Insights into the efficient mineralization of antibiotic trimethoprim in aqueous media by Fe2+ catalytically enhanced vacuum-UV irradiation: Kinetics, mechanisms, and toxicity evaluation.

The widespread existence of antibiotics in the environment has attracted growing concerns regarding the potential adverse effects on aquatic organisms, ecosystems, and human health even at low concentrations. Extensive efforts have been devoted to developing new methods for effective elimination of antibiotics from wastewater. Herein, a novel process of Fe2+ catalytically enhanced vacuum ultraviolet (VUV) irradiation was proposed as a promising approach for the removal of antibiotic trimethoprim (TMP) in water. Compared with UVC photolysis, VUV photolysis, and UVC/Fe2+, VUV/Fe2+ could increase the pseudo-first-order reaction rate constant of TMP removal by 6.6-38.4 times and the mineralization rate by 36.5%-59.9%. The excellent performance might originate from the synergistic effect of VUV and Fe2+, i.e., VUV irradiation could effectively split water and largely accelerate the Fe3+/Fe2+ cycle to generate more reactive oxygen species (ROS). EPR results indicated that •OH and O2•- were identified as the main ROS in the UVC/Fe2+ and VUV/Fe2+ processes, while •OH, O2•-, and 1O2 were involved in the VUV process. The operating parameters, such as Fe2+ dosage and initial TMP contents, were evaluated and optimized. Up to 8 aromatic intermediates derived from hydroxylation, demethylation, carbonylation, and methylene group cleavage were identified by UPLC-QTOF-MS/MS technique, the possible pathways of TMP degradation were proposed. Finally, the acute and chronic toxicity of intermediates formed during TMP degradation in the VUV/Fe2+ process were also evaluated.

Synergistic degradation of trimethoprim and its phytotoxicity via the UV/chlorine process: Influencing factors on removal and kinetic.

Occurrence of trimethoprim (TMP), recalcitrant antibiotic, and its adverse effect on ecosystem have been reported in several countries. The study aims to remove the TMP and its phytotoxicity via a UV/chlorine process, compared with chlorination and UV irradiation alone. Various treatment conditions including chlorine doses, pHs, and TMP concentrations was conducted with synthetic waters and effluent waters. The UV/chlorine process exhibited a synergistic effect on the TMP removal, compared with chlorination and UV irradiation alone. The UV/chlorine process was the most effective in removing TMP, followed by chlorination. The UV irradiation slightly affected the TMP removal (less than 5%). The UV/chlorine process completely removed TMP by 15 min contact time, while chlorination for 60 min could achieve 71% of TMP removal. The TMP removal fitted well with the pseudo first-order kinetics, and the rate constant (k') increased with higher chlorine doses, lower TMP concentrations and low pH. HO• was the major oxidant affecting the TMP removal and its degradation rate, compared with other reactive chlorine species (e.g., Cl•, OCl•). The TMP exposure increased the phytotoxicity by decreasing a germination rate of Lactuca sativa and Vigna radiata seeds. The use of UV/chlorine process could effectively detoxify the TMP, resulting in the phytotoxicity level of treated waters equivalent or lower than those of TMP-free effluent water. The detoxification level depended on the TMP removal, and it was about 0.43-0.56 times of TMP removal. The findings indicated the potential use of UV/chlorine process in removing TMP residual and its phytotoxicity.

Effects of ciprofloxacin, trimethoprim, and amoxicillin on microbial structure and growth as emerging pollutants reaching crop soils.

