Biotransformation of diclofenac by Stenotrophomonas humi strain DIC_5 and toxicological examination of the resulting metabolites.

The widely used non-steroidal anti-inflammatory drug, diclofenac, detected in increasing concentrations in freshwater ecosystems, is among the most pressing environmental problems today. In this study, the bacterial isolate Stenotrophomonas humi strain DIC_5 was capable of degrading diclofenac. It eliminated 75.1% of diclofenac at an initial concentration of 1.5 mg/L after 8 days in the presence of glucose (3.0 g/L). During the process, nitro-diclofenac was identified as a resulting metabolite, whose concentration increased significantly in the bacterial medium from the 7th day of the experiment, while the concentration of diclofenac decreased correspondingly. The ecotoxicological tests on Aliivibrio fischeri and zebrafish embryos showed that the bacterial metabolites without diclofenac have a higher toxicity (up to 35.5% bacterial bioluminescence inhibition and 36.7% embryo mortality) than the diclofenac degradation residues (28% and 26.7%, respectively). Based on these results, neither diclofenac nor its degradation products exhibit toxic effects on the test organisms. Conversely, the toxic effect caused by the bacteria was reduced in the presence of diclofenac. Our work highlights the importance of using biotic controls in biotransformation trials, especially when the foreign material is applied in intermediate or environmentally relevant concentration ranges. KEY POINTS: • Biotransformation of diclofenac by bacteria isolated from a bacterial biofilm. • Biotransformation of diclofenac led to the formation of nitro-diclofenac. • Microorganisms are alternatives for reducing the concentration of diclofenac in water.

Response of crop seed germination and primary root elongation to a binary mixture of diclofenac and naproxen.

Non-steroidal anti-inflammatory drugs, diclofenac (DCF) and naproxen (NPX), represent a group of environmental contaminants often detected in various water and soil samples. This work aimed to assess possible phytotoxic effects of DCF and NPX in concentrations 0.1, 1 and 10 mg/L, both individually and in binary mixtures, on the seed germination and primary root elongation of crops, monocots Allium porrum and Zea mays, and dicots Lactuca sativa and Pisum sativum. Results proved that the seed germination was affected by neither individual drugs nor their mixture. The response of primary root length in monocot and dicot species to the same treatment was different. The Inhibition index (%) comparing the root length of drug-treated plants to controls proved to be approximately 10% inhibition in the case of dicots lettuce and pea, and nearly 20% inhibition in monocot leek, but almost 20% stimulation in monocot maize. Assessment of the binary mixture effect confirmed neither synergistic nor antagonistic interaction of DCF and NPX on early plant development in the applied concentration range.

Elevated temperature exacerbates pharmaceutical-induced oxidative stress in zebrafish (Danio rerio) larvae.

There is growing evidence that rising global temperatures resulting from climate change may exacerbate the toxic effect of pollutants and heterotherms, including fish, in which homestatic mechanisms are directly influenced by environmental temperature will be most affected. Pharmaceuticals discharged into the environment are potentially harmful to wildlife as many of their drug targets are conserved across divergent phyla. Oxidative stress (OS) is a major mechanism by which many pharmaceutical contaminants can induce toxicity but this has received little consideration in the context of effects in wildlife. Further, these mechanisms are relatively poorly understood, particularly regarding multiple stressor interactions. We used transgenic TG(EpRE:mCherry) zebrafish, developed in our laboratory for detecting OS, as our experimental model. We show that the oxidative effects of high concentrations of pharmaceuticals from three different therapeutic classes (paracetamol, diclofenac and doxorubicin) are increased at temperatures elevated by 2-5 °C above those for zebrafish standard husbandry and relevant to their current natural environment (and predicted under the IPCC 2023 scenarios for intermediate to very high greenhouse gas emissions). These OS responses were primarily seen in the pronephros, liver, and gastrointestinal tract. The increase in OS at the increased water temperature may have resulted from the elevated temperature acting as a direct additive physiological stressor to the OS imposed by the drugs and/or via the temperature increasing the chemicals oxidative effect. For paracetamol, it appeared that the elevated responses at the higher temperature of 33 °C were in part due to an increase in uptake of the drug. Our data illustrate that risk assessments for chemicals inducing OS in fish (and likely other heterotherms) should consider the influence of temperature to ensure environmental protection in future environments.

