Long-term exposure of Allonais inaequalis to a mixture of antibiotics in freshwater and synthetic wastewater matrices: Reproduction, recovery, and swimming responses.

Antibiotics from sulfonamide, fluoroquinolone, and diaminopyrimidine classes are widely used in human and veterinary medicine, and their combined occurrence in the aquatic environment is increasing around the world. In parallel, the understanding of how mixtures of these compounds affect non-target species from tropical freshwaters is scarce. Thus, this work aimed to study the long-term reproductive, recovery, and swimming effects of mixtures of 12 antibiotics from three different classes (up to 10 µg L-1 ) added to freshwater (FWM) and synthetic wastewater (SWM) matrices on freshwater worm Allonais inaequalis. Results revealed that at the reproduction level, the exposure to antibiotics in the SWM matrix does not cause a significant toxic effect on species after 10 days. On the other hand, exposures to initial dose mixtures (10 µg L-1 each) in FWM caused a significant reduction of offspring by 19.2%. In addition, recovery bioassays (10 days in an antibiotic-free environment) suggested that A. inaequalis has reduced offspring production due to previous exposure to antibiotic mixtures in both matrices. Furthermore, despite slight variation in swimming speed over treatments, no significant differences were pointed out. Regarding antibiotics in the water matrices after 10-day exposures, the highest concentrations were up to 2.7, 7.8, and 4.2 µg L-1 for antibiotics from sulfonamide, fluoroquinolone, and diaminopyrimidine classes, respectively. These findings suggest that a species positioned between primary producers and secondary consumers may experience late reproductive damage even in an antibiotic-free zone, after previous 10-day exposure to antibiotic mixtures. PRACTITIONER POINTS: A mixture of sulfonamide, fluoroquinolone, and diaminopyrimidine antibiotics in freshwater affects the offspring production of A. inaequalis after 10 days. After the 10-day antibiotic exposure, the reproduction of A. inaequalis remains affected in an antibiotic-free environment over the recovery period. The swimming speed of the worms does not change after 10 days of exposure to the antibiotic mixture. The concentration of dissolved solids can limit the natural degradation of sulfonamide, fluoroquinolone, and diaminopyrimidine antibiotics in the aquatic environment.

Toxicity of fluoroquinolones on the cladoceran Daphnia magna.

This study evaluates the acute and chronic toxicological effects of six fluoroquinolones on the mortality and growth of Daphnia magna. The NOECs calculated with the multivariate Probit regression model for the chronic study were 56 µg/L ciprofloxacin, 63 µg/L enrofloxacin, 78 µg/L levofloxacin, 85 µg/L marbofloxacin, 69 µg/L norfloxacin, and 141 µg/L ofloxacin. The risk quotients were determined using the measure environmental concentrations reported in water sources from different countries. The risks were low and moderate in water samples from rivers and lakes, although concentrations of ciprofloxacin, norfloxacin, and ofloxacin reported in some countries can cause toxicological damage to D. magna. In addition, urban wastewater and hospital wastewater samples constitute a threat to D. magna (high and moderate risks), requiring the treatment of these wastewater. PRACTITIONER POINTS: The NOECs calculated with the multivariate Probit model for the six fluoroquinolonas are between 56 µg/L ciprofloxacin and 141 µg/L ofloxacin. The levels of ciprofloxacin, norfloxacin, and ofloxacin in urban wastewater and hospital wastewater produce moderate and high risks for D. magna. Water and river samples from some countries containing ciprofloxacin, norlfoxacin, and ofloxacin present high risks for D. magna.

Fluoroquinolone-contaminated poultry litter strongly affects earthworms as verified through lethal and sub-lethal evaluations.

Poultry litter is one of the main sources of fluoroquinolones (FQs) in agricultural soils. In this study, our main goal was to investigate FQ-contaminated poultry litter effects on Eisenia andrei earthworms. To achieve this, acute and chronic tests covered several endpoints, such as avoidance, biomass, lethality, reproduction and changes to immune cells. FQs (enrofloxacin and ciprofloxacin) were determined in a poultry litter sample through high performance liquid chromatography with a fluorescence detector. The avoidance test indicates that poultry litter strongly repels earthworms, even at the lowest concentration (50 g kg-1). In the acute test, the lethal concentration of poultry litter to 50% of the earthworms (LC50), was estimated at 28.5 g kg-1 and a significant biomass loss (p < 0.05) occurred at 40 g kg-1. In the chronic test, a significant reproduction effect was observed at 20 g kg-1. Cell typing, density and feasibility indicated significant effects ranging from 5 to 20 g kg-1. A high risk quotient was estimated based on recommended poultry litter applications in field studies. Although FQ contamination in poultry litter and soils has been widely reported in previous studies, this is, to the best of our knowledge, the first toxicological assessment concerning earthworms exposed to FQ-contaminated poultry litter.

