Human Therapeutic Plasma Concentrations Research Articles

Exposure effects of synthetic glucocorticoid drugs on skeletal developmental and immune cell function in zebrafish

Synthetic glucocorticoids (GCs) are used to treat a wide range of human health conditions and as such are frequently detected in the aquatic environment. This, together with the highly conserved nature of the glucocorticoid system across vertebrates means that the potential for biological effects of GCs in fish is relatively high. Here, we found that exposure of zebrafish (Danio rerio) to environmentally relevant concentrations of 4 of the most widely used synthetic GCs (beclomethasone dipropionate, budesonide, fluticasone propionate, and prednisolone), from 0 to 4 days post fertilisation (dpf), resulted in no effects on embryo-larval development or bone and cartilage formation. However, after exposure to equivalents of human therapeutic plasma levels, developmental abnormalities were observed that included pericardial oedema, blood pooling and alterations in jaw cartilage. Furthermore, using a double transgenic zebrafish osteoblast and chondrocyte reporter line, exposure up to 10 dpf resulted in alterations to lower jaw cartilage and bone development for all compounds at, and above, human therapeutic plasma concentrations. In the case of beclomethasone dipropionate, a reduction in lower jaw intercranial distance was observed at the environmentally relevant concentration of 0.1 μg/L. Using further transgenic reporter lines with fluorescently tagged neutrophils and macrophages, we also show exposure of embryo-larvae (0–4 dpf) to the GCs tested resulted in altered immune cell migration, but only at relatively high exposure concentrations. Collectively, our findings show GC exposure impacts embryo-larval zebrafish development, immune function, and skeletal formation, but predominantly at concentrations greater than those currently reported for the aquatic environment. Despite this, however, it is suggested that studies with longer exposure times, and to mixtures of multiple GCs (many GCs act via the same mechanism of action) are warranted before we can confidently assert that these commonly detected contaminants do not pose a risk to fish in the wild.

Understanding pharmaceutical exposure and the potential for effects in marine biota: A survey of bonefish (Albula vulpes) across the Caribbean Basin

Most research on pharmaceutical presence in the environment to date has focused on smaller scale assessments of freshwater and riverine systems, relying mainly on assays of water samples, while studies in marine ecosystems and of exposed biota are sparse. This study investigated the pharmaceutical burden in bonefish (Albula vulpes), an important recreational and artisanal fishery, to quantify pharmaceutical exposure throughout the Caribbean Basin. We sampled 74 bonefish from five regions, and analyzed them for 102 pharmaceuticals. We assessed the influence of sampling region on the number of pharmaceuticals, pharmaceutical assemblage, and risk of pharmacological effects. To evaluate the risk of pharmacological effects at the scale of the individual, we proposed a metric based on the human therapeutic plasma concentration (HTPC), comparing measured concentrations to a threshold of 1/3 the HTPC for each pharmaceutical. Every bonefish had at least one pharmaceutical, with an average of 4.9 and a maximum of 16 pharmaceuticals in one individual. At least one pharmaceutical was detected in exceedance of the 1/3 HTPC threshold in 39% of bonefish, with an average of 0.6 and a maximum of 11 pharmaceuticals exceeding in a Key West individual. The number of pharmaceuticals (49 detected in total) differed across regions, but the risk of pharmacological effects did not (23 pharmaceuticals exceeded the 1/3 HTPC threshold). The most common pharmaceuticals were venlafaxine (43 bonefish), atenolol (36), naloxone (27), codeine (27), and trimethoprim (24). Findings suggest that pharmaceutical detections and concentration may be independent, emphasizing the need to monitor risk to biota regardless of exposure diversity, and to focus on risk quantified at the individual level. This study supports the widespread presence of pharmaceuticals in marine systems and shows the utility of applying the HTPC to assess the potential for pharmacological effects, and thus quantify impact of exposure at large spatial scales.

The Potential Mechanisms behind Loperamide-Induced Cardiac Arrhythmias Associated with Human Abuse and Extreme Overdose.