Mitochondria dysfunction is one of the causes of diclofenac toxicity in the green alga Chlamydomonas reinhardtii.

Background: Non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac (DCF), form a significant group of environmental contaminants. When the toxic effects of DCF on plants are analyzed, authors often focus on photosynthesis, while mitochondrial respiration is usually overlooked. Therefore, an in vivo investigation of plant mitochondria functioning under DCF treatment is needed. In the present work, we decided to use the green alga Chlamydomonas reinhardtii as a model organism. Methods: Synchronous cultures of Chlamydomonas reinhardtii strain CC-1690 were treated with DCF at a concentration of 135.5 mg × L-1, corresponding to the toxicological value EC50/24. To assess the effects of short-term exposure to DCF on mitochondrial activity, oxygen consumption rate, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS) production were analyzed. To inhibit cytochrome c oxidase or alternative oxidase activity, potassium cyanide (KCN) or salicylhydroxamic acid (SHAM) were used, respectively. Moreover, the cell's structure organization was analyzed using confocal microscopy and transmission electron microscopy. Results: The results indicate that short-term exposure to DCF leads to an increase in oxygen consumption rate, accompanied by low MMP and reduced mtROS production by the cells in the treated populations as compared to control ones. These observations suggest an uncoupling of oxidative phosphorylation due to the disruption of mitochondrial membranes, which is consistent with the malformations in mitochondrial structures observed in electron micrographs, such as elongation, irregular forms, and degraded cristae, potentially indicating mitochondrial swelling or hyper-fission. The assumption about non-specific DCF action is further supported by comparing mitochondrial parameters in DCF-treated cells to the same parameters in cells treated with selective respiratory inhibitors: no similarities were found between the experimental variants. Conclusions: The results obtained in this work suggest that DCF strongly affects cells that experience mild metabolic or developmental disorders, not revealed under control conditions, while more vital cells are affected only slightly, as it was already indicated in literature. In the cells suffering from DCF treatment, the drug influence on mitochondria functioning in a non-specific way, destroying the structure of mitochondrial membranes. This primary effect probably led to the mitochondrial inner membrane permeability transition and the uncoupling of oxidative phosphorylation. It can be assumed that mitochondrial dysfunction is an important factor in DCF phytotoxicity. Because studies of the effects of NSAIDs on the functioning of plant mitochondria are relatively scarce, the present work is an important contribution to the elucidation of the mechanism of NSAID toxicity toward non-target plant organisms.

Thunbergia's Flowers Secondary Metabolites a Natural Armor Against Kidney Damage by Diclofenac.

Nephrotoxicity is a common side effect arising from exposure to drugs or toxins. The study investigates the therapeutic effects of Thunbergia alata and Thunbergia erecta flowers on diclofenac-induced renal injury. Secondary metabolite characterization by positive mode high-resolution-ESI (LC-HR-ESI-MS) was followed by assessing their renal protection against diclofenac-induced damage and molecular docking studies. Using positive LC-HR-ESI-MS, 18 compounds from T. erecta and T. alata were identified. Diclofenac administration induced significant deterioration of all parameters in the kidney in addition to renal tissue contents of several inflammatory markers. The flower extracts of T. alata and T. erecta showed a clear improvement in the treated groups compared to the diclofenac-control group. The results were confirmed by histopathological examinations followed by immunohistochemical determination of vascular endothelial growth factor (VEGF), nuclear factor erythroid 2-related factor 2 (Nrf2), and transforming growth factor beta 1 (TGF-ß1) expression. Furthermore, a protein-protein network to understand the complex interplay between the target proteins and their counterparts was done in addition to a molecular docking study of the de-replicated compounds in the active sites of NF-κB, TGF-ß1, and VEGFR.

