study show that polyacrylate fibers can be used to quantify sorption to DOC of neutral and ionic contaminants, having multiple functional groups and spanning a wide hydrophobicity range (log Kow = 2.5-7.5).

Differential Roles of Phase I and Phase II Enzymes in 3,4-Methylendioxymethamphetamine-Induced Cytotoxicity

Metabolism plays an important role in the toxic effects caused by 3,4-methylenedioxymethamphetamine (MDMA). Most research has focused on the involvement of CYP2D6 enzyme in MDMA bioactivation, and less is known about the contribution of other cytochrome P450 (P450) and phase II metabolism. In this study, we researched the differential roles of phase I P450 enzymes CYP1A2, CYP3A4, and CYP2D6 and phase II enzymes glutathione S-transferase (GST) and catechol-O-methyltransferase (COMT) on the toxic potential of MDMA. MDMA acts as inhibitor of its own metabolism with a relative potency of inhibition of CYP2D>CYP3A≫ CYP1A in rat liver microsomes and in human liver [immortalized human liver epithelial cells (THLE)] cells transfected with individual CYP1A2, CYP3A4, or CYP2D6. Cytotoxicity measurements [by 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] in THLE cells showed that the inhibition of phase I enzymes CYP1A2 by α-naphthoflavone and CYP3A4 by troleandomycin does not affect MDMA-induced cytotoxicity. MDMA metabolism by CYP2D6 significantly increased cytotoxicity, which was counteracted by CYP2D6 inhibition by quinidine. Inhibition of COMT by 2′-fluoro-3,4-dihydroxy-5-nitrobenzophenone (Ro-41-0960) and GST by buthionine sulfoximine showed that COMT is mainly involved in detoxification of CYP2D6-formed MDMA metabolites, whereas glutathione (GSH) is mainly involved in detoxification of CYP3A4-formed MDMA metabolites. Liquid chromatography/tandem mass spectrometry analyses of MDMA-metabolites in the THLE cell culture media confirmed formation of the specific MDMA metabolites and corroborated the observed cytotoxicity. Our data suggest that CYP2D6 as well as CYP3A4 play an important role in MDMA bioactivation. In addition, further studies are needed to address the differential roles of CYP3A4 and GSH/GST in MDMA bioactivation and detoxification.

Ceftiofur derivates in serum and endometrial tissue after intramuscular administration in healthy mares.

Endometritis is one of the major problems in the horse breeding industry. The use of antibiotics for treatment of endometritis in the mare is recommended as best practice. The intrauterine application of antibiotics, however, has been under discussion over the last years because of concerns about its efficacy. The systemic use of antibiotics has been considered more effective because of its better distribution within the uterus. The objective of the present study was to determine the concentration of ceftiofur derivates in serum and endometrial tissue after intramuscular administration. Specifically, the authors tested the hypothesis that ceftiofur concentrations in serum and endometrial tissue remain above the minimum inhibitory concentration (MIC) for common uterine pathogens for 24 h. Nine mares in estrus received a single dose of 2.2 mg/kg ceftiofur hydrochloride intramuscular per kg of body weight. Blood samples and endometrial tissue were obtained immediately before treatment (-1 h) and 2 h and 24 h after treatment. Endometrial tissue was collected with a Kevorkian biopsy punch. Additional blood samples were collected 4 h and 10 h after treatment from the jugular veins. For determination of ceftiofur derivates in serum and endometrial tissue a high performance liquid chromatography (HPLC) assay was used. Results in serum and uterine tissue revealed greatest concentration of ceftiofur at 2 h and lowest concentrations at 24 h after treatment. Concentrations of ceftiofur at 2 and 24 h after treatment were significantly greater in serum than in endometrial tissue, but remained above the reported MIC for Streptococcus equi zooepidemicus and Escherichia coli in both serum and endometrial tissue until 24 h after treatment.