Electrochemical treatment of penicillin, cephalosporin, and fluoroquinolone antibiotics via active chlorine: evaluation of antimicrobial activity, toxicity, matrix, and their correlation with the degradation pathways.

Antibiotics are pharmaceuticals widely consumed and frequently detected in environmental water, where they can induce toxic effects and development of resistant bacteria. Their structural variety makes the problem of antibiotics in natural water more complex. In this work, six highly used antibiotics (at 40 µmol L-1) belonging to three different classes (penicillins, cephalosporins, and fluoroquinolones) were treated using an electrochemical system with a Ti/IrO2 anode and a Zr cathode in the presence of NaCl (0.05 µmol L-1). The attack of electrogenerated active chlorine was found to be the main degradation route. After only 20 min of treatment, the process decreased more than 90% of the initial concentration of antibiotics, following the degradation order: fluoroquinolones > penicillins > cephalosporins. The primary interactions of the degrading agent with fluoroquinolones occurred at the cyclic amine (i.e., piperazyl ring) and the benzene ring. Meanwhile, the cephalosporins and penicillins were initially attacked on the ß-lactam and sulfide groups. However, the tested penicillins presented an additional reaction on the central amide. In all cases, the transformations of antibiotics led to the antimicrobial activity decreasing. On the contrary, the toxicity level showed diverse results: increasing, decreasing, and no change, depending on the antibiotic type. In fact, due to the conservation of quinolone nucleus in the fluoroquinolone by-products, the toxicity of the treated solutions remained unchanged. With penicillins, the production of chloro-phenyl-isoxazole fragments increased the toxicity level of the resultant solution. However, the opening of ß-lactam ring of cephalosporin antibiotics decreased the toxicity level of the treated solutions. Finally, the application of the treatment to synthetic hospital wastewater and seawater containing a representative antibiotic showed that the high amount of chloride ions in seawater accelerates the pollutant degradation. In contrast, the urea and ammonium presence in the hospital wastewater retarded the removal of this pharmaceutical.

Toxicity and Genotoxicity of Three Antimicrobials Commonly Used in Veterinary Medicine.

The toxicity of chlortetracycline (CTC), oxytetracycline (OTC) and enrofloxacin (ENF) was tested on two green algal species: the international standard Pseudokirchneriella subcapitata and the native Argentine species Ankistrodesmus fusiformis. All three antibiotics inhibited the algal growth. The most sensitive species was P. subcapitata, for which the EC50 for CTC, OTC and ENF were 1.19 ± 0.53, 0.92 ± 0.30 and 5.18 ± 3.80 mg L-1, respectively. The EC50 for A. fusiformis, were 3.23 ± 0.53, 7.15 ± 2.69 and 10.6 ± 1.28 mg L-1, respectively. The genotoxicity of these veterinary antibiotics was also assessed using chromosome aberration (CA) and micronuclei (MN) induction in Allium cepa roots. Three concentrations were tested (0.1, 1 and 10 mg L-1). Only ENF at 1 and 10 mg L-1 showed any significant MN induction. These data revealed that CTC, OTC and ENF could cause toxicity on green algae, whereas ENF could cause genotoxicity on A. cepa plants.

Ecotoxicity of veterinary enrofloxacin and ciprofloxacin antibiotics on anuran amphibian larvae.