Proteome profiling, biochemical and histological analysis of diclofenac-induced liver toxicity in Yersinia enterocolitica and Lactobacillus fermentum fed rat model: a comparative analysis.

Diclofenac is a hepatotoxic non-steroidal anti-inflammatory drug (NSAID) that affects liver histology and its protein expression levels. Here, we studied the effect of diclofenac on rat liver when co-administrated with either Yersinia enterocolitica strain 8081 serotype O:8 biovar 1B (D*Y) or Lactobacillus fermentum strain 9338 (D*L). Spectroscopic analysis of stool samples showed biotransformation of diclofenac. When compared with each other, D*Y rats lack peaks at 1709 and 1198 cm-1, while D*L rats lack peaks at 1411 cm-1. However, when compared to control, both groups lack peaks at 1379 and 1170 cm-1. Assessment of serum biomarkers of hepatotoxicity indicated significantly altered activities of AST (D*Y: 185.65 ± 8.575 vs Control: 61.9 ± 2.607, D*L: 247.5 ± 5.717 vs Control: 61.9 ± 2.607), ALT (D*Y: 229.8 ± 6.920 vs Control: 70.7 ± 3.109, D*L: 123.75 ± 6.068 vs Control: 70.7 ± 3.109), and ALP (D*Y: 276.4 ± 18.154 vs Control: 320.6 ± 9.829, D*L: 298.5 ± 12.336 vs Control: 320.6 ± 9.829) in IU/L. The analysis of histological alterations showed hepatic sinusoidal dilation with vein congestion and cell infiltration exclusively in D*Y rats along with other histological changes that are common to both test groups, thereby suggesting more pronounced alterations in D*Y rats. Further, LC-MS/MS based label-free quantitation of proteins from liver tissues revealed 74.75% up-regulated, 25.25% down-regulated in D*Y rats and 51.16% up-regulated, 48.84% down-regulated in D*L experiments. The proteomics-identified proteins majorly belonged to metabolism, apoptosis, stress response and redox homeostasis, and detoxification and antioxidant defence that demonstrated the potential damage of rat liver, more pronounced in D*Y rats. Altogether the results are in favor that the administration of lactobacilli somewhat protected the rat hepatic cells against the diclofenac-induced toxicity.

Functional knockout of the Oatp1d1 membrane transporter affects toxicity of diclofenac in zebrafish embryos.

Organic anion transporting polypeptides (OATPs) facilitate the cellular uptake of a large number of compounds. Zebrafish Oatp1d1 matches the functional capabilities of human OATP orthologs, particularly in hormone and drug transport. It is highly expressed in the liver and later stages of embryonic development, indicating its critical role in zebrafish physiology and development. Data from previous in vitro analyses have shown a high affinity of zebrafish Oatp1d1 for pharmaceuticals and xenobiotics, providing the basis for further in vivo studies on its defence and developmental functions. Using CRISPR-Cas9 technology, we have generated an Oatp1d1 zebrafish mutant that has highly reduced Oatp1d1 expression in embryos and adult tissues compared to wild type (WT). The absence of Oatp1d1 was confirmed using custom-made antibodies. To evaluate its ecotoxicological relevance, mutant and WT embryos were exposed to increasing concentrations of diclofenac, an NSAID known for its wide and frequent use, environmental pseudo-persistence and ecological implications. WT embryos showed developmental delays and malformations such as spinal curvature, cardiac edema and blood pooling at higher diclofenac concentrations, whereas the Oatp1d1 mutant embryos showed marked resilience, with milder developmental defects and delayed toxic effects. These observations suggest that the absence of Oatp1d1 impedes the efficient entry of diclofenac into hepatocytes, thereby slowing its biotransformation into potentially more toxic metabolites. In addition, the changes in transcript expression of other uptake transporters revealed a highly probable and complex network of compensatory mechanisms. Therefore, the results of this study point to the importance of Oatp1d1-mediated transport of diclofenac, as demonstrated for the first time in vivo using an Oatp1 deficient zebrafish line. Finally, our data indicates that the compensatory role of other transporters with overlapping substrate preferences needs to be considered for a reliable understanding of the physiological and/or defensive role(s) of membrane transporters.