Loperamide has been a safe and effective treatment for diarrhea for many years. However, many cases of cardiotoxicity with intentional abuse of loperamide ingestion have recently been reported. We evaluated loperamide in in vitro and in vivo cardiac safety models to understand the mechanisms for this cardiotoxicity. Loperamide slowed conduction (QRS-duration) starting at 0.3 µM [~1200-fold (×) its human Free Therapeutic Plasma Concentration; FTPC] and reduced the QT-interval and caused cardiac arrhythmias starting at 3 µM (~12,000× FTPC) in an isolated rabbit ventricular-wedge model. Loperamide also slowed conduction and elicited Type II/III A-V block in anesthetized guinea pigs at overdose exposures of 879× and 3802× FTPC. In ion-channel studies, loperamide inhibited hERG (IKr), INa, and ICa currents with IC50 values of 0.390 µM, 0.526 µM, and 4.091 µM, respectively (i.e., >1560× FTPC). Additionally, in silico trials in human ventricular action potential models based on these IC50s confirmed that loperamide has large safety margins at therapeutic exposures (≤600× FTPC) and confirmed repolarization abnormalities in the case of extreme doses of loperamide. The studies confirmed the large safety margin for the therapeutic use of loperamide but revealed that at the extreme exposure levels observed in human overdose, loperamide can cause a combination of conduction slowing and alterations in repolarization time, resulting in cardiac proarrhythmia. Loperamide's inhibition of the INa channel and hERG-mediated IKr are the most likely basis for this cardiac electrophysiological toxicity at overdose exposures. The cardiac toxic effects of loperamide at the overdoses could be aggravated by co-medication with other drug(s) causing ion channel inhibition.

Effects of carbamazepine on the central nervous system of zebrafish at human therapeutic plasma levels

The fish plasma model (FPM) facilitated the environmental risk assessment of human drugs by using existing data on human therapeutic plasma concentrations (HTPCs) and predicted fish plasma concentrations (FPCs). However, studies on carbamazepine (CMZ) with both the mode of action (MOA) based biological effects at molecular level (such as neurotransmitter and gene level) and measured FPCs are lacking. Bioconcentration of CMZ in adult zebrafish demonstrated that the FPM underestimated the bioconcentration factors (BCFs) in plasma at environmental CMZ exposure concentrations (1-100μg/L). CMZ significantly increased Glu and GABA, decreased ACh and AChE as well as inhibited the transcription levels of gabra1, grin1b, grin2b, gad1b, and abat when the actual FPCs were in the ranges of 1/1000 HTPC to HTPC. It is the first read-across study of CMZ integrating MOA-based biological effects at molecular level and FPCs. This study facilitates model performance against a range of different drug classes.

Accumulation and risk prioritization of psychoactive substances in the critically endangered Yangtze finless porpoise.

Psychoactive substances have been identified as a kind of emerging contaminants in aquatic environment and pose potential adverse effects on aquatic animals. Yangtze finless porpoise, a critically endangered species in China, is also facing the threat of psychoactive substances. In this study, the accumulation characteristics and risk prioritization of psychoactive substances were investigated in Yangtze finless porpoise collected from Poyang Lake (PYL) and Tian-E-Zhou Oxbow (TZO) in Yangtze River basin. The levels of psychoactive substances were detected in the range of below method detection limits (MDLs) to 98.4ng/mL in the serum of Yangtze finless porpoise. Codeine (COD) and methamphetamine were identified as the major substances due to the highest residual levels with a median concentration of 0.72ng/mL and 0.33ng/mL, respectively. The total concentrations of psychoactive substances in the porpoise collected from TZO was significantly higher than those from PYL. Risk analysis based on effect ratio derived from the ratio of steady-state psychoactive substance serum concentration in the porpoise and human therapeutic plasma concentration revealed that COD was the substance with the highest risk among the psychoactive substances detected, followed by lysergic acid diethylamide (LSD), morphine, alprazolam (ALPZ) and lormetazepam. Location-specific risk prioritization of psychoactive substances found that the top 3 substances are LSD, lorazepam (LORZ) and ALPZ in PYL, and COD, LSD and LORZ in TZO. The results disclose the accumulation of psychoactive substances in Yangtze finless porpoise and suggest that the potential adverse effects should be concerned.

Salidroside and isorhamnetin attenuate urotensin II-induced inflammatory response in vivo and in vitro: Involvement in regulating the RhoA/ROCK II pathway.

