Synthesis of novel hapten and production of generic monoclonal antibody for immunoassay of penicillins residues in milk

The objective of this study was to produce a generic monoclonal antibody for determination of penicillins residues in milk. The compound 6-aminopenicillanic acid was used as the template to synthesize two novel generic haptens that were used to produce the monoclonal antibodies. The obtained monoclonal antibodies simultaneously recognized 11 penicillin drugs (amoxicillin, ampicillin, penicillin G, penicillin V, sulbenicillin, carbencillin, methicillin, cloxacillin, dicloxacillin, oxacillin, and nafcillin). After evaluation of different reagent combinations, a heterologous indirect competitive enzyme immunoassay was developed to multi-determine the 11 drugs in milk. The crossreactivities to the 11 drugs were in a range of 16%–117% and the limits of detection were in a range of 0.7–9.3 ng/mL depending on the drug. The recoveries from the fortified blank milk were in a range of 77.6%–99.4% with coefficients of variation lower than 13.5%. This method could be used as a rapid screen tool for routine monitoring the residues of the 11 penicillin drugs in animal derived foods.     

Production of the broad specific monoclonal antibody against sarafloxacin for rapid immunoscreening of 12 fluoroquinolones in meat

The objective of this study was to produce a generic antibody for immunoassay of fluoroquinolone drugs in meat. Two novel haptens of sarafloxacin were synthesized that were used to produce the monoclonal antibodies. The obtained monoclonal antibodies simultaneously recognized 12 fluoroquinolone drugs (sarafloxacin, diflocaxin, marbofloxacin, ofloxacin, ciprofloxacin, enrofloxacin, norfloxacin, pefloxacin, lomefloxacin, amifloxacin, enofloxacin and danofloxacin). After evaluation of different coating antigen/antibody combinations, a heterologous competitive indirect enzyme linked immunosorbent assay (ELISA) was developed to determine the 12 drugs. The crossreactivities to these analytes were in the range of 18%–113% and the limits of detection were in the range of 0.8–6.5 ng/mL depending on the compound. Eight fluoroquinolones licensed as veterinary drugs in China were fortified into blank chicken for ELISA analysis. The recoveries were in the range of 67.6%–94.6% with coefficients of variation lower than 12.4%. Therefore, this method could be used as a screen tool for routine monitoring of the residues of these fluoroquinolone drugs in animal derived foods.     

Production of monoclonal antibody against doxycycline for immunoassay of seven tetracyclines in bovine muscle and milk

The objective of this study was to produce a generic monoclonal antibody for multi-determination of the residues of tetracycline drugs in bovine muscle and milk. Two new immunogens of doxycycline were prepared that were used to produce the monoclonal antibodies. Results showed the obtained antibodies simultaneously recognized seven tetracycline drugs (doxycycline, tetracycline, chlortetracycline, oxytetracycline, minocycline, methacycline, demeclocycline). The obtained antibodies and three coating antigens were arranged into six combinations to optimize the reagents combination. After comparison of the performances of these combinations, a heterologous indirect competitive ELISA was then used to determine the seven tetracyclines in bovine muscle and milk. The crossreactivities to the seven analytes were in the range of 47%–102% and the limits of detection were in the range of 1.5–6.9 ng/mL depending on the compound. The recoveries of the seven drugs from fortified blank samples were in the range of 75.3%–106.8% with coefficients of variation lower than 10.9%. Therefore, this method could be used as a multi-analytes screen tool for routine monitoring of the residues of these tetracycline drugs in bovine muscle and milk.     