Safety Assessment of a Novel Intravenous Diclofenac Sodium Formulation Following 28-Day Repeated Administration in Wistar Rats.

These toxicity studies aimed to assess the safety and tolerability of a novel intravenous diclofenac sodium (37.5 mg/mL) formulation containing povidone K12 (80 mg/mL) as the key excipient in Wistar rats. This formulation was tested at doses of 3, 7, and 15 mg/kg/day and was administered daily for 28 days by intravenous route. Toxicokinetic estimation revealed a dose-proportional increase in plasma exposure to diclofenac. The formulation was well tolerated in males; however, mortality was observed in females (2/15) at the highest dose (15 mg/kg/day). Adverse gastrointestinal events related to NSAIDS and a few other treatment-related effects on clinical and anatomic pathology were noted at the 15 mg/kg/day dose, which normalized at the end of the 2-week recovery period. In addition, the excipient povidone K12 was present in a higher amount than the approved Inactive Ingredient Database (IID) limit in the proposed novel formulation. It was qualified through a separate 28-day repeated dose toxicity study by intravenous route in Wistar rats. Povidone K12 was found to be well tolerated and safe up to a dose of 165 mg/kg/day. No treatment-related adverse effects were observed in this study. In conclusion, repeated administration of a novel intravenous formulation containing diclofenac sodium was found to be safe up to the dose of 7 mg/kg/day in female rats and 15 mg/kg/day in male rats.

Melatonin protects against diclofenac induced oxidative stress mediated myocardial toxicity in rats: A mechanistic insight.

Diclofenac, a traditional non-steroidal anti-inflammatory drug, is commonly used for treating chronic pain and inflammation. Recently, a number of articles have highlighted the toxicities associated with diclofenac. The current study explores the molecular mechanism of diclofenac induced cardiac toxicity following oxidative stress. Diclofenac inhibits catalase, disrupts the redox balance in cardiac tissue, accelerates the monoamine oxidase induced hydroperoxide generation and eventually inhibits crucial mitochondrial enzyme, viz., aldehyde dehydrogenase, thereby causing myocardial injury. Melatonin, the pineal indoleamine with high antioxidative efficacy, is well known for its cardio-protective properties and its dietary consumption has profound impact on cardiac health. The present study demonstrates perhaps for the first time, that apart from ameliorating oxidative load in the cardiac tissue, melatonin also attenuates the inhibition of catalase and aldehyde dehydrogenase, and prevents stress mediated stimulation of monoamine oxidase. Moreover, favourable binding of diclofenac with melatonin may protect the myocardium from the deleterious effects of this drug. The results indicate toward a novel mechanism of protection by melatonin, having future therapeutic relevance.

Transcriptional and biochemical changes in mouse liver following exposure to a metal/drug cocktail. Attenuating effect of a selenium-enriched diet.

Real-life pollution usually involves simultaneous co-exposure to different chemicals. Metals and drugs are frequently and abundantly released into the environment, where they interact and bioaccumulate. Few studies analyze potential interactions between metals and pharmaceuticals in these mixtures, although their joint effects cannot be inferred from their individual properties. We have previously demonstrated that the mixture (PC) of the metals Cd and Hg, the metalloid As and the pharmaceuticals diclofenac (DCF) and flumequine (FLQ) impairs hepatic proteostasis. To gain a deeper vision of how PC affects mouse liver homeostasis, we evaluated here the effects of PC exposure upon some biochemical and morphometric parameters, and on the transcriptional profiles of selected group of genes. We found that exposure to PC caused oxidative damage that exceeded the antioxidant capacity of cells. The excessive oxidative stress response resulted in an overabundance of reducing equivalents, which hindered the metabolism and transport of metabolites, including cholesterol and bile acids, between organs. These processes have been linked to metabolic and inflammatory disorders, cancer, and neurodegenerative diseases. Therefore, our findings suggest that unintended exposure to mixtures of environmental pollutants may underlie the etiology of many human diseases. Fortunately, we also found that a diet enriched with selenium mitigated the harmful effects of this combination of toxicants.