Urotensin II (UII), a vital vasoconstrictor peptide, causes an inflammatory response in the pathogenesis of atherosclerosis. Previous studies have reported that the Ras homolog gene family, member A (RhoA)/Rho kinases (ROCK) pathway modulates the inflammatory response of the atherosclerotic process. However, to the best of our knowledge, whether the RhoA/ROCK pathway mediates the inflammatory effect of UII has not been previously elucidated. Salidroside and isorhamnetin are two early developed antioxidant Tibetan drugs, both displaying cardioprotective effects against atherosclerosis. Therefore, the aim of the present study was to investigate the protective effects of salidroside, isorhamnetin or combination of these two drugs on the UII-induced inflammatory response in vivo (rats) or in vitro [primary vascular smooth muscle cells (VSMCs)], as well as to examine the role of the RhoA/ROCK pathway in these processes. The levels of inflammatory markers were measured via ELISA. The mRNA and protein expression levels of RhoA and ROCK II were detected using reverse transcription-quantitative PCR assay and western blot analysis. It was demonstrated that salidroside, isorhamnetin and both in combination decreased the levels of the serum pro-inflammatory cytokines TNF-α and IL-1β, as well as increased the levels of the anti-inflammatory cytokine IL-10 and macrophage migration inhibitory factor in rats with subacute infusion of UII and in the culture supernatant from primary VSMCs-exposed to UII. Moreover, salidroside, isorhamnetin and both in combination attenuated the mRNA and protein expression levels of RhoA and ROCK II in vivo and in vitro, at concentrations corresponding to human therapeutic blood plasma concentrations. Thus, these drugs could inhibit the RhoA/ROCK II pathway under UII conditions. The combination of salidroside and isorhamnetin did not display a stronger inhibitory effect on the inflammatory response and the RhoA/ROCK II pathway compared with salidroside and isorhamnetin in isolation. Collectively, the results indicated that salidroside, isorhamnetin and both in combination inhibited the RhoA/ROCK II pathway, which then attenuated the inflammatory response under UII-induced conditions, resulting in cardioprotection in atherosclerosis.

Trout and Human Plasma Protein Binding of Selected Pharmaceuticals Informs the Fish Plasma Model.

Concerns are increasing that pharmaceuticals released into the environment pose a risk to nontarget organism such as fish. The fish plasma model is a read-across approach that uses human therapeutic blood plasma concentrations for estimating likely effects in fish. However, the fish plasma model neglects differences in plasma protein binding between fish and humans. Because binding data for fish plasma are scarce, the binding of 12 active pharmaceutical ingredients (APIs; acidic, basic, and neutral) to rainbow trout (Oncorhynchus mykiss) and human plasma was measured using solid-phase microextraction (SPME). The plasma/water distribution ratios (D plasma/w ) of neutral and basic APIs were similar for trout and human plasma, differing by no more than a factor of 2.7 for a given API. For the acidic APIs, the D plasma/w values of trout plasma were much lower than for human plasma, by up to a factor of 71 for naproxen. The lower affinity of the acidic APIs to trout plasma compared with human plasma suggests that the bioavailability of these APIs is higher in trout. Read-across approaches like the fish plasma model should account for differences in plasma protein binding to avoid over- or underestimation of effects in fish. For the acidic APIs, the effect ratio of the fish plasma model would increase by a factor of 5 to 60 if the unbound plasma concentrations were used to calculate the effect ratio. Environ Toxicol Chem 2022;41:559-568. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Neuroactive drugs and other pharmaceuticals found in blood plasma of wild European fish

To gain a better understanding of which pharmaceuticals could pose a risk to fish, 94 pharmaceuticals representing 23 classes were analyzed in blood plasma from wild bream, chub, and roach captured at 18 sites in Germany, the Czech Republic and the UK, respectively. Based on read across from humans, we evaluated the risks of pharmacological effects occurring in the fish for each measured pharmaceutical. Twenty-three compounds were found in fish plasma, with the highest levels measured in chub from the Czech Republic. None of the German bream had detectable levels of pharmaceuticals, whereas roach from the Thames had mostly low concentrations. For two pharmaceuticals, four individual Czech fish had plasma concentrations higher than the concentrations reached in the blood of human patients taking the corresponding medication. For nine additional compounds, determined concentrations exceeded 10% of the corresponding human therapeutic plasma concentration in 12 fish. The majority of the pharmaceuticals where a clear risk for pharmacological effects was identified targets the central nervous system. These include e.g. flupentixol, haloperidol, and risperidone, all of which have the potential to affect fish behavior. In addition to identifying pharmaceuticals of environmental concern, the results emphasize the value of environmental monitoring of internal drug levels in aquatic wildlife, as well as the need for more research to establish concentration-response relationships.