Analyses of combined effects of cytostatic drugs on micronucleus formation in the <Emphasis Type="Italic">Tradescantia</Emphasis>

Recent experiments showed that 5-fluorouracil (5FU), cisplatin (CDDP), etoposide (ET), and imatinib mesylate (IM), which are currently among the most widely used anticancer drugs, cause damage of the genetic material in higher plants. The aim of the present study was to determine whether mixtures of these drugs cause synergistic or antagonistic effects which may have an impact on their environmental safety. Therefore, the effects of binary mixtures of these anticancer drugs on the induction of micronuclei (MN) which reflect structural and numerical chromosomal aberrations were assessed in Tradescantia tetrads. Synergistic/antagonistic effects were determined by comparison with single exposures that would be equally effective in a reference model of independent action. This comparison was performed at two distinct effect sizes. We found clear evidence for synergisms in combination experiments with IM and antagonism in a high-dose experiment with ET and 5FU. Our findings indicate that IM increases the genotoxic effects of other anticancer drugs. The maximal effects which we found were in the range between 19 and 38 % in the excess of effect sizes predicted under independent action. These effects may have an impact on the overall genotoxic activities of untreated hospital waste waters but not on the environment in general as the predicted environmental concentrations of the studied drugs are several orders of magnitude lower as the levels which are required to cause induction of MN in higher plants.     

Evaluation of antimicrobial agents for veterinary use in the ecotoxicity test using microalgae

The influence of antimicrobial agents approved as veterinary drugs in Japan on the growth of green algae, Selenastrum capricornutum and Chlorella vulgaris, was studied in accordance with the OECD guidelines for testing chemicals. Among the agents tested, growth inhibitory activity was very varied, i.e. erythromycin showed the strongest activity (EC₅₀, 50% effective concentration, =0.037 mg/l), sulfa drugs had activity to some extent (EC₅₀s of sulfamethoxazole, sulfadiazine, and sulfadimethoxine were 1.5, 2.2, and 2.3 mg/l, respectively), but ampicillin and cefazolin did not inhibit growth (EC₅₀s > 1000 mg/l). We also investigated synergistic effect of combining sulfa drugs with trimethoprim or pyrimethamine, which are commonly used as a combined drug. By adding trimethoprim, the growth inhibitory activity of sulfamethoxazole and sulfadiazine was significantly enhanced. Growth inhibition by sulfa drugs was reduced by the addition of folic acid, indicating that they inhibit folate synthesis in green algae.     

Competitive removal of pharmaceuticals from environmental waters by adsorption and photocatalytic degradation

This work explores the competitive removal of pharmaceuticals from synthetic and environmental waters by combined adsorption-photolysis treatment. Five drugs usually present in waterways have been used as target compounds, some are pseudo-persistent pollutants (carbamazepine, clofibric acid, and sulfamethoxazole) and others are largely consumed (diclofenac and ibuprofen). The effect of the light source on adsorption of drugs onto activated carbons followed by photolysis with TiO₂ was assessed, being UV-C light the most effective for drug removal in both deionized water and river water. Different composites prepared from titania nanoparticles and powdered activated carbons were tested in several combined adsorption-photocatalysis assays. The composites prepared by calcination at 400 °C exhibited much better performance than those synthesized at 500 °C, being the C400 composite the most effective one. Furthermore, some synthetic waters containing dissolved species and environmental waters were used to investigate the effect of the aqueous matrix on each drug removal. In general, photocatalyst deactivation was found in synthetic and environmental waters. This was particularly evident in the experiments performed with bicarbonate ions as well as with wastewater effluent. In contrast, tests conducted in seawater showed adsorption and photocatalytic degradation yields comparable to those obtained in deionized water. Considering the peculiarities of substrate competition in each aqueous matrix, the combined adsorption-photolysis treatment generally increased the overall elimination of drugs in water.     

Illicit drugs in Canadian municipal wastewater and estimates of community drug use

In this study of wastewater treatment plants in three Canadian cities, selected illicit drugs, including cocaine and its major metabolite, benzoylecgonine (BE), amphetamine, methamphetamine and ecstasy (i.e. MDMA) were detected in untreated wastewater. Cocaine was the most widely used illicit drug at a median level for the 3 cities of 15.7 doses per day per 1000 people. For the other drugs, the median doses per day per 1000 people were 1.8 for amphetamine, 4.5 for methamphetamine and 0.4 for ecstasy. Methamphetamine use was highest in the largest city and cocaine use was lowest in the smallest city. Removal of the illicit drugs by wastewater treatment was generally >50%, except in a WWTP that uses primary treatment. The community consumption estimate for ecstasy in the present study is far below published estimates of the prevalence of ecstasy use among the Canadian population, which may be due to only occasional use of ecstasy.     