Mytilus galloprovincialis: A valuable bioindicator species for understanding the effects of diclofenac under warming conditions.

Drugs are chemical compounds used to treat and improve organic dysfunctions caused by diseases. These include analgesics, antibiotics, antidepressants, and antineoplastics. They can enter aquatic environments through wastewater streams, where their physico-chemical properties allow metabolites to distribute and accumulate. Current climate change and associated extreme weather events may significantly impact these substances' toxicity and aquatic organisms' sensitivity. Among the chemicals present in aquatic environments is the non-steroidal anti-inflammatory drug diclofenac (DIC), which the EU monitors due to its concentration levels. This study investigated the influence of temperature (control at 17 °C vs. 21 °C) on the effects of DIC (0 µg/L vs. 1 µg/L) in the mussel species Mytilus galloprovincialis. Significant results were observed between 17 and 21 °C. Organisms exposed to the higher temperature showed a decrease in several parameters, including metabolic capacity and detoxification, particularly with prolonged exposure. However, in some parameters, after 21 days, the M. galloprovincialis showed no differences from the control, indicating adaptation to the stress. The results of this study confirm that DIC concentrations in the environment, particularly when combined with increased temperatures, can produce oxidative stress and adversely affect M. galloprovincialis biochemical and physiological performance. This study also validates this species as a bioindicator for assessing environmental contamination with DIC. Beyond its direct impact on aquatic organisms, the presence of pharmaceuticals like DIC in the environment highlights the interconnectedness of human, animal, and ecosystem health, underscoring the One Health approach to understanding and mitigating environmental pollution.

Electrochemical degradation of diclofenac generates unexpected thyroidogenic transformation products: Implications for environmental risk assessment.

Diclofenac (DCF) is an environmentally persistent, nonsteroidal anti-inflammatory drug (NSAID) with thyroid disrupting properties. Electrochemical advanced oxidation processes (eAOPs) can efficiently remove NSAIDs from wastewater. However, eAOPs can generate transformation products (TPs) with unknown chemical and biological characteristics. In this study, DCF was electrochemically degraded using a boron-doped diamond anode. Ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry was used to analyze the TPs of DCF and elucidate its potential degradation pathways. The biological impact of DCF and its TPs was evaluated using the Xenopus Eleutheroembryo Thyroid Assay, employing a transgenic amphibian model to assess thyroid axis activity. As DCF degradation progressed, in vivo thyroid activity transitioned from anti-thyroid in non-treated samples to pro-thyroid in intermediately treated samples, implying the emergence of thyroid-active TPs with distinct modes of action compared to DCF. Molecular docking analysis revealed that certain TPs bind to the thyroid receptor, potentially triggering thyroid hormone-like responses. Moreover, acute toxicity occurred in intermediately degraded samples, indicating the generation of TPs exhibiting higher toxicity than DCF. Both acute toxicity and thyroid effects were mitigated with a prolonged degradation time. This study highlights the importance of integrating in vivo bioassays in the environmental risk assessment of novel degradation processes.

Impact of cadmium and diclofenac exposure on biochemical responses, transcriptome, gut microflora, and growth performance in grass carp (Ctenopharyngodonidella).