Uptake and Effects of the Beta-Adrenergic Agonist Salbutamol in Fish: Supporting Evidence for the Fish Plasma Model.

The fish plasma model (FPM) predicts the fish blood plasma concentration of a pharmaceutical from the water concentration to which the fish is exposed and compares it with the human therapeutic plasma concentration (Hther PC) with the postulate that no adverse toxic effects occur below the Hther PC. The present study provides several lines of evidence supporting the FPM for the beta-adrenergic agonist salbutamol, a small cationic molecule at ambient pH. Salbutamol exhibited very low acute toxicity to early and adult life stages of fish. Biomass reduction in fish early life stages was the most sensitive apical endpoint, with no-observed-effect concentrations (NOECs) in the low mg/L range after continuous exposure for up to 120 d. Given that predicted and measured environmental concentrations are at least 1000-fold lower, the risk of salbutamol in freshwater is deemed very low. Increase in heart beat rate and decrease in total triglyceride content in fish also occurred at the low mg/L range and resembled effects known from humans. This finding supports the FPM assumption of conserved targets in fish with similar functionality. Plasma concentrations measured in adult and juvenile fish exposed to water concentrations at approximately the NOECs exceeded Hther PC and even approached plasma concentrations toxic to humans. This result confirms for salbutamol the FPM hypothesis that no adverse (i.e., population-relevant) toxic effects occur in fish below the Hther PC. Environ Toxicol Chem 2019;38:2509-2519. © 2019 SETAC.

Biomonitoring of pesticides, pharmaceuticals and illicit drugs in a freshwater invertebrate to estimate toxic or effect pressure

Multiple classes of environmental contaminants have been found in aquatic environments, globally. Understanding internalised concentrations in the organism could further improve the risk assessment process. The present study is concerned with the determination of several contaminant classes (107 compounds) in Gammarus pulex collected from 15 sites covering 5 river catchments across Suffolk, UK. Quantitative method performance was acceptable for 67 compounds including pharmaceuticals, pesticides, illicit drugs and drugs of abuse. A total of 56 compounds were detectable and ranged from <LOQ to 45.3 ng g−1, with cocaine and lidocaine being the most frequently detected compounds present in all biota samples (n = 66). For surface water, 50 compounds were detectable and ranged from <LOQ to 382.2 ng L−1. Additionally, some pesticides currently not approved for use were detected, including fenuron that reached a maximum of 16.1 ng g−1. The internal concentrations of pesticides were used to estimate toxic pressure which showed that for the measured pesticides toxic pressure was low ranging from logTU ≤−7 to ≤−2. This methodology was extended to pharmaceuticals and drugs of abuse in a novel approach that proposed the use of pharmacological data (human therapeutic plasma concentrations) to estimate the likelihood of an effect (or effect pressure) to occur based on the internal exposure of the organism. The quantified effect pressure ranged from logEU ≤−9 to ≤1 with haloperidol showing the largest likelihood for an effect. The approach showed that several pharmaceuticals have the potential to elicit effects but further investigation surrounding thresholds for effects would be required. This new approach presented showed potential to be used to improve risk assessment for pharmaceuticals in the environment.