The presence of emerging pollutants, and specifically antibiotics, in agricultural soils has increased notably in recent decades, causing growing concern as regards potential environmental and health issues. With this in mind, the current study focuses on evaluating the toxicity exerted by three antibiotics (amoxicillin, trimethoprim, and ciprofloxacin) on the growth of soil bacterial communities, when these pollutants are present at different doses, and considered in the short, medium, and long terms (1, 8 and 42 days of incubation). Specifically, the research was carried out in 12 agricultural soils having different physicochemical characteristics and was performed by means of the leucine (3H) incorporation method. In addition, changes in the structure of soil microbial communities at 8 and 42 days were studied in four of these soils, using the phospholipids of fatty acids method for this. The main results indicate that the most toxic antibiotic was amoxicillin, followed by trimethoprim and ciprofloxacin. The results also show that the toxicity of amoxicillin decreases with time, with values of Log IC50 ranging from 0.07 ± 0.05 to 3.43 ± 0.08 for day 1, from 0.95 ± 0.07 to 3.97 ± 0.15 for day 8, and from 2.05 ± 0.03 to 3.18 ± 0.04 for day 42, during the incubation period. Regarding trimethoprim, 3 different behaviors were observed: for some soils the growth of soil bacterial communities was not affected, for a second group of soils trimethoprim toxicity showed dose-response effects that remained persistent over time, and, finally, for a third group of soils the toxicity of trimethoprim increased over time, being greater for longer incubation times (42 days). As regards ciprofloxacin, this antibiotic did not show a toxicity effect on the growth of soil bacterial communities for any of the soils or incubation times studied. Furthermore, the principal component analysis performed with the phospholipids of fatty acids results demonstrated that the microbial community structure of these agricultural soils, which persisted after 42 days of incubation, depended mainly on soil characteristics and, to a lesser extent, on the dose and type of antibiotic (amoxicillin, trimethoprim or ciprofloxacin). In addition, it was found that, in this research, the application of the three antibiotics to soils usually favored the presence of fungi and Gram-positive bacteria.

A model-based approach to designing developmental toxicology experiments using sea urchin embryos.

The key aim of this paper is to suggest a more quantitative approach to designing a dose-response experiment, and more specifically, a concentration-response experiment. The work proposes a departure from the traditional experimental design to determine a dose-response relationship in a developmental toxicology study. It is proposed that a model-based approach to determine a dose-response relationship can provide the most accurate statistical inference for the underlying parameters of interest, which may be estimating one or more model parameters or pre-specified functions of the model parameters, such as lethal dose, at maximal efficiency. When the design criterion or criteria can be determined at the onset, there are demonstrated efficiency gains using a more carefully selected model-based optimal design as opposed to an ad-hoc empirical design. As an illustration, a model-based approach was theoretically used to construct efficient designs for inference in a developmental toxicity study of sea urchin embryos exposed to trimethoprim. This study compares and contrasts the results obtained using model-based optimal designs versus an ad-hoc empirical design.

Maladaptive DNA repair is the ultimate contributor to the death of trimethoprim-treated cells under aerobic and anaerobic conditions.

The bactericidal effects of antibiotics are undoubtedly triggered by target-specific interactions, but there is growing evidence that an important aspect of cytotoxicity results from treatment-induced metabolic perturbations. In this study, we characterized molecular mechanisms whereby trimethoprim treatment results in cell death, using Escherichia coli as the model organism. E. coli cells grown in rich medium that contained all amino acids and low amounts of thymidine were treated with trimethoprim under aerobic and anaerobic conditions. Under these growth conditions, accelerated thymine depletion is the primary trigger of the processes leading to cell death. Thymine depletion-induced DNA replication stress leads to the production of reactive oxygen species under aerobic conditions and of the DNA-damaging byproducts of nitrate respiration under anaerobic conditions. Lowering the DNA replication initiation rate by introducing the dnaA(Sx) allele or by overexpressing Hda protein reduces the number of active replication forks, which reduces the consumption of thymidine and increases resistance to trimethoprim under both aerobic and anaerobic conditions. Analysis of the involvement of DNA repair enzymes in trimethoprim-induced cytotoxicity clearly indicates that different amounts and/or different types of DNA lesions are produced in the presence or absence of oxygen. Maladaptive processing of the DNA damage by DNA repair enzymes, in particular by MutM and MutY DNA glycosylases, ultimately contributes to cell death.

Indirect Photodegradation of Sulfamethoxazole and Trimethoprim by Hydroxyl Radicals in Aquatic Environment: Mechanisms, Transformation Products and Eco-Toxicity Evaluation.

The bacteriostatic antibiotics, sulfamethoxazole (SMX) and trimethoprim (TMP), have frequently been found in wastewater and surface water, which raises the concerns about their ecotoxicological effects. The indirect photochemical transformation has been proven to be an efficient way to degrade SMX and TMP. In this study, the reaction mechanisms of the degradation by SMX and TMF by OH radicals were investigated by theoretical calculations. Corresponding rate constants were determined and the eco-toxicity of SMX and TMP and its degradations products were predicted using theoretical models. The results indicate that the most favorable pathways for the transformation of SMX and TMP are both •OH-addition reaction of benzene ring site with lowest Gibbs free energy barriers (6.86 and 6.21 kcal mol-1). It was found that the overall reaction rate constants of •OH-initial reaction of SMX and TMP are 1.28 × 108 M-1 s-1 and 6.21 × 108 M-1 s-1 at 298 K, respectively. When comparing the eco-toxicity of transformation products with parent SMX and TMP, it can be concluded that the acute and chronic toxicities of the degraded products are reduced, but some products remain harmful for organisms, especially for daphnid (toxic or very toxic level). This study can give greater insight into the degradation of SMX and TMP by •OH through theoretical calculations in aquatic environment.