In recent years, the concentrations of cadmium (Cd) and diclofenac (DCF) in water have frequently exceeded the standard; however, the toxic effects of these two pollutants on grass carp under single and combined exposure are unknown. In this study, the concentrations of pollutants in different tissues were detected, and the toxicities of the two pollutants to grass carp under different exposure conditions were compared based on growth traits, biochemical responses, gut microbiome, and transcriptomes. Based on these findings, the brain showed the lowest levels of Cd and DCF accumulation. Oxidative stress and pathological damage were observed in the brain and intestines. Changes in the structure and abundance of the gut microflora affect the synthesis of neurotransmitters, such as GABA and steroids. Differentially expressed genes in the brain were enriched in circadian rhythm functions. The expression of PER, CLOCK,1L-1ß, 1L-17, and other genes are related to the abundance of Akkermansia, which indicates that the disorder of gut microflora will affect the normal circadian rhythm of the brain. All indices in the recovery group showed an increasing trend. Overall, the toxicity of Cd and DCF showed antagonism, and a single exposure had a stronger effect on gut microorganisms and circadian rhythm, which provided a scientific basis for exploring the comprehensive effects of different pollutants.

The Importance of Including Variable Exposure Concentrations When Assessing Toxicity of Sediment-Associated Pharmaceuticals to an Amphipod.

Pharmaceuticals have been classified as an environmental concern due to their increasing consumption globally and potential environmental impact. We examined the toxicity of sediment-associated diclofenac and citalopram administered as both single compounds and in a mixture to the sediment-living amphipod Corophium volutator. This laboratory-based study addressed the following research questions: (1) What is the toxicity of sediment-associated diclofenac and citalopram to C. volutator? (2) Can the mixture effect be described with either of the two mixture models: concentration addition (CA) or independent action (IA)? (3) What is the importance of the choice of (i) exposure measure (start concentration, time-weighted average [TWA], full exposure profile) and (ii) effect model (concentration-response vs. the toxicokinetic-toxicodynamic model general unified threshold model for survival in its reduced form [GUTS-RED]) for the derived effect concentration values? Diclofenac was more toxic than citalopram to C. volutator as a single compound (10-day exposure). Diclofenac exposure to C. volutator provided median lethal concentrations (LC50s) within the same range (11 µg g-1 dry wt sediment) using concentration-response based on TWA and both GUTS-RED models. However, concentration-response based on measured start concentrations provided an approximately 90% higher LC50 (21.6 ± 2.0 µg g-1 dry wt sediment). For citalopram, concentration-response parameters were similar regardless of model or concentration used (LC50 85-97 µg g-1 dry wt sediment), however, GUTS-RED with the assumption of individual tolerance resulted in a lower LC50 (64.9 [55.3-74.8] µg g-1 dry wt sediment). The mixture of diclofenac and citalopram followed the CA quite closely, whereas the result was synergistic when using the IA prediction. In summary, concentration-response based on TWA and GUTS-RED provided similar and reasonably good fits compared to the data set. The implications are that GUTS-RED will provide a more flexible model, which, in principle, can extend beyond the experimental period and make predictions based on variable exposure profiles (toxicity at different time frames and at different variable exposure scenarios) compared to concentration-response, which provides contaminant toxicity at one point in time. Environ Toxicol Chem 2024;43:1767-1777. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Removal of pharmaceutical compounds and toxicology study in wastewater using Malaysian fungal Ganoderma lucidum.

Elevated usage of pharmaceutical products leads to the accumulation of emerging contaminants in sewage. In the current work, Ganoderma lucidum (GL) was used to remove pharmaceutical compounds (PCs), proposed as a tertiary method in sewage treatment plants (STPs). The PCs consisted of a group of painkillers (ketoprofen, diclofenac, and dexamethasone), psychiatrists (carbamazepine, venlafaxine, and citalopram), beta-blockers (atenolol, metoprolol, and propranolol), and anti-hypertensives (losartan and valsartan). The performance of 800 mL of synthetic water, effluent STP, and hospital wastewater (HWW) was evaluated. Parameters, including treatment time, inoculum volume, and mechanical agitation speed, have been tested. The toxicity of the GL after treatment is being studied based on exposure levels to zebrafish embryos (ZFET) and the morphology of the GL has been observed via Field Emission Scanning Electron Microscopy (FESEM). The findings conclude that GL can reduce PCs from <10% to >90%. Diclofenac and valsartan are the highest (>90%) in the synthetic model, while citalopram and propranolol (>80%) are in the real wastewater. GL effectively removed pollutants in 48 h, 1% of the inoculum volume, and 50 rpm. The ZFET showed GL is non-toxic (LC50 is 209.95 mg/mL). In the morphology observation, pellets GL do not show major differences after treatment, showing potential to be used for a longer treatment time and to be re-useable in the system. GL offers advantages to removing PCs in water due to their non-specific extracellular enzymes that allow for the biodegradation of PCs and indicates a good potential in real-world applications as a favourable alternative treatment.