Determination of ceftiofur derivatives in serum, endometrial tissue, and lochia in puerperal dairy cows after subcutaneous administration of ceftiofur crystalline free acid

Puerperal uterine infections are often associated with decreased reproductive performance in dairy cows. Routine treatment protocols include the systemic administration of antibiotics. Antibiotic drugs, however, should be administered daily over at least 5 d. The objective of this study was to determine concentrations of ceftiofur derivatives in serum, endometrial tissue, and lochia after subcutaneous administration of ceftiofur crystalline free acid in 6 clinically healthy puerperal dairy cows with normal parturition. Samples were taken immediately before treatment, 2 h after, and then every 24 h over a 7-d period. Concentrations of ceftiofur derivatives were quantified using an HPLC assay. In serum and endometrial tissue, ceftiofur derivatives could be detected above the reported minimum drug concentrations required to inhibit relevant pathogens such as Escherichia coli and Arcanobacterium pyogenes over a 7-d period. Concentrations of desfuroylceftiofuracetamide at 5 d after administration of ceftiofur crystalline free acid were 1.21±0.61 μg/mL in serum, 0.86±0.61 μg/mg in endometrial tissue, and 0.96±1.15 μg/mL in lochia. In lochia, mean concentrations of ceftiofur derivatives also remained above the minimal inhibitory concentration of relevant pathogens, but showed greater variations between cows.

Proficiency studies on the determination of paralytic shellfish poisoning toxins in shellfish

Paralytic shellfish poisoning toxins are produced by dinoflagellates. Shellfish filtering these unicellular algae will accumulate the toxins and pose a health risk when consumed by man. In the European Union, paralytic shellfish poisoning toxins in bivalve molluscs are regulated at a maximum content of 80 μg/100 g (91/492/EEC). The current reference method in the European Union is the mouse bioassay, but alternative methods including the liquid chromatography methodology are preferred for ethical reasons. Analyses of suspected shellfish batches revealed, however, unacceptable differences in results reported by a small group of Dutch laboratories all using liquid chromatography methods with precolumn derivatization, followed by fluorescence detection. Therefore, a series of proficiency studies were undertaken among these laboratories. In the first three studies, participants were more or less allowed their own choice of method execution details. This approach yielded unsatisfactory results. A fourth study was then initiated in which a standardized method was mandatory. Two types of test material were used in the fourth study: lyophilized Cardium tuberculatum material containing saxitoxin (STX) and decarbamoyl-saxitoxin (dc-STX), and lyophilized mussel material containing dc-STX. The latter material was investigated in an interlaboratory study involving 15 participants and was considered as the reference material. Among the four laboratories, coefficients of variation (ANOVA) for C. tuberculatum material were 10% (n = 11) and 9% (n = 12) for STX and dc-STX, respectively, and for the reference material was 8% (n = 12) for dc-STX. The joint efforts showed that variability in analysis results between laboratories that all apply more or less the same method can be drastically improved if the methodology is rigorously standardized.

HPLC determination of residues of spectinomycin in various tissue types from husbandry animals

An HPLC method was developed for the determination of bacteriostatic aminocyclitol spectinomycin (SP) in animal tissue products. These products included chicken eggs and edible fat, kidney, liver, muscle tissues from calf, poultry, pig and sheep. Residues of SP were extracted from homogenized tissue and egg-derived material with 25 mM citrate of pH 4.0, trichloroacetic acid and dichloromethane. The extract was purified and concentrated over a carboxylic acid-bonded solid-phase extraction (SPE) column. The SPE-eluate was analysed by cation-exchange HPLC involving a two-column switching system, post-column derivatization and fluorescence detection. Spectinomycin could be successfully determined at levels of 0.05 mg kg-1 and higher. Recoveries from spiked tissue material and from spiked egg material were in excess of 74% and did not show a concentration or tissue-type dependence. Precision of the elution position and signal response was better than 2%. Matrix effects and interference from lincomycin were less than 7 and 2%, respectively, on the signal response. Spectinomycin was shown to be stable at -20 degrees C in combined egg yolk and white over a test period of 12 weeks and in calf and sheep muscle tissue over a test period of 10 days. SP was, however, not stable at this temperature over a period of 12 months in chicken muscle tissue. Incurred SP residues were successfully determined in kidney and muscle tissue at the injection site of pigs administered with two doses of 15 mg kg-1 body weight SP with an intermittent withdrawal period of 15 days. Kidney showed higher concentrations and more persistent residues of SP than muscle tissue at the injection site.