Impact of common cytostatic drugs on pollen fertility in higher plants

Cytostatic drugs are among the most toxic chemicals which are produced. Many of them cause damage of the genetic material which may affect the fertility of higher organisms. To study the impact of the widely used anticancer drugs [cisplatin (CisPt), etoposide (Et), and 5-fluorouracil (5-FU)] on the reproduction of higher plants, pollen abortion experiments were conducted with species which belong to major plant families, namely with Tradescantia paludosa (Commelinaceae), Arabidopsis thaliana (Brassicaceae), Chelidonium majus (Papaveraceae), and Alisma plantago-aquatica (Alismataceae). All compounds increased the frequencies of abortive grains. The lowest effective doses were in general in a narrow range (i.e., 1 and 10 mg/kg of dry soil). The effects of the individual drugs were similar in T. paludosa, A. plantago-aquatica, and Ch. majus, while A. thaliana was consistently less sensitive. The highest abortion rate was obtained in most experiments with CisPt, followed by 5-FU and Et. Comparisons of the doses which caused effects in the present experiments in the different species with the predicted environment concentrations and with the levels of the cytostatics which were detected in hospital wastewaters show that the realistic environmental concentrations of the drugs are 4–6 orders of magnitude lower. Therefore, it is unlikely that these drugs affect the fertility of higher plants in aquatic and terrestrial ecosystems.     

Development of an enzyme linked immunosorbent assay for the determination of phenothiazine drugs in meat and animal feeds

In this study, 2-chlorophenothiazine was used to synthesize a hapten for production of monoclonal antibody. The obtained monoclonal antibody showed high crossreactivities to chlorpromazine, promethazine and perphenazine, and showed low crossreactivities to acepromazine and fluphenazine. After evaluation of three coating antigens, a heterologous competitive indirect enzyme linked immunosorbent assay was developed to determine the five phenothiazines in animal feeds and the residues of chlorpromazine, promethazine and perphenazine in meat. The crossreactivities to the five analytes were in a range of 2.4%–98%. The limits of detection for the five drugs in feeds were in a range of 0.1–3.0 μg g^?1, and that for chlorpromazine, promethazine and perphenazine in meat were in a range of 0.5–0.8 ng g^?1. Their recoveries from standards fortified blank samples (chicken, pork and feeds) were in a range of 74.1%–96.5% with coefficients of variation of 6.4%–15.1%. Therefore, this method could be used as a rapid screen tool to determine phenothiazine drugs in animal feeds and animal derived foods.     

Illicit drugs in wastewater of the city of Zagreb (Croatia) - Estimation of drug abuse in a transition country

A comprehensive study of various psychoactive substances and their metabolites was performed in the wastewater treatment plant of the city of Zagreb (780 000 inhabitants) using liquid chromatography/tandem mass spectrometry (LC-MS-MS). The estimation of drug abuse for five different illicit drugs, including heroin, cocaine, marijuana, amphetamine and ecstasy, was made on the basis of their representative excretion rates, which were determined over a period of 8 months. Marijuana (1000 kg/year), heroin (75 kg/year) and cocaine (47 kg/year) were found to be the most frequently consumed illicit drugs, while the consumption of amphetamine-type drugs was much lower (1-3 kg/year). A comparison with other reports indicated that drug abuse profiles in transition countries might be different from those reported for Western Europe, in particular with respect to the comparatively increased consumption of heroin. Enhanced consumption of stimulating drugs (cocaine and ectasy) was systematically detected during weekends.     

Detection and quantitative analysis of 21 veterinary drugs in river water using high-pressure liquid chromatography coupled to tandem mass spectrometry

Introduction

The use of veterinary drugs in food production focuses on the control and improvement of animal health. The disadvantage of this practice is that pharmaceuticals and their metabolites are released into the environment, finding their way to natural water systems and becoming a potential risk to non-target organism.

Methods

This paper reports the development and validation of a quantitative method, based on high-performance liquid chromatography coupled to tandem mass spectrometry, for the simultaneous analysis of 21 veterinary drugs, antimicrobials, corticosteroids, coccidiostats and antifungal agents, in surface water.