Reversible Control of Protein Localization in Living Cells Using a Photocaged-Photocleavable Chemical Dimerizer.

Many dynamic biological processes are regulated by protein-protein interactions and protein localization. Experimental techniques to probe such processes with temporal and spatial precision include photoactivatable proteins and chemically induced dimerization (CID) of proteins. CID has been used to study several cellular events, especially cell signaling networks, which are often reversible. However, chemical dimerizers that can be both rapidly activated and deactivated with high spatiotemporal resolution are currently limited. Herein, we present a novel chemical inducer of protein dimerization that can be rapidly turned on and off using single pulses of light at two orthogonal wavelengths. We demonstrate the utility of this molecule by controlling peroxisome transport and mitotic checkpoint signaling in living cells. Our system highlights and enhances the spatiotemporal control offered by CID. This tool addresses biological questions on subcellular levels by controlling protein-protein interactions.

Effects of erythromycin, trimethoprim and clindamycin on attached microbial communities from an effluent dominated prairie stream.

In this study, differing metrics were utilized to measure effects of erythromycin (ER), trimethoprim (TR) and clindamycin (CL) on the structure and function of attached Wascana Creek, SK microbial communities. All three test antibiotics, especially ER, affected community structure and function of biofilms grown in rotating annular reactors. Biofilm thickness, bacterial biomass, and lectin binding biovolume (exopolymeric substances) were consistently less in ER treated biofilms when compared to the control. As well negative effects on protozoan numbers, and carbon utilization were detected. Finally, PCA analyses of DGGE results indicated that bacterial community diversity in ER exposed biofilms was always different from the control. ER exhibited toxic effects even at lower concentrations. Observations on TR and CL exposed biofilms indicated that bacterial biomass, lectin binding biovolume and carbon utilization were negatively affected as well. In terms of bacterial community diversity, however, CL exposed biofilms tended to group with the control while TR grouped with nutrient additions suggesting both nutritive and toxic effects. This study results represent an important step in understanding antibiotic effects, especially ER, on aquatic microbial communities. And because ER is so ubiquitous in receiving water bodies worldwide, the Wascana study results suggest the possibility of ecosystem disturbance elsewhere. CAPSULE ABSTRACT: Erythromycin (ER) is ubiquitous in waterbodies receiving sewage effluent. Structure and function of microbial communities from an effluent dominated stream were negatively affected by ER, at realistic concentrations.

Chemical Morphing of DNA Containing Four Noncanonical Bases.

The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate in vitro and to serve as genetic templates in vivo was evaluated. A nucleotide triphosphate set of 5-chloro-2'-deoxyuridine, 7-deaza-2'-deoxyadenosine, 5-fluoro-2'-deoxycytidine, and 7-deaza-2'deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo-) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional in vivo.

Two-generation reproduction and teratology studies of feeding aditoprim in Wistar rats.

Aditoprim, a new bacteriostatic agent that belongs to diaminopyrimidines, has a broad antimicrobial spectrum, good antibacterial activity and excellent pharmacokinetics. To evaluate the reproductive toxicity and teratogenic potential of aditoprim, different concentrations of aditoprim were administered to Wistar rats by feeding diets containing 0, 20, 100 and 1000 mg kg(-1) , respectively. Each group consisting of 18 males and 25 females (F0 ) was treated with different concentrations of aditoprim through a 13-week period before mating and during mating, gestation, parturition and lactation. At weaning, 20 males and 25 females of the F1 generation weanlings per group were selected randomly as parents for the F2 generation. Selected F1 weanlings were exposed to the same diet and treatment as their parents. At 1000 mg kg(-1) dose group, body weights in F0 and F1 rats, fetal body weight on day 21 (0, 4 and 21) after birth and number of viable fetuses in the F0 and F1 generation significantly decreased. Teratogenicity study was performed in combination with the F1 generation of a two-generation reproduction study. F1 parents of the reproduction study were mated after weaning of the F2a pups. Pregnant female rats were subjected to cesarean section on gestational day 20 for teratogenic examination. At 1000 mg kg(-1) group, body weights, fetal body lengths, tail lengths, litter weights and number of viable fetuses were significantly decreased. No obvious external, skeletal or visceral malformations in fetuses were noted in any groups in the teratogenic test. The no-observed-adverse-effect level for reproduction/development toxicity of aditoprim was 100 mg kg(-1) diet (about 7.89-9.25 mg kg(-1) body weight day(-1) ).