Determination of ceftiofur derivatives in serum, endometrial tissue, and lochia in puerperal dairy cows with fever or acute puerperal metritis after subcutaneous administration of ceftiofur crystalline free acid

Acute puerperal metritis (APM) is one of the most common diseases during the puerperal period. Systemic administration of ceftiofur for 5 consecutive days has been shown to be effective for treatment of APM. The objective of this study was to determine concentrations of ceftiofur derivatives in serum, endometrial tissue, and lochia of cows with fever postpartum or APM 4 to 6d after treatment with a single subcutaneous dose of 6.6 mg of ceftiofur crystalline free acid (CCFA)/kg of estimated BW at the base of the ear. In the first experiment, samples from CCFA-treated cows with fever postpartum or APM (n=42) were taken on d 4, 5, or 6 after treatment. Concentrations of ceftiofur derivatives were quantified using an HPLC assay. Concentrations of active ceftiofur metabolite desfuroylceftiofuracetamide (DCA) were greatest at d 4 after treatment with CCFA in all samples, but they were considerably lower than the concentrations of DCA in healthy postpartum cows treated with the same dose of CCFA. The concentrations of DCA in serum, endometrial tissue, and lochia were affected by odor of vaginal discharge before treatment with CCFA. Mean concentrations of DCA could be detected above the reported minimal drug concentrations (minimum inhibitory concentrations, MIC) required to inhibit relevant pathogens such as Escherichia coli and Arcanobacterium pyogenes in serum on all days and in endometrial tissue and lochia only on d 4 in CCFA-treated cows with fetid vaginal discharge before treatment. In the second experiment, samples from CCFA-treated cows with APM (n=8) were taken on d 0 (before treatment) and d 4, 5, and 6 after treatment. Mean concentrations of DCA in serum and lochia were similar on d 4 to 6 in both laboratories. Furthermore, determined concentrations of DCA from both laboratories were correlated for serum and lochia. Mean concentrations of DCA could be detected above the reported MIC in serum and lochia only on d 4. Our 2 experiments demonstrated that in postpartum cows with fever postpartum or APM concentrations above the MIC for relevant bacteria (>0.5 μg/mL or >0.5 μg/g) of DCA could be sustained only for 4 (serum: 15/17; endometrial tissue: 2/17; lochia: 1/16) to 5d (serum: 10/13; endometrial tissue: 1/13; lochia: 2/12) after a single treatment with CCFA only in a certain proportion of cows. Overall, our data provide first pharmacological evidence that a single subcutaneous administration of 6.6g of CCFA/kg of BW might not be sufficient to efficaciously treat APM in postpartum dairy cows.

Induction of glutathione synthesis and conjugation by 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-dihydroxymethamphetamine (HHMA) in human and rat liver cells, including the protective role of some antioxidants.