Results

The precision of the method was established by calculating the mean recoveries, which were in the range of 94?C101%. The developed method was employed to conduct the first monitoring study on the presence of veterinary drugs in the Galicia region, Northwest of Spain and was applied to 235 surface water samples. Eleven veterinary drugs were detected at concentrations from below the limit of quantification to 2,978.6?ng?L^?1. Limits of detection and quantification were in the range of 6.2 (betamethasone, cortisone, decoquinate, dexamethasone, maduramycin, monensin, narasin, salinomycin, sulfachloropyridazine, sulfamethoxypyridazine and trimethoprim) to 12.5?ng?L^?1 (for the rest of the selected drugs) and 12.5 (betamethasone, cortisone, decoquinate, dexamethasone, maduramycin, monensin, narasin, salinomycin, sulfachloropyridazine, sulfamethoxypyridazine and trimethoprim) to 25.0?ng?L^?1 (for the remaining pharmaceuticals), respectively.

Conclusion

Sulfonamides were the group most frequently found, which are widely used in veterinary medicine.     

Toxicity of the mixture of selected antineoplastic drugs against aquatic primary producers

The residues of antineoplastic drugs are considered as new and emerging pollutants in aquatic environments. Recent experiments showed relatively high toxicity of 5-fluorouracil (5-FU), imatinib mesylate (IM), etoposide (ET) and cisplatin (CP) that are currently among most widely used antineoplastic drugs, against phytoplankton species. In this study, we investigated the toxic potential of the mixture of 5-FU?+?IM?+?ET against green alga Pseudokirchneriella subcapitata and cyanobacterium Synechococcus leopoliensis, and the stability and sorption of these drugs to algal cells. Toxic potential of the mixture was predicted by the concepts of ‘concentration addition’ and ‘independent action’ and compared to the experimentally determined toxicity. In both test species, the measured toxicity of the mixture was at effects concentrations EC₁₀–EC₅₀ higher than the predicted, whereas at higher effect concentration (EC₉₀), it was lower. In general, P. subcapitata was more sensitive than S. leopoliensis. The stability studies of the tested drugs during the experiment showed that 5-FU, IM and CP are relatively stable, whereas in the cultures exposed to ET, two transformation products with the same mass as ET but different retention time were detected. The measurements of the cell-linked concentrations of the tested compounds after 72 h exposure indicated that except for CP (1.9 % of the initial concentration), these drugs are not adsorbed or absorbed by algal cells. The results of this study showed that in alga and cyanobacteria exposure to the mixture of 5-FU?+?ET?+?IM, in particular at low effect concentration range, caused additive or synergistic effect on growth inhibition, and they suggest that single compound toxicity data are not sufficient for the proper toxicity prediction for aquatic primary producers.     

Genotoxic potential of selected cytostatic drugs in human and zebrafish cells

Due to their increasing use, the residues of anti-neoplastic drugs have become emerging pollutants in aquatic environments. Most of them directly or indirectly interfere with the cell’s genome, which classifies them into a group of particularly dangerous compounds. The aim of the present study was to conduct a comparative in vitro toxicological characterisation of three commonly used cytostatics with different mechanisms of action (5-fluorouracil [5-FU], cisplatin [CDDP] and etoposide [ET]) towards zebrafish liver (ZFL) cell line, human hepatoma (HepG2) cells and human peripheral blood lymphocytes (HPBLs). Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and acridine orange/ethidium bromide staining. All three drugs induced time- and dose-dependent decreases in cell viability. The sensitivity of ZFL and HepG2 cells towards the cytotoxicity of 5-FU was comparable (half maximal inhibitory concentration (IC₅₀) 5.3 to 10.4 μg/mL). ZFL cells were more sensitive towards ET- (IC₅₀ 0.4 μg/mL) and HepG2 towards CDDP- (IC₅₀ 1.4 μg/mL) induced cytotoxicity. Genotoxicity was determined by comet assay and cytokinesis block micronucleus (CBMN) assay. ZFL cells were the most sensitive, and HPBLs were the least sensitive. In ZFL cells, induction of DNA strand breaks was a more sensitive genotoxicity endpoint than micronuclei (MNi) induction; the lowest effective concentration (LOEC) for DNA strand break induction was 0.001 μg/mL for ET, 0.01 μg/mL for 5-FU and 0.1 μg/mL for CDDP. In HepG2 cells, MNi induction was a more sensitive genotoxicity endpoint. The LOEC values were 0.01 μg/mL for ET, 0.1 μg/mL for 5-FU and 1 μg/mL for CDDP. The higher sensitivity of ZFL cells to cytostatic drugs raises the question of the impact of such compounds in aquatic ecosystem. Since little is known on the effect of such drugs on aquatic organisms, our results demonstrate that ZFL cells provide a relevant and sensitive tool to screen genotoxic potential of environmental pollutant in the frame of hazard assessment.     