Safety assessment of aditoprim acute, subchronic toxicity and mutagenicity studies.

Aditoprim (ADP), a new developed dihydrofolate reductase (DHFR) inhibitor, has great potential in clinical veterinary medicine because of its greater pharmacokinetic properties than structural analogs. Preclinical toxicology studies were performed to assess the safety of ADP including an acute oral toxicity test, a subchronic toxicity test and five mutagenicity tests. In the acute oral toxicity test, ADP was administered singly by oral gavage to Wistar rats and Kunming mice. The LD50 calculated was 1400 mg kg(-1) body weight (BW) day(-1) in rats and 1130 mg kg(-1) BW day(-1) in mice. In a subchronic study, Wistar rats were administered ADP at dose levels of 0, 20, 100 and 1000 mg kg(-1) diet for 90 days. Significant decreases were observed on body weight and food efficiency in the high-dose group. Treatment-related changes in clinical serum biochemistry were found in the medium- and high-dose groups. Significant increases in the relative weights of livers and kidneys in females and testis in males in the 1000 mg kg(-1) diet, and significant decrease in relative weights of livers in males in the 100 mg kg(-1) diet were noted. Histopathological observations revealed that the 1000 mg kg(-1) ADP diet could induce lymphocytic infiltration and hepatocytic necrosis near the hepatic portal area. The genotoxicity of ADP was negative in tests, such as the bacterial reverse mutation assay, mice bone marrow erythrocyte micronucleus assay, in vitro chromosomal aberration test, in vitro cho/hgprt mammalian cell mutagenesis assay and mice testicle cells chromosome aberration. Based on the subchronic study, the no-observed-adverse-effect level for ADP was a 20 mg kg(-1) diet, which is about 1.44-1.53 mg kg(-1) BW day(-1) in rats.

Effects of enrofloxacin, ciprofloxacin, and trimethoprim on two generations of Daphnia magna.

Multigenerational tests on Daphnia magna were performed exposing two subsequent generation to enrofloxacin (EFX) and its metabolite ciprofloxacin (CPX), and to trimethoprim (TMP). Mortality rate of 100% and 50% was detected in F0 at concentrations of ≥ 13 mgL(-1) (EFX) and 50 mgL(-1) (TMP), respectively. In F1 with respect to F0, both for growth and reproduction, a worsening trend of the response with EFX, a similar response with CPX and an attenuating trend with TMP was observed. Furthermore, the lowest EC20 for reproduction inhibition (1.3 mgL(-1)) was calculated for F1 exposed to EFX. However, other experimentations, longer and more complex, are necessary in order to confirm that EFX is more hazardous to daphnids than CPX and TMP. EC50 measured for the three assayed antibacterials were in the 6.5-37 mgL(-1) range therefore environmental unrealistic, except in case of exceptional contaminations that may occur in relation to poorly controlled wastewaters from pharmaceutical factories or excessive use of prophylactic treatments in aquaculture.

From bacteria to fish: ecotoxicological insights into sulfamethoxazole and trimethoprim.