Improving the clinical relevance of a mouse pregnancy model of antiretroviral toxicity; a pharmacokinetic dosing-optimization study of current HIV antiretroviral regimens

Animal models can be useful tools for the study of HIV antiretroviral (ARV) safety/toxicity in pregnancy and the mechanisms that underlie ARV-associated adverse events. The utility and translatability of animal model-based ARV safety/toxicity data is improved if ARVs are tested in clinically relevant concentrations. The objective of this work was to improve the clinical relevance of our mouse pregnancy model of ARV toxicity, by determining the doses of currently prescribed ARV regimens that would yield human therapeutic plasma concentrations. Pregnant mice were administered increasing doses of ARV combinations by oral gavage, followed by measurement of drug concentrations in the maternal plasma and amniotic fluid. Concentrations of ten different ARVs in maternal plasma and amniotic fluid samples of pregnant mice are presented, with dosing optimization to yield human pregnancy-relevant plasma drug concentrations. We have proposed optimal dosing for different regimen component drugs to achieve human therapeutic plasma levels, so that a clinically relevant standard dosing is established. A review of related ARV pharmacokinetic studies in (pregnant/non-pregnant) rodents and human pregnancy is also shown. We hope these data will inform and encourage the use of mouse pregnancy models in the study of ARV safety/toxicity.

Concentrating mixtures of neuroactive pharmaceuticals and altered neurotransmitter levels in the brain of fish exposed to a wastewater effluent

Fish can be exposed to a variety of neuroactive pharmaceuticals via the effluent discharges from wastewater treatment plants and concerns have arisen regarding their potential impacts on fish behaviour and ecology. In this study, we investigated the uptake of 14 neuroactive pharmaceuticals from a treated wastewater effluent into blood plasma and brain regions of roach (Rutilus rutilus) after exposure for 15days. We show that a complex mixture of pharmaceuticals including, 6 selective serotonin reuptake inhibitors, a serotonin-noradrenaline reuptake inhibitor, 3 atypical antipsychotics, 2 tricyclic antidepressants and a benzodiazepine, concentrate in different regions of the brain including the telencephalon, hypothalamus, optic tectum and hindbrain of effluent-exposed fish. Pharmaceuticals, with the exception of nordiazepam, were between 3–40 fold higher in brain compared with blood plasma, showing these neuroactive drugs are readily uptaken, into brain tissues in fish. To assess for the potential for any adverse ecotoxicological effects, the effect ratio was calculated from human therapeutic plasma concentrations (HtPCs) and the measured or predicted fish plasma concentrations of pharmaceuticals. After accounting for a safety factor of 1000, the effect ratios indicated that fluoxetine, norfluoxetine, sertraline, and amitriptyline warrant prioritisation for risk assessment studies. Furthermore, although plasma concentrations of all the pharmaceuticals were between 33 and 5714-fold below HtPCs, alterations in serotonin, glutamate, acetylcholine and tryptophan concentrations were observed in different brain regions of effluent-exposed fish. This study highlights the importance of determining the potential health effects arising from the concentration of complex environmental mixtures in risk assessment studies.

Anti-Epileptic Drug Combination Efficacy in an In Vitro Seizure Model - Phenytoin and Valproate, Lamotrigine and Valproate.

In this study, we investigated the relative efficacy of different classes of commonly used anti-epileptic drugs (AEDs) with different mechanisms of action, individually and in combination, to suppress epileptiform discharges in an in vitro model. Extracellular field potential were recorded in 450 μm thick transverse hippocampal slices prepared from juvenile Wistar rats, in which “epileptiform discharges” (ED’s) were produced with a high-K+ (8.5 mM) bicarbonate-buffered saline solution. Single and dual recordings in stratum pyramidale of CA1 and CA3 regions were performed with 3–5 MΩ glass microelectrodes. All drugs—lamotrigine (LTG), phenytoin (PHT) and valproate (VPA)—were applied to the slice by superfusion at a rate of 2 ml/min at 32°C. Effects upon frequency of ED’s were assessed for LTG, PHT and VPA applied at different concentrations, in isolation and in combination. We demonstrated that high-K+ induced ED frequency was reversibly reduced by LTG, PHT and VPA, at concentrations corresponding to human therapeutic blood plasma concentrations. With a protocol using several applications of drugs to the same slice, PHT and VPA in combination displayed additivity of effect with 50μM PHT and 350μM VPA reducing SLD frequency by 44% and 24% individually (n = 19), and together reducing SLD frequency by 66% (n = 19). 20μM LTG reduced SLD frequency by 32% and 350μM VPA by 16% (n = 18). However, in combination there was a supra-linear suppression of ED’s of 64% (n = 18). In another independent set of experiments, similar results of drug combination responses were also found. In conclusion, a combination of conventional AEDs with different mechanisms of action, PHT and VPA, displayed linear additivity of effect on epileptiform activity. More intriguingly, a combination of LTG and VPA considered particularly efficacious clinically showed a supra-additive suppression of ED’s. This approach may be useful as an in vitro platform for assessing drug combination efficacy.