Fate and transport of atorvastatin and simvastatin drugs during conventional wastewater treatment

This research investigates the environmental behavior of two widely prescribed cholesterol-lowering statin drugs that are expected to be present at significant concentrations in wastewater influents, namely: atorvastatin and simvastatin. Batch biodegradation experiments suggest that both statins are well degraded during secondary treatment, and removal rates exhibit a substrate-enhancement model reflecting elements of both first-order behavior and cometabolism. Resulting biodegradation parameters are used in conjunction with literature sorption parameters to construct a mass-balance model of statin concentrations during conventional treatment. Model results exhibit excellent accuracy compared to measurements from a medium-sized WWTP in the Southeastern USA. Influent concentrations of 1.56 μg L(-1) and 1.23 μg L(-1) were measured for atorvastatin and simvastatin. Results also suggest that 85-90% of each drug is removed during conventional treatment, with sorption accounting for less than 10% of overall removal. Expected effluent concentrations are orders of magnitude less than previously reported ecotoxicity thresholds for both drugs. Overall, results suggest statin active ingredients do not pose a significant environmental threat. It is recommended that future work characterize the fate of statin metabolites and that the same mass-balance modeling approach be used to assess other highly-prescribed pharmaceutical drugs.     

Influence of selected anti-cancer drugs on the induction of DNA double-strand breaks and changes in gene expression in human hepatoma HepG2 cells

In chemotherapy, various anti-cancer drugs with different mechanisms of action are used and may represent different risk of undesirable delayed side effects in treated patients as well as in occupationally exposed populations. The aim of the present study was to evaluate genotoxic potential of four widely used anti-cancer drugs with different mechanisms of action: 5-fluorouracil (5-FU), cisplatin (CDDP) and etoposide (ET) that cause cell death by targeting DNA function and imatinib mesylate (IM) that inhibits targeted protein kinases in cancer cells in an experimental model with human hepatoma HepG2 cells. After 24 h of exposure all four anti-cancer drugs at non-cytotoxic concentrations induced significant increase in formation of DNA double strand breaks (DSBs), with IM being the least effective. The analysis of the changes in the expression of genes involved in the response to DNA damage (CDKN1A, GADD45A, MDM2), apoptosis (BAX, BCL2) and oncogenesis (MYC, JUN) showed that 5-FU, CDDP and ET upregulated the genes involved in DNA damage response, while the anti-apoptotic gene BCL2 and oncogene MYC were downregulated. On the contrary, IM did not change the mRNA level of the studied genes, showing different mechanism of action that probably does not involve direct interaction with DNA processing. Genotoxic effects of the tested anti-cancer drugs were observed at their therapeutic concentrations that may consequently lead to increased risk for development of delayed adverse effects in patients. In addition, considering the genotoxic mechanism of action of 5-FU, CDDP and ET an increased risk can also not be excluded in occupationally exposed populations. The results also indicate that exposure to 5-FU, CDDP and ET represent a higher risk for delayed effects such as cancer, reproductive effects and heritable disease than exposure to IM.     