Sulfamethoxazole (SMX) and trimethoprim (TRIM) are two of the most used antibiotics in the last 50 years, to prevent and treat bacterial infections; however, the available literature about toxicity to non-target organisms is quite discrepant and incomplete. This study aims to assess the SMX and TRIM ecotoxicological effects in standard species: Aliivibrio fischeri (bioluminescence inhibition), Escherichia coli ATCC 25922 (growth inhibition), Lemna minor (growth inhibition and biochemical biomarkers), Daphnia magna (immobilization/mortality, life history traits, and biochemical biomarkers), and Danio rerio (survival, hatching, abnormalities, and biochemical biomarkers). The species tested showed different acute sensitivities to SMX (A. fischeri < D. magna < E. coli < L. minor) and TRIM (L. minor < A. fischeri < D. magna < E. coli). Overall, TRIM reveals less toxicity than SMX, except for E. coli (Ecotoxicological approach based on Antimicrobial Susceptibility Testing - EcoAST procedure). Both antibiotics affect individually (e.g., growth and survival) and sub-individually (e.g., antioxidant defenses) L. minor, D. magna, and D. rerio. This study allowed us to generate relevant data and fill gaps in the literature regarding the effects of SMX and TRIM in aquatic organisms. The here-obtained results can be used to (i) complete and re-evaluate the Safety Data Sheet to improve the assessment of environmental safety and management of national and international entities; (ii) clarify the environmental risks of these antibiotics in aquatic ecosystems reinforcing the inclusion in the 4th Watch List of priority substances to be monitored in whole inland waters by the Water Framework Directive; and (iii) combat the development of antimicrobial resistance, as well as supporting the definition of environmental measurements in the context of European One Health Action Plan. However, it is essential to continue studying these antibiotics to better understand their toxicity at ecologically relevant concentrations and their long-term effects under different climatic change scenarios.

Effects of sulfamonomethoxine and trimethoprim co-exposures at different environmentally relevant concentrations on microalgal growth and nutrient assimilation.

In aquaculture around the world, sulfamonomethoxine (SMM), a long-acting antibiotic that harms microalgae, is widely employed in combination with trimethoprim (TMP), a synergist. However, their combined toxicity to microalgae under long-term exposures at environmentally relevant concentrations remains poorly understood. Therefore, we studied the effects of SMM single-exposures and co-exposures (SMM:TMP=5:1) at concentrations of 5 µg/L and 500 µg/L on Chlorella pyrenoidosa within one aquacultural drainage cycle (15 days). Photosynthetic activity and N assimilating enzyme activities were employed to evaluate microalgal nutrient assimilation. Oxidative stress and flow cytometry analysis for microalgal proliferation and death jointly revealed mechanisms of inhibition and subsequent self-adaptation. Results showed that exposures at 5 µg/L significantly inhibited microalgal nutrient assimilation and induced oxidative stress on day 7, with a recovery to levels comparable to the control by day 15. This self-adaptation and over 95 % removal of antibiotics jointly contributed to promoting microalgal growth and proliferation while reducing membrane-damaged cells. Under 500 µg/L SMM single-exposure, microalgae self-adapted to interferences on nutrient assimilation, maintaining unaffected growth and proliferation. However, over 60 % of SMM remained, leading to sustained oxidative stress and apoptosis. Remarkably, under 500 µg/L SMM-TMP co-exposure, the synergistic toxicity of SMM and TMP significantly impaired microalgal nutrient assimilation, reducing the degradation efficiency of SMM to about 20 %. Consequently, microalgal growth and proliferation were markedly inhibited, with rates of 9.15 % and 17.7 %, respectively, and a 1.36-fold increase in the proportion of cells with damaged membranes was observed. Sustained and severe oxidative stress was identified as the primary cause of these adverse effects. These findings shed light on the potential impacts of antibiotic mixtures at environmental concentrations on microalgae, facilitating responsible evaluation of the ecological risks of antibiotics in aquaculture ponds.

Superiority of solar Fenton oxidation over TiO2 photocatalysis for the degradation of trimethoprim in secondary treated effluents.

The overall aim of this work was to examine the degradation of trimethoprim (TMP), which is an antibacterial agent, during the application of two advanced oxidation process (AOP) systems in secondary treated domestic effluents. The homogeneous solar Fenton process (hv/Fe(2+)/H2O2) and heterogeneous photocatalysis with titanium dioxide (TiO2) suspensions were tested. It was found that the degradation of TMP depends on several parameters such as the amount of iron salt and H2O2, concentration of TiO2, pH of solution, solar irradiation, temperature and initial substrate concentration. The optimum dosages of Fe(2+) and H2O2 for homogeneous ([Fe(2+)] = 5 mg L(-1), [H2O2] = 3.062 mmol L(-1)) and TiO2 ([TiO2] = 3 g L(-1)) for heterogeneous photocatalysis were established. The study indicated that the degradation of TMP during the solar Fenton process is described by a pseudo-first-order reaction and the substrate degradation during the heterogeneous photocatalysis by the Langmuir-Hinshelwood kinetics. The toxicity of the treated samples was evaluated using a Daphnia magna bioassay and was finally decreased by both processes. The results indicated that solar Fenton is more effective than the solar TiO2 process, yielding complete degradation of the examined substrate within 30 min of illumination and dissolved organic carbon (DOC) reduction of about 44% whereas the respective values for the TiO2 process were ∼70% degradation of TMP within 120 min of treatment and 13% DOC removal.