Anti-anxiety drugs and fish behavior: Establishing the link between internal concentrations of oxazepam and behavioral effects.

Psychoactive drugs are frequently detected in the aquatic environment. The evolutionary conservation of the molecular targets of these drugs in fish suggests that they may elicit mode of action-mediated effects in fish as they do in humans, and the key open question is at what exposure concentrations these effects might occur. In the present study, the authors investigated the uptake and tissue distribution of the benzodiazepine oxazepam in the fathead minnow (Pimephales promelas) after 28 d of waterborne exposure to 0.8 μg L-1 , 4.7 μg L-1 , and 30.6 μg L-1 . Successively, they explored the relationship between the internal concentrations of oxazepam and the effects on fish exploratory behavior quantified by performing 2 types of behavioral tests, the novel tank diving test and the shelter-seeking test. The highest internal concentrations of oxazepam were found in brain, followed by plasma and liver, whereas muscle presented the lowest values. Average concentrations measured in the plasma of fish from the 3 exposure groups were, respectively, 8.7 ± 5.7 μg L-1 , 30.3 ± 16.1 μg L-1 , and 98.8 ± 72.9 μg L-1 . Significant correlations between plasma and tissue concentrations of oxazepam were found in all 3 groups. Exposure of fish to 30.6 µg L-1 in water produced plasma concentrations within or just below the human therapeutic plasma concentration (HT PC) range in many individuals. Statistically significant behavioral effects in the novel tank diving test were observed in fish exposed to 4.7 μg L-1 . In this group, plasma concentrations of oxazepam were approximately one-third of the lowest HT PC value. No significant effects were observed in fish exposed to the lowest and highest concentrations. The significance of these results is discussed in the context of the species-specific behavior of fathead minnow and existing knowledge of oxazepam pharmacology. Environ Toxicol Chem 2016;35:2782-2790. © 2016 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

The ABCG2 efflux transporter from rabbit placenta: Cloning and functional characterization

In human placenta, the ATP-binding cassette efflux transporter ABCG2 is highly expressed in syncytiotrophoblast cells and mediates cellular excretion of various drugs and toxins. Hence, physiological ABCG2 activity substantially contributes to the fetoprotective placenta barrier function during gestation. Developmental toxicity studies are often performed in rabbit. However, despite its toxicological relevance, there is no data so far on functional ABCG2 expression in this species. Therefore, we cloned ABCG2 from placenta tissues of chinchilla rabbit. Sequencing showed 84–86% amino acid sequence identity to the orthologues from man, rat and mouse. We transduced the rabbit ABCG2 clone (rbABCG2) in MDCKII cells and stable rbABCG2 gene and protein expression was shown by RT-PCR and Western blot analysis. The rbABCG2 efflux activity was demonstrated with the Hoechst H33342 assay using the specific ABCG2 inhibitor Ko143. We further tested the effect of established human ABCG2 (hABCG2) drug substrates including the antibiotic danofloxacin or the histamine H2-receptor antagonist cimetidine on H33342 accumulation in MDCKII-rbABCG2 or -hABCG2 cells. Human therapeutic plasma concentrations of all tested drugs caused a comparable competitive inhibition of H33342 excretion in both ABCG2 clones. Altogether, we first showed functional expression of the ABCG2 efflux transporter in rabbit placenta. Moreover, our data suggest a similar drug substrate spectrum of the rabbit and the human ABCG2 efflux transporter.