MDMA (3,4-methylenedioxymethamphetamine) metabolism is a major cause of MDMA-mediated hepatotoxicity. In this study the effects of MDMA and its metabolites on the glutathione system were evaluated. Glutathione (GSH/GSSG) levels and gene expression of glutamate cysteine ligase catalytic subunit (GCLC), glutathione-S-transferase (GST) and pregnane X receptor (PXR) were compared in the immortalized human liver epithelial cell line THLE-Neo lacking phase I metabolism and primary rat hepatocytes expressing both phase I and II metabolism. Furthermore, we evaluated the potential protective effects of two antioxidants, N-acetyl-cysteine (NAC) and sulforaphane (SFN) in these cell systems. In THLE-Neo cells, the MDMA metabolite 3,4-dihydroxymetamphetamine (HHMA) significantly decreased cell viability and depleted GSH levels, resulting in an increased expression of GCLC and GST up to 3.4- and 2.2-fold, respectively. In primary rat hepatocytes, cell viability or GSH levels were not significantly affected upon MDMA exposure. GCLC expression levels where not significantly altered either, although GST expression was increased 2.3-fold. NAC counteracted MDMA-induced cytotoxicity and restored GSH levels. Phase II enzyme expression was also reverted. Conversely, SFN increased MDMA-induced cytotoxicity and GSH depletion, while GCLC and GST expression were significantly induced. In addition, PXR expression decreased after HHMA and MDMA exposure, while co-exposure to SFN induced it up to 3.6- and 3.9-fold compared to vehicle-control in the THLE-Neo cells and rat hepatocytes, respectively. Taken together, these data indicate that HHMA is a major factor in the MDMA-mediated hepatotoxicity through interaction with the glutathione system. The results of our study show that for MDMA intoxication the treatment with an antioxidant such as NAC may counteract the potentially hepatotoxicity. However, SFN supplementation should be considered with care because of the indications of possible drug-drug interactions.

Differences in hepatic cytochrome P450 activity correlate with the strain-specific biotransformation of medetomidine in AX/JU and IIIVO/JU inbred rabbits

Medetomidine is an alpha(2)-adrenoceptor agonist with sedative and analgesic properties. Previously we demonstrated significant differences in the response to medetomidine between two inbred rabbit strains, denoted IIIVO/JU and AX/JU. The aim of the present study was twofold: first, to compare the hepatic CYP450 enzyme activities between these rabbit strains [n = 13(male male,7 female female)/strain]. To this end, liver microsomes were incubated with known fluorescent substrates for the major drug-metabolizing CYP450 isoforms. A comparison of the obtained results indicated significant gender differences as well as differences between the two rabbit inbred strains. Secondly, the biotransformation rate of medetomidine in liver microsomes of both rabbit strains was determined using liquid chromatography coupled to tandem mass spectrometry. The rate of hydroxymedetomidine and medetomidine carboxylic acid formation was found to be significantly higher in the AX/JU strain. Specific CYP2D and CYP2E inhibitors could decrease the formation of both metabolites. Significant correlations were found between the rate of biotransformation of medetomidine and the activities of CYP2D and CYP2E, as well as between CYP450 enzyme activities and the anaesthetic response to medetomidine.

Critical micelle concentration values for different surfactants measured with solid-phase microextraction fibers

The amphiphilic nature of surfactants drives the formation of micelles at the critical micelle concentration (CMC). Solid-phase microextraction (SPME) fibers were used in the present study to measure CMC values of 12 nonionic, anionic, cationic, and zwitterionic surfactants. The SPME-derived CMC values were compared to values determined using a traditional surface tension method. At the CMC of a surfactant, a break in the relationship between the concentration in SPME fibers and the concentration in water is observed. The CMC values determined with SPME fibers deviated by less than a factor of 3 from values determined with a surface tension method for 7 out of 12 compounds. In addition, the fiber-water sorption isotherms gave information about the sorption mechanism to polyacrylate-coated SPME fibers. A limitation of the SPME method is that CMCs for very hydrophobic cationic surfactants cannot be determined when the cation exchange capacity of the SPME fibers is lower than the CMC value. The advantage of the SPME method over other methods is that CMC values of individual compounds in a mixture can be determined with this method. However, CMC values may be affected by the presence of compounds with other chain lengths in the mixture because of possible mixed micelle formation. Environ Toxicol Chem 2016;35:2173-2181.