The ecological risks posed by two ß-diketone antibiotics (DKAs, enrofloxacin, ENR and ciprofloxacin, CPX), characterized by their long persistence in aqueous environments and known deleterious effect on model organisms such as zebrafish were analysed using Rhinella arenarum larvae. Sublethal tests were conducted using environmentally relevant concentrations of both ENR and CPX (1-1000µgL-1) under standard laboratory conditions for 96h. Biological endpoints and biomarkers evaluated were body size, shape, development and growth rates, and antioxidant enzymes (glutathione-S-transferase, GST; Catalase, CAT). Risk assessment was analysed based on ration quotients (RQ). The size and shape measurements of the larvae exposed to concentrations greater than 10µgL-1 of CPX were lower compared to controls (Dunnett post hoc p<0.05) and presented signs of emaciation. Concentrations of 1000µgL-1of CPX induced GST activity, in contrast with inhibited GST and CAT of larvae exposed to ENR. Risk assessments indicated that concentrations greater than or equal to10µgL-1 of CPX and ENR are ecotoxic for development, growth, detoxifying, and oxidative stress enzymes. It is suggested that additional risk assessments may provide evidence of bioaccumulation of CPX and ENR in tissues or organs of amphibian larvae by mesocosm sediment test conditions. Finally, intestinal microbiome studies should be considered to establish the mechanisms of action of both antibiotics.

Antimicrobial activity and acute toxicity of ozonated lomefloxacin solution.

Lomefloxacin (LOM) is a synthetic antimicrobial from the fluoroquinolone family (FQ) used as a veterinary and human drug. Once in the environment, LOM may pose a risk to aquatic and terrestrial microorganisms due to its antimicrobial activity. This study evaluated the effect of ozonation of LOM (500 µg L-1), the residual antimicrobial activity against Escherichia coli and acute toxicity against Vibrio fischeri. In addition, degradation products were investigated by UHPLC-MS/MS and proposed. Ozonation was carried out varying the applied ozone dose from 0 to 54.0 mg L-1 O3 and pH values of 3, 7, and 11. Ozonation was most efficient at pH 11 and led to 92.8% abatement of LOM in a 9-min reaction time (54.0 mg L-1 O3 applied ozone dose). Ozonation at pH 3 was able to degrade 80.4% of LOM. At pH 7, 74.3% of LOM was degraded. Although the LOM concentration and the antimicrobial activity of the solution dropped as ozone dose increased (antimicrobial activity reduction of 95% at pH 11), toxicity to V. fischeri increased for pH 7 and 11 (i.e., 65% at pH 7 and 75% at pH 11). The reduction in antimicrobial activity may be related to the oxidation of piperazinyl and the quinolone moiety. The formation of intermediates depended on the oxidant (hydroxyl radicals or/and molecular O3) that acted the most in the process.

Phototoxic Effect of Topical Fluoroquinolones Administered Before Corneal Crosslinking in a Murine Model.

PURPOSE: Corneal crosslinking by UV light (UV-CXL) has become a popular treatment for keratoconus and corneal ectasia. Fluoroquinolones (FQs), commonly administered topically before UV-CXL, are known to be phototoxic to the skin and lens. The purpose of this study was to investigate phototoxic effects of topical FQ treatment on murine corneas before UV-CXL, in which the corneal epithelium was kept intact. METHODS: Murine corneas were treated with various antibiotics with or without riboflavin before UV-CXL. At 24 h, the animals were sacrificed, and the corneas were analyzed for histologic evidence of inflammation and apoptosis and for expression of apoptosis markers BAX and caspases 3 and 9 and for expression of matrix metalloproteinase 9 (MMP-9). Spectrofluorometric analysis was performed. RESULTS: Corneas treated with topical FQ with or without riboflavin before UV-CXL showed mild corneal stromal inflammation, apoptosis by both terminal deoxynucleotidyl transferase dUTP nick end labeling staining and increased expression of BAX gene and caspases 3 and 9 by densitometric analysis. Untreated corneas, corneas treated with azithromycin before UV-CXL, and corneas undergoing UV-CXL without any antibiotic or riboflavin pretreatment showed normal histology, no staining for apoptosis, and no increased production of apoptosis markers by polymerase chain reaction. CONCLUSIONS: The phototoxic effects of FQs on the cornea may lead surgeons to consider another antibiotic class for prophylaxis against infectious keratitis in UV-CXL. These effects, along with the known cytotoxic effects of FQs independent of UV radiation, may contribute to some of the complications of corneal UV-CXL. Dosage studies may be warranted.

Antimicrobial activity against Gram-positive and Gram-negative bacteria during gatifloxacin degradation by hydroxyl radicals.