From single chemicals to mixtures—Reproductive effects of levonorgestrel and ethinylestradiol on the fathead minnow

The aquatic environment is polluted with thousands of chemicals. It is currently unclear which of these pose a significant threat to aquatic biota. The typical exposure scenario is now represented by a widespread blanket of contamination composed of myriads of individual pollutants—each typically present at a low concentration. The synthetic steroids, 17α-ethinylestradiol and levonorgestrel, have been widely reported to be present in the aquatic environment in the low ng to sub-ng/l range. They are widely used in contraceptive formulations, both individually and in combination. Our research employed the fathead minnow (Pimephales promelas) 21 day ‘pair-breeding’ assay to assess reproductive output when pairs of fish were exposed to the single chemicals at low environmentally relevant concentrations, and then to a binary mixture of them. A variety of endpoints were assessed, including egg production, which was inhibited in a concentration-dependent manner by both the individual chemicals and the mixture. Significant, sex specific effects were also seen with both chemicals, at differing levels of biological organisation. Plasma concentrations of EE2 and levonorgestrel were predicted and in the case of levonorgestrel measured, and compared with the human therapeutic plasma concentrations (Read-Across approach) to support the interpretation of the results. A novel quantitative method was developed for the data analysis, which ensured a suitable endpoint for the comparative mixture assessment. This approach compares the reproductive performance from individual pairs of fish during chemical exposure to its pre-treatment performance. The responses from the empirical mixture study were compared to predictions derived from the single substance data. We hypothesised combined responses which were best described by the concept of concentration addition, and found no clear indications against this additivity expectation. However, the effect profiles support the current knowledge that both compounds act in different ways to reduce egg production in fish, and suggest that probably response addition (also called Independent action) is the more appropriate mixture model in this case.

Understanding the biokinetics of ibuprofen after single and repeated treatments in rat and human in vitro liver cell systems

Common in vitro toxicity testing often neglects the fate and intracellular concentration of tested compounds, potentially limiting the predictability of in vitro results for in vivo extrapolation. We used in vitro long-term cultures of primary rat (PRH) and human hepatocytes (PHH) and HepaRG cells to characterise and model the biokinetic profile of ibuprofen (IBU) after single and daily repeated exposure (14 days) to two concentrations. A cross-model comparison was carried out at 100μM, roughly corresponding to the human therapeutic plasma concentration. Our results showed that IBU uptake was rapid and a dynamic equilibrium was reached within 1 or 2 days. All three cell systems efficiently metabolised IBU. In terms of species-differences, our data mirrored known in vivo results. Although no bioaccumulation was observed, IBU intracellular concentration was higher in PRH due to a 10-fold lower metabolic clearance compared to the human-derived cells. In HepaRG cells, IBU metabolism increased over time, but was not related to the treatment. In PHH, a low CYP2C9 activity, the major IBU-metabolising CYP, led to an increased cytotoxicity. A high inter-individual variability was seen in PHH, whereas HepaRG cells and PRH were more reproducible models. Although the concentrations of IBU in PRH over time differed from the concentrations found in human cells under similar exposure conditions.

Quantitative cross-species extrapolation between humans and fish: the case of the anti-depressant fluoxetine.

Fish are an important model for the pharmacological and toxicological characterization of human pharmaceuticals in drug discovery, drug safety assessment and environmental toxicology. However, do fish respond to pharmaceuticals as humans do? To address this question, we provide a novel quantitative cross-species extrapolation approach (qCSE) based on the hypothesis that similar plasma concentrations of pharmaceuticals cause comparable target-mediated effects in both humans and fish at similar level of biological organization (Read-Across Hypothesis). To validate this hypothesis, the behavioural effects of the anti-depressant drug fluoxetine on the fish model fathead minnow (Pimephales promelas) were used as test case. Fish were exposed for 28 days to a range of measured water concentrations of fluoxetine (0.1, 1.0, 8.0, 16, 32, 64 µg/L) to produce plasma concentrations below, equal and above the range of Human Therapeutic Plasma Concentrations (HTPCs). Fluoxetine and its metabolite, norfluoxetine, were quantified in the plasma of individual fish and linked to behavioural anxiety-related endpoints. The minimum drug plasma concentrations that elicited anxiolytic responses in fish were above the upper value of the HTPC range, whereas no effects were observed at plasma concentrations below the HTPCs. In vivo metabolism of fluoxetine in humans and fish was similar, and displayed bi-phasic concentration-dependent kinetics driven by the auto-inhibitory dynamics and saturation of the enzymes that convert fluoxetine into norfluoxetine. The sensitivity of fish to fluoxetine was not so dissimilar from that of patients affected by general anxiety disorders. These results represent the first direct evidence of measured internal dose response effect of a pharmaceutical in fish, hence validating the Read-Across hypothesis applied to fluoxetine. Overall, this study demonstrates that the qCSE approach, anchored to internal drug concentrations, is a powerful tool to guide the assessment of the sensitivity of fish to pharmaceuticals, and strengthens the translational power of the cross-species extrapolation.