Multi-biomarker response of cyanobacteria Synechocystis salina and Microcystis aeruginosa to diclofenac.

The cyanobacterial response to pharmaceuticals is less frequently investigated compared to green algae. Pharmaceuticals can influence not only the growth rate of cyanobacteria culture, but can also cause changes at the cellular level. The effect of diclofenac (DCF) as one of the for cyanobacteria has been rarely tested, and DCF has never been applied with cellular biomarkers. The aim of this work was to test the response of two unicellular cyanobacteria (Synechocystis salina and Microcystis aeruginosa) toward DCF (100 mg L-1) under photoautotrophic growth conditions. Such endpoints were analyzed as cells number, DCF uptake, the change in concentrations of photosynthetic pigments, the production of toxins, and chlorophyll a in vivo fluorescence. It was noted that during a 96 h exposure, cell proliferation was not impacted. Nevertheless, a biochemical response was observed. The increased production of microcystin was noted for M. aeruginosa. Due to the negligible absorption of DCF into cells, it is possible that the biochemical changes are induced by an external signal. The application of non-standard biomarkers demonstrates the effect of DCF on microorganism metabolism without a corresponding effect on biomass. The high resistance of cyanobacteria to DCF and the stimulating effect of DCF on the secretion of toxins raise concerns for environment biodiversity.

Antagonistic effects of a COX1/2 inhibitor drug in human HepG2 cells exposed to an environmental carcinogen.

Understanding interactions between legacy and emerging environmental contaminants has important implications for risk assessment, especially when mutagens and carcinogens are involved, whose critical effects are chronic and therefore difficult to predict. The current work aimed to investigate potential interactions between benzo[a]pyrene (B[a]P), a carcinogenic polycyclic aromatic hydrocarbon and legacy pollutant, and diclofenac (DFC), a non-steroidal anti-inflammatory drug and pollutant of emerging concern, and how DFC affects B[a]P toxicity. Exposure to binary mixtures of these chemicals resulted in substantially reduced cytotoxicity in human HepG2 cells compared to single-chemical exposures. Significant antagonistic effects were observed in response to high concentrations of B[a]P in combination with DFC at IC50 and ⅕ IC50. While additive effects were found for levels of intracellular reactive oxygen species, antagonistic mixture effects were observed for genotoxicity. B[a]P induced DNA strand breaks, γH2AX activation, and micronuclei formation at ½ IC50 concentrations or lower, whereas DFC induced only low levels of DNA strand breaks. Their mixture caused significantly lower levels of genotoxicity by all three endpoints compared to those expected based on concentration additivity. In addition, antagonistic mixture effects on CYP1 enzyme activity suggested that the observed reduced genotoxicity of B[a]P was due to its reduced metabolic activation as a result of enzymatic inhibition by DFC. Overall, the findings further support the growing concern that co-exposure to environmental toxicants and their non-additive interactions may be a confounding factor that should not be neglected in environmental and human health risk assessment.

Gut-gonad crosstalk in mice exposed to a "chemical cocktail" combining metabolomics and microbial profile by amplicon sequencing.