Sublethal effects of trimethoprim on four freshwater organisms.

Sublethal effects of trimethoprim (TMP) were evaluated in four freshwater organisms: Pseudokirchneriella subcapitata and Lemna minor (growth inhibition), Daphnia magna (reproduction and growth inhibition) and Poecilia reticulata (swimming activity inhibition). Cytochrome P4501A induction was also evaluated in P. reticulata. TMP showed varying levels of toxicity in the four test performed, with NOEC for the various endpoints in the range of 3.12-25 mg L(-1). The compound was active on P. reticulata at concentration ≥ 50 mg L(-1) causing inhibition of swimming activity. In the same organism an induction of CYP1A protein, mainly in kidney, gills and intestine, was also detected. L. minor was more sensitive than unicellular algae to TMP, with a NOEC of 12.5 mg L(-1). The lowest NOEC (3.12 mg L(-1)) was obtained in D. magna reproduction test and then a Risk Quotient of <0.03 was calculated by comparing the PNEC (31.2 µg L) and the TMP concentrations usually detected in freshwater (<1 µg L(-1)). However, based on recently reported data, it was concluded that while TMP concentrations normally detected in surface water are below those able to evoke appreciable biological effects in the various aquatic organisms, TMP concentrations in aquaculture and hospital effluents can be one to three orders of magnitude higher. Furthermore, the co-occurrence and additive effects of other antifolic agents should be taken into account for a cautious risk assessment of the drug.

Antibiotics removal from aquaculture effluents by ozonation: chemical and toxicity descriptors.

Antibiotics are often applied in aquaculture to prevent fish diseases. These substances can cause disturbances on receiving waters, when not properly eliminated from the aquaculture effluents. In this work, ozone (O3) was investigated as a possible oxidizing agent to remove fishery antibiotics from aquaculture effluents: florfenicol (FF), oxytetracycline (OTC), sulfadimethoxine (SDM), sulfamethoxazole (SMX), and trimethoprim (TMP). Batch experiments were performed using ultrapure water and aquaculture effluents spiked with a mixture of target antibiotics at relatively high concentrations (10 mg L-1 each). OTC, SMX and TMP were fully removed (< 30 min) regardless of the tested conditions, mainly by O3 direct attack. In contrast, FF was partially removed in 30 min (∼ 10 and 60%, in aquaculture effluents and ultrapure water, respectively), but only in the presence of hydroxyl radicals (HO•), the FF concentrations reaching levels below the detection limits in ultrapure water after 60 min. In the case of SDM, its degradation was highly influenced by the selected water matrix, but with removals always higher than 68%. In continuous-flow experiments applying more environmentally relevant antibiotic concentrations (100 ng L-1 each) and low O3 doses (1.5 mg L-1), ozonation highly removed (> 98%) all tested antibiotics from aquaculture effluents with a hydraulic retention time (HRT) of 10 min, except FF (68%). Although by-products were detected in treated samples, zebrafish (Danio rerio) embryotoxicity tests did not show a toxicity increase by applying this ozonation treatment. Ozonation is thus a possible solution to remove antibiotics from aquaculture effluents. Still, full-scale studies in aquaculture farms are needed, and generation of HO• may be favoured to readily oxidize the FF antibiotic.

The antibacterials ciprofloxacin, trimethoprim and sulfadiazine modulate gene expression, biomarkers and metabolites associated with stress and growth in gilthead sea bream (Sparus aurata).

The high consumption and subsequent input of antibacterial compounds in marine ecosystems has become a worldwide problem. Their continuous presence in these ecosystems allows a direct interaction with aquatic organisms and can cause negative effects over time. The objective of the present study was to evaluate the effects of exposure to three antibacterial compounds of high consumption and presence in marine ecosystems (Ciprofloxacin CIP, Sulfadiazine SULF and Trimethoprim TRIM) on the physiology of the gilthead sea bream, Sparus aurata. Plasma parameters, enzymatic biomarkers of oxidative stress and damage and expression of genes related to stress and growth were assessed in exposed S. aurata specimens. For this purpose, sea bream specimens were exposed to individual compounds at concentrations of 5.2 ± 2.1 µg L-1 for CIP, 3.8 ± 2.7 µg L-1 for SULF and 25.7 ± 10.8 µg L-1 for TRIM during 21 days. Exposure to CIP up-regulated transcription of genes associated with the hypothalamic-pituitary-thyroid (HPT) (thyrotropin-releasing hormone, trh) and hypothalamic-pituitary-interrenal (HPI) axes (corticotropin-releasing hormone-binding protein, crhbp) in the brain, as well as altering several hepatic stress biomarkers (catalase, CAT; glutathione reductase, GR; and lipid peroxidation, LPO). Similar alterations at the hepatic level were observed after exposure to TRIM. Overall, our study indicates that S. aurata is vulnerable to environmentally relevant concentrations of CIP and TRIM and that their exposure could lead to a stress situation, altering the activity of antioxidant defense mechanisms as well as the activity of HPT and HPI axes.