Exposure and food web transfer of pharmaceuticals in ospreys (Pandion haliaetus): Predictive model and empirical data.

The osprey (Pandion haliaetus) is a well-known sentinel of environmental contamination, yet no studies have traced pharmaceuticals through the water-fish-osprey food web. A screening-level exposure assessment was used to evaluate the bioaccumulation potential of 113 pharmaceuticals and metabolites, and an artificial sweetener in this food web. Hypothetical concentrations in water reflecting "wastewater effluent dominated" or "dilution dominated" scenarios were combined with pH-specific bioconcentration factors (BCFs) to predict uptake in fish. Residues in fish and osprey food intake rate were used to calculate the daily intake (DI) of compounds by an adult female osprey. Fourteen pharmaceuticals and a drug metabolite with a BCF greater than 100 and a DI greater than 20 µg/kg were identified as being most likely to exceed the adult human therapeutic dose (HTD). These 15 compounds were also evaluated in a 40 day cumulative dose exposure scenario using first-order kinetics to account for uptake and elimination. Assuming comparable absorption to humans, the half-lives (t1/2 ) for an adult osprey to reach the HTD within 40 days were calculated. For 3 of these pharmaceuticals, the estimated t1/2 in ospreys was less than that for humans, and thus an osprey might theoretically reach or exceed the HTD in 3 to 7 days. To complement the exposure model, 24 compounds were quantified in water, fish plasma, and osprey nestling plasma from 7 potentially impaired locations in Chesapeake Bay. Of the 18 analytes detected in water, 8 were found in fish plasma, but only 1 in osprey plasma (the antihypertensive diltiazem). Compared to diltiazem detection rate and concentrations in water (10/12 detects, <method detection limits [MDL]-173 ng/L), there was a lower detection frequency in fish (31/233 detects, <MDL-2400 ng/L); however when present in fish, all values exceeded the maximum diltiazem concentration found in water. Diltiazem was found in all 69 osprey plasma samples (540-8630 ng/L), with 41% of these samples exceeding maximum concentrations found in fish. Diltiazem levels in fish and osprey plasma were below the human therapeutic plasma concentration (30000 ng/L). Effect thresholds for diltiazem are unknown in ospreys at this time, and there is no evidence to suggest adverse effects. This screening-level exposure model can help identify those compounds that warrant further investigation in high-trophic level species.

Endocrine disrupting compounds affecting corticosteroid signaling pathways in Czech and Swiss waters: potential impact on fish.

This study investigated the occurrence of corticosteroid signaling disruptors in wastewaters and rivers in the Czech Republic and in Switzerland. 36 target compounds were detected using HPLC-MS/MS, with up to 6.4 μg/L for azole antifungals that indirectly affect corticosteroid signaling. Glucocorticoid receptor (GR)-mediated activity was determined using the GR-CALUX bioassay with dexamethasone equivalent concentrations ranging from <LOD-2.6, 19-37, and 78-542 ng/L for river water, treated, and untreated wastewater, respectively. For most samples, the chemically predicted GR-mediated response was higher than that determined by the bioassay. Correspondingly, antiglucocorticoid activity was observed in some fractions. The fish plasma model (FPM), which predicts plasma concentrations, was applied to evaluate the potential of detected pharmaceuticals to cause receptor-mediated effects in fish. With one exception, medroxyprogesterone, the FPM applied to individual compounds predicted fish plasma concentrations to be below the level of human therapeutic plasma concentrations. To account for the activity of the sum of GR-active compounds, we introduce the "cortisol equivalents fish plasma concentration" approach, through which an increase in fish glucocorticoid plasma levels comparable to 0.9-83 ng/mL cortisol after exposure to the analyzed river waters was estimated. The results suggest that these chemicals may impact wild fish.