Testes are very prone to be damaged by environmental pollutants, but there is a lack of information about the impact of "chemical cocktails" (CC) on the testicular metabolome and the possible influence in the gut-gonad crosstalk. For this, BALB/c mice were given flumequine and diclofenac orally in food and potentially toxic trace elements (Cd, Hg, As) in drinking water. A mice group was supplemented with selenium, a well-known antagonist against many pollutants. Our results revealed that the steroid 5-alpha-androstan-17-beta-ol propionate, suggested as a parameter of androgenicity independent of testosterone levels, proline that improves reproductive indicators in male rabbits affected by environmental stress) among others metabolites are only present after CC exposure with rodent and selenium supplemented diet. Selenium also antagonized the up-or down-regulation of anandamide (20:l, n-9) (p < 0.001 and FC 0.54 of CC vs C but p > 0,05 and FC 0.74 of CC-Se vs C), that regulates gonadotropin-releasing hormones in mammals, 2,3-dinor-11b-PGF2a (p < 0.001 and FC 0.12 of CC vs C but p > 0,05 and FC 0.34 of CC-Se vs C), which has been related with reproductive hormones, besides others testicular metabolites altered by the exposure to the CC and reversed the levels to control. Moreover, numerous significant associations between gut microbes and testicular metabolites indicated a possible impact of pollutants in the testes mediated by gut microbiota due to a gut-gonad crosstalk.

Polystyrene nanoplastics synergistically exacerbate diclofenac toxicity in embryonic development and the health of adult zebrafish.

In this study, we investigated the possible ecotoxicological effect of co-exposure to polystyrene nanoplastics (PS-NPs) and diclofenac (DCF) in zebrafish (Danio rerio). After six days of exposure, we noticed that the co-exposure to PS-NP (100 µg/L) and DCF (at 50 and 500 µg/L) decreased the hatching rate and increased the mortality rate compared to the control group. Furthermore, we noted that larvae exposed to combined pollutants showed a higher frequency of morphological abnormalities and increased oxidative stress, apoptosis, and lipid peroxidation. In adults, superoxide dismutase and catalase activities were also impaired in the intestine, and the co-exposure groups showed more histopathological alterations. Furthermore, the TNF-α, COX-2, and IL-1ß expressions were significantly upregulated in the adult zebrafish co-exposed to pollutants. Based on these findings, the co-exposure to PS-NPs and DCF has shown an adverse effect on the intestinal region, supporting the notion that PS-NPs synergistically exacerbate DCF toxicity in zebrafish.

Systemic absorption and gastrointestinal adverse effects from topical ketorolac and diclofenac ophthalmic solutions in healthy dogs.

OBJECTIVE: To investigate systemic absorption and gastrointestinal (GI) adverse effects of topical ketorolac 0.5% and diclofenac 0.1% ophthalmic solutions. ANIMALS: 11 healthy purpose-bred Beagles. METHODS: Dogs were randomly assigned to receive either ketorolac (n = 6) or diclofenac (5), 1 drop in both eyes 4 times daily for 28 days. Upper GI endoscopy was performed on days 0 and 29 with mucosal lesion scores (0 to 7) assigned to each region evaluated. Plasma samples were collected on days 14, 21, and 28 for measurement of diclofenac and ketorolac using high-performance liquid chromatography-mass spectrometry. RESULTS: GI erosions and/or ulcers developed in all ketorolac-treated dogs and 1 of 5 diclofenac-treated dogs. Post-treatment mucosal lesion score for the antrum was higher in the ketorolac group than in the diclofenac group (P = .006) but not significantly different for any other region. Post-treatment antral mucosal lesion scores were significantly related to plasma ketorolac concentrations (P < .001). Ketorolac and diclofenac were detected in the plasma at all time points (median ketorolac day 14, 191 ng/mL; day 21, 173.5 ng/mL; and day 28, 179.5 ng/mL; and median diclofenac day 14, 21.1 ng/mL; day 21, 20.6 ng/mL; day 28, 27.5 ng/mL). Vomiting and decreased appetite events were observed uncommonly and were not significantly different between treatment groups. CLINICAL RELEVANCE: GI ulceration and erosion developed after ophthalmic administration of ketorolac and diclofenac, with higher plasma concentrations and more severe GI lesions associated with ketorolac. Clients should be alerted to this potential risk with ophthalmic use and informed to watch for systemic clinical signs that would warrant veterinary reevaluation.