Relative sensitivity of fish and mammalian cells to the antibiotic, trimethoprim: cytotoxic and genotoxic responses as determined by neutral red retention, Comet and micronucleus assays.

Relative cytotoxicity and genotoxicity of a widely used antibiotic, trimethoprim (TRIMP) was evaluated under in vitro conditions using rainbow trout gonad-2 (RTG-2) and Chinese hamster ovary-K1 (CHO-K1) cells. Whilst cytotoxicity was determined using neutral red retention (NRR) assay, the genotoxicity was determined using single cell gel electrophoresis or the Comet assay and cytokinesis-block micronucleus (CBMN) assay. For NRR assay, concentration-dependent cytotoxic effect was observed for both the cell lines (estimated EC(50) values: 671.82 ± 21.78 and 611.6 ± 20.4 µg ml(-1) for RTG-2 and CHO-K1 cells, respectively). There was no statistically significant difference between the two cell lines for this assay. For the Comet assay, standard 6 h exposure to TRIMP did not show any positive response for any of the cell types used. However, 48 h exposure to RTG-2 cells showed a concentration-dependent induction of DNA damage (r = 0.86). The highest concentration of TRIMP used (i.e. 100 µg ml(-1)) showed relatively higher DNA damage, compared to ethyl methane sulfonate (EMS; 1 µg ml(-1) or 8 mM), a reference genotoxic agent, used concurrently. In contrast, 24 h exposure time for CHO-K1 cells did not show any concentration-dependent increase for this assay. For MN assay, a significant correlation was found between the MN induction and TRIMP concentration for both the cell lines (RTG-2: r = 0.68; CHO-K1: r = 0.79), although only the highest concentration used showed a significant increase for binucleated (BN) cell with micronuclei (BNMN). The study suggests that whilst the cells of different origin could exhibit similar cytotoxicity, they could display differential genotoxic effects. Furthermore, genotoxic effects of TRIMP are primarily exposure period dependent phenomena and, in addition to inhibiting the action of dihydrofolate reductase, oxidative stress could also contribute for the observed toxic effects, fish cells in general being more sensitive for genotoxic effects.

Investigating the Mechanism of Trimethoprim-Induced Skin Rash and Liver Injury.

Trimethoprim (TMP)-induced skin rash and liver injury are likely to involve the formation of reactive metabolites. Analogous to nevirapine-induced skin rash, 1 possible reactive metabolite is the sulfate conjugate of α-hydroxyTMP, a metabolite of TMP. We synthesized this sulfate and found that it reacts with proteins in vitro. We produced a TMP-antiserum and found covalent binding of TMP in the liver of TMP-treated rats. However, we found that α-hydroxyTMP is not a substrate for human sulfotransferases, and we did not detect covalent binding in the skin of TMP-treated rats. Although less reactive than the sulfate, α-hydroxyTMP was found to covalently bind to liver and skin proteins in vitro. Even though there was covalent binding to liver proteins, TMP did not cause liver injury in rats or in our impaired immune tolerance mouse model that has been able to unmask the ability of other drugs to cause immune-mediated liver injury. This is likely because there was much less covalent binding of TMP in the livers of TMP-treated mice than TMP-treated rats. It is possible that some patients have a sulfotransferase that can produce the reactive benzylic sulfate; however, α-hydroxyTMP, itself, has sufficient reactivity to covalently bind to proteins in the skin and may be responsible for TMP-induced skin rash. Interspecies and interindividual differences in TMP metabolism may be 1 factor that determines the risk of TMP-induced skin rash. This study provides important data required to understand the mechanism of TMP-induced skin rash and drug-induced skin rash in general.