Gatifloxacin, an antimicrobial drug belonging to the fluoroquinolone family, is active against Gram-positive and Gram-negative bacteria and is extensively used for the control of infections in humans. The presence of the drug in environmental matrices has already been reported. This study investigated the degradation of gatifloxacin in water by hydroxyl radicals generated by the UV254 nm/H2O2 process ([Formula: see text] 0.4-2.4 mmol L-1) and evaluated the capacity of the radicals to reduce the antimicrobial activity against Gram-positive and Gram-negative bacteria. Acute toxicity assays were performed with Vibrio fischeri, and the degradation products were proposed. The hydroxyl radicals formed in the processes were able to degrade the fluoroquinolone and remove the antimicrobial activity from the aqueous solution. Approximately 97 % gatifloxacin degradation was observed after applying 2.4 mmol L-1 of initial H2O2 concentration and 20 min of UVC254nm irradiation (130 J s-1). The acute toxicity assays showed that the toxicity of the treated solution for V. fischeri increased as the gatifloxacin concentration in the solution decreased.

Toxicity of Intracameral Injection of Fourth-Generation Fluoroquinolones on the Corneal Endothelium.

PURPOSE: The aim of this study was to compare the cellular susceptibility patterns and morphologic changes in the corneal endothelium associated with the use of fourth-generation fluoroquinolones. METHOD: Endothelial susceptibility was assessed through intracameral injection of besifloxacin, gatifloxacin, and moxifloxacin. Human umbilical vein endothelial cells (HUVECs) were used as the standard cellular lineage to assess the quantitative toxicity of each antibiotic solution. Qualitative changes in the morphologic character of the corneal structure and the endothelial layer were generated using a combination of ex vivo and in vivo assays. Experimental assays were conducted in triplicate, and the results were statistically analyzed. RESULTS: At 1 hour of exposure, all HUVECs exposed to antibiotics showed viability above 85%, after 3 hours of exposure to besifloxacin, gatifloxacin, and moxifloxacin, the percentages of viable cells were 68.3 ± 4.0 (P < 0.001), 90.7 ± 4.2 (P < 0.05), and 93.3 ± 1.5 (P > 0.05), respectively. All fluoroquinolones tested showed toxicity to HUVECs, resulting in significant (P < 0.001) loss of cellular viability after 24 hours of drug exposure. Giant endothelial cells were observed in animals treated with the 3 fluoroquinolones in contrast to the absence of these abnormal cells in the untreated group. Early cellular detachment was seen in the endothelial layer after exposure to gatifloxacin and moxifloxacin. CONCLUSIONS: We concluded that injection of fourth-generation fluoroquinolones in the aqueous humor did not adversely affect the corneal endothelium. However, these results suggested that prophylactic intracameral injection of besifloxacin, gatifloxacin, or moxifloxacin, if needed, should be administered as a last therapeutic resource in clinical practice, with careful and constant monitoring of corneal endothelium.

Trovafloxacin enhances lipopolysaccharide-stimulated production of tumor necrosis factor-α by macrophages: role of the DNA damage response.

Trovafloxacin (TVX) is a drug that has caused idiosyncratic, drug-induced liver injury (IDILI) in humans. In a murine model of IDILI, otherwise nontoxic doses of TVX and the inflammagen lipopolysaccharide (LPS) interacted to produce pronounced hepatocellular injury. The liver injury depended on a TVX-induced, small but significant prolongation of tumor necrosis factor-α (TNF) appearance in the plasma. The enhancement of TNF expression by TVX was reproduced in vitro in RAW 264.7 murine macrophages (RAW cells) stimulated with LPS. The current study was designed to identify the molecular target of TVX responsible for this response in RAW cells. An in silico analysis suggested a favorable binding profile of TVX to eukaryotic topoisomerase II-α (TopIIα), and a cell-free assay revealed that TVX inhibited eukaryotic TopIIα activity. Topoisomerase inhibition is known to lead to DNA damage, and TVX increased the DNA damage marker phosphorylated histone 2A.X in RAW cells. Moreover, TVX induced activation of the DNA damage sensor kinases, ataxia telangiectasia mutated (ATM) and Rad3-related (ATR). The ATR inhibitor NU6027 [6-(cyclohexylmethoxy)-5-nitrosopyrimidine-2,4-diamine] prevented the TVX-mediated increases in LPS-induced TNF mRNA and protein release, whereas a selective ATM inhibitor [2-(4-morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one (KU55933)] was without effect. TVX prolonged TNF mRNA stability, and this effect was largely attenuated by NU6027. These results suggest that TVX can inhibit eukaryotic topoisomerase, leading to activation of ATR and potentiation of TNF release by macrophages, at least in part through increased mRNA stability. This off-target effect might contribute to the ability of TVX to precipitate IDILI in humans.