Psychoactive drugs: occurrence in aquatic environment,analytical methods,and ecotoxicity—a review

This review focused on seven psychoactive drugs being six benzodiazepines (alprazolam, bromazepam, clonazepam, diazepam, lorazepam, and oxazepam) and one antidepressant (citalopram) widely consumed by modern society and detected in different aqueous matrices (drinking water, surface water, groundwater, seawater, estuary water, influent and effluent of wastewater treatment plants). The review included 219 selected scientific papers from which 1642 data/entries were obtained, each entry corresponding to one target compound in one aqueous matrix. Concentrations of all investigated drugs in all aqueous matrices varied from 0.14 to 840,000 ng L^?1. Citalopram presented the highest concentrations in the aqueous matrices. Based on the Wilcoxon-Mann-Whitney test, differences between wastewater influents and effluents were not significant for most wastewater categories, suggesting that conventional wastewater treatment systems as such do not remove or remove partially these compounds. High-income countries showed much lower concentrations in surface water than the group formed by upper-middle-, lower-middle-, and low-income countries. Regarding analytical methods, solid-phase extraction (SPE) was by far the most used extraction method (83%) and performance liquid chromatography (HPLC) (73%) coupled to mass spectrometry (99%) the most common analytical method. Changes in behavior and in survival rates were the most common effects reported on bioindicators (aquatic species) due to the presence of these drugs in water. Concentrations of psychoactive drugs found in surface waters were most of the time within the range that caused measurable toxic effects in ecotoxicity assays.     

Investigation of drugs of abuse and relevant metabolites in Dutch sewage water by liquid chromatography coupled to high resolution mass spectrometry

An extensive study on the presence of illicit drugs and pharmaceuticals with potential for abuse in sewage waters was made for the first time in the Netherlands. A total number of 24 target drugs were investigated in influent and effluent wastewater using liquid chromatography coupled to a high resolution Orbitrap mass spectrometer. This powerful analyzer has allowed not only the detection and identification of the compounds under investigation, but also their quantification at very low levels, which is highly innovative in the field of drugs of abuse. Samples were taken from five sewage treatment plants (STPs) during a whole week. The selected STPs served four cities of different size and an international airport. Daily variances of drug loads were demonstrated and removal efficiencies calculated for each drug and STP individually. Twelve target compounds were found in at least one influent or effluent, and highest concentrations were observed in influents collected from more urbanized areas. The compounds more frequently detected were amphetamine, benzoylecgonine, cocaine and THCCOOH together with the pharmaceuticals codeine, oxazepam and temazepam. Established week trends in consumption of drugs showed distinct differences between individual drugs. A slightly different occurrence pattern was observed in wastewaters from the airport. Thus, methamphetamine was only detected at Schiphol, a fact that was interpreted to be caused by consumption of this drug by travelers. Despite the fact that the Netherlands has frequently been criticized for its liberal drug policy the results from this study did not reveal higher drug consumption than found elsewhere, with the exception of cannabis.     

Investigation of drugs of abuse and relevant metabolites in Dutch sewage water by liquid chromatography coupled to high resolution mass spectrometry

An extensive study on the presence of illicit drugs and pharmaceuticals with potential for abuse in sewage waters was made for the first time in the Netherlands. A total number of 24 target drugs were investigated in influent and effluent wastewater using liquid chromatography coupled to a high resolution Orbitrap mass spectrometer. This powerful analyzer has allowed not only the detection and identification of the compounds under investigation, but also their quantification at very low levels, which is highly innovative in the field of drugs of abuse. Samples were taken from five sewage treatment plants (STPs) during a whole week. The selected STPs served four cities of different size and an international airport. Daily variances of drug loads were demonstrated and removal efficiencies calculated for each drug and STP individually. Twelve target compounds were found in at least one influent or effluent, and highest concentrations were observed in influents collected from more urbanized areas. The compounds more frequently detected were amphetamine, benzoylecgonine, cocaine and THCCOOH together with the pharmaceuticals codeine, oxazepam and temazepam. Established week trends in consumption of drugs showed distinct differences between individual drugs. A slightly different occurrence pattern was observed in wastewaters from the airport. Thus, methamphetamine was only detected at Schiphol, a fact that was interpreted to be caused by consumption of this drug by travelers. Despite the fact that the Netherlands has frequently been criticized for its liberal drug policy the results from this study did not reveal higher drug consumption than found elsewhere, with the exception of cannabis.