Environmental contamination by fluoroquinolones

Over the past few decades, a high number of pharmaceuticals have been detected in surface, ground and drinking waters. This contamination comes from domestic sewage, livestock, hospitals and chemical-pharmaceutical industries. Typical examples of these pollutants are the fluoroquinolones - powerful antibiotics used in human and veterinary medicine. The presence of fluoroquinolones in the environment can pose a serious threat to the ecosystem and to human health due to their high consumption globally: in 1998, around 120 tons were produced. Even at low environmental concentrations, antibiotics stimulate bacterial resistance. The consequences of the presence of fluoroquinolones in the environment are not fully understood, but are known to be toxic to plants and aquatic organisms. Approximately 85% of the fluoroquinolones present in influents can be removed by conventional wastewater treatment plants, but the removed fraction is frequently accumulated in the sludge, which is sometimes used as fertilizer, representing an additional input route into the environment. The removal of fluoroquinolones by biological treatment is ineffective, and it is believed that only advanced oxidation technologies are able to destroy these emerging pollutants.

Nas últimas décadas, um grande número de fármacos tem sido identificado em águas superficiais, subterrâneas e potáveis. Tal contaminação advém do esgoto doméstico, hospitais, criação de animais e das indústrias químico-farmacêuticas. Exemplos típicos desses poluentes são as fluoroquinolonas – potentes antibióticos empregados na medicina humana e veterinária. A presença de fluoroquinolonas no meio ambiente pode representar uma séria ameaça para o ecossistema e para a saúde humana devido ao alto consumo mundial: em 1998 foram produzidas, aproximadamente, 120 toneladas. Mesmo em baixas concentrações, antibióticos podem estimular a resistência bacteriana. As consequências da presença de fluoroquinolonas no ambiente não são completamente compreendidas, mas sabe-se que são tóxicas para plantas e organismos aquáticos. Aproximadamente 85% das fluoroquinolonas presentes em efluentes podem ser removidos em estações de tratamento de efluentes convencionais, porém a fração removida é frequentemente acumulada no lodo, muitas vezes usado como fertilizante, o que representa uma rota adicional de entrada desses compostos no ambiente. A remoção de fluoroquinolonas por meio de tratamento biológico não é eficiente, e acredita-se que somente as tecnologias de oxidação avançada sejam capazes de degradar esses poluentes emergentes.

Biological safety studies of gemifloxacin mesylate and related substances.

PURPOSE: The aim of this study was to evaluate the cytotoxic, phototoxic, genotoxic and photogenotoxic potential of gemifloxacin mesylate (GFM), its main synthetic impurity (SI) and one isolated and structurally elucidated degradation product (DP). METHODS: The neutral red uptake (NRU) and reduction of 2,5-diphenyl-3,-(4,5-dimethyl-2-thiazolyl)tetrazolium bromide (MTT) assays were performed as in vitro endpoints to evaluate cytotoxicity and phototoxicity in a 3T3 cell line, and predict toxicity and/or phototoxicity after systemic administration of the drug. The in vitro alkaline single-cell electrophoresis (comet) assay was used to evaluate the genotoxic and photogenotoxic potential of the substances using the same cell line. RESULTS: The results showed that the SI and the DP are more cytotoxic and phototoxic than the drug GFM using the 3T3 cell line. In the comet assay, the drug GFM was found to be more genotoxic and photogenotoxic than its related substances. CONCLUSIONS: Our findings highlight the relevance of the biological safety studies to increase the knowledge regarding the toxic potential of the related substances, which can be associated with the drug side effects and toxicity.

Recombinagenic and mutagenic activities of fluoroquinolones in Drosophila melanogaster.

Fluoroquinolones are widely used in human and in veterinary medicine due to their broad-spectrum antibacterial activity. They act by inhibiting type II DNA topoisomerases (gyrase and topoisomerase IV). Because of the sequence homology between prokaryotic and eukaryotic topoisomerases II, fluoroquinolones can pose a hazard to eukaryotic cells. However, published information concerning the genotoxic profiles of these drugs in vivo is sparse and inconsistent. We have assessed the activities of three fluoroquinolones, ciprofloxacin, enrofloxacin and norfloxacin, in the Drosophila melanogaster Somatic Mutation and Recombination Test (SMART) and measured their mutagenic and recombinagenic potentials. Norfloxacin was non-genotoxic. Ciprofloxacin and enrofloxacin induced significant increases in spot frequencies in trans-heterozygous flies. To test the roles of somatic recombination and mutation in the observed genotoxicity, balancer-heterozygous flies were also analyzed. Ciprofloxacin and enrofloxacin were preferential inducers of homologous recombination in proliferative cells, an event linked to loss of heterozygosity.

Intoxicação por enrofloxacina em um cão da raça Pinscher Miniatura: relato de caso / Enrofloxacin poisoning in a Miniature Pinscher: a case report

Uma cadela da raça Pinscher Miniatura foi medicada pelo proprietário com enrofloxacina, na dose de 50mg/kg, uma vez ao dia, por dois dias (dose diária 10 vezes maior que a prescrita). Ao exame clínico o animal apresentou-se deprimido, em cifose lombar, hipotérmico, com mucosas pálidas, dispnéia, sialorréia, vômitos e anúria, evoluindo para parada respiratória, convulsões e coma. Foi realizado tratamento sintomático. O fluxo urinário retornou ao normal em 12 horas e as convulsões foram controladas, mas o animal permaneceu em coma, morrendo 72 horas após o início do tratamento. A necropsia e o histopatológico confirmaram insuficiência renal e hepática agudas, e choque hipovolêmico, compatível com intoxicação por enrofloxacina.(AU)

A Miniature Pinscher bitch was treated by the owner with enrofloxacin at dose of 50 mg/kg, once a day, for two days (daily dose rate 10 times greater than prescribed). Physical examination showed depression, lumbar cifosis, hipotermia, pale mucosa, dispneia, drewling, vomiting and anuria, followed by respiratory failure, seizures, and coma. Symptomatic treatment was performed, and the urinary flow returned to normal in 12 hours, and seizures were controlled. However, the animal stayed in coma, and died 72 hours after the beginning of the treatment. Acute renal and hepatic failure, and hipovolemic shock, compatible with enrofloxacin intoxication, were observed through necropsia and histopatology.(AU)

Intoxicação por enrofloxacina em um cão da raça Pinscher Miniatura: relato de caso / Enrofloxacin poisoning in a Miniature Pinscher: a case report

Uma cadela da raça Pinscher Miniatura foi medicada pelo proprietário com enrofloxacina, na dose de 50mg/kg, uma vez ao dia, por dois dias (dose diária 10 vezes maior que a prescrita). Ao exame clínico o animal apresentou-se deprimido, em cifose lombar, hipotérmico, com mucosas pálidas, dispnéia, sialorréia, vômitos e anúria, evoluindo para parada respiratória, convulsões e coma. Foi realizado tratamento sintomático. O fluxo urinário retornou ao normal em 12 horas e as convulsões foram controladas, mas o animal permaneceu em coma, morrendo 72 horas após o início do tratamento. A necropsia e o histopatológico confirmaram insuficiência renal e hepática agudas, e choque hipovolêmico, compatível com intoxicação por enrofloxacina.

A Miniature Pinscher bitch was treated by the owner with enrofloxacin at dose of 50 mg/kg, once a day, for two days (daily dose rate 10 times greater than prescribed). Physical examination showed depression, lumbar cifosis, hipotermia, pale mucosa, dispneia, drewling, vomiting and anuria, followed by respiratory failure, seizures, and coma. Symptomatic treatment was performed, and the urinary flow returned to normal in 12 hours, and seizures were controlled. However, the animal stayed in coma, and died 72 hours after the beginning of the treatment. Acute renal and hepatic failure, and hipovolemic shock, compatible with enrofloxacin intoxication, were observed through necropsia and histopatology.