Luigi Margiotta-Casaluci

The Read-Across Hypothesis and Environmental Risk Assessment of Pharmaceuticals

Pharmaceuticals in the environment have received increased attention over the past decade, as they are ubiquitous in rivers and waterways. Concentrations are in sub-ng to low μg/L, well below acute toxic levels, but there are uncertainties regarding the effects of chronic exposures and there is a need to prioritise which pharmaceuticals may be of concern. The read-across hypothesis stipulates that a drug will have an effect in non-target organisms only if the molecular targets such as receptors and enzymes have been conserved, resulting in a (specific) pharmacological effect only if plasma concentrations are similar to human therapeutic concentrations. If this holds true for different classes of pharmaceuticals, it should be possible to predict the potential environmental impact from information obtained during the drug development process. This paper critically reviews the evidence for read-across, and finds that few studies include plasma concentrations and mode of action based effects. Thus, despite a large number of apparently relevant papers and a general acceptance of the hypothesis, there is an absence of documented evidence. There is a need for large-scale studies to generate robust data for testing the read-across hypothesis and developing predictive models, the only feasible approach to protecting the environment.

Pharmaceuticals in the Aquatic Environment: Steroids and Anti-Steroids as High Priorities for Research

ABSTRACT Many human pharmaceuticals, with many modes of action, are present in the aquatic environment. It is very difficult to know which of these might pose a threat to aquatic organisms, and which are of no environmental concern. Hence, it is difficult to design a research strategy that most effectively maximizes the possibility of identifying those pharmaceuticals that do pose the greatest risk. We consider that steroidal pharmaceuticals, and drugs that inhibit the actions of endogenous steroids, merit more research than they have yet received so far. We analyzed a comprehensive U.K. database covering all prescriptions of pharmaceuticals to determine the annual usage of all steroid and anti-steroid pharmaceuticals. We found that both progestogens and glucocorticoids are used in much greater amounts than estrogens, whereas androgens are used in similar amounts to estrogens. There is also very significant use of anti-estrogens, anti-androgens, and anti-mineralocorticoids. All these pharmaceuticals are likely to be constantly entering the aquatic environment in effluents from sewage treatment works. River concentrations of these pharmaceuticals will be very low; probably in the ng or sub-ng/l range. However, such low concentrations mat still pose a threat to aquatic organisms, especially fish.

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.

5α-Dihydrotestosterone is a potent androgen in the fathead minnow (Pimephales promelas)

Dihydrotestosterone (DHT) is one of the most physiologically important androgens in many male vertebrates, with the exception of teleost fish, in which 11-ketotestosterone (KT) is generally considered the major circulating male androgen. In the present study, we investigated the effects of KT and DHT on fathead minnow juveniles (Pimephales promelas), with the aim to compare the effects of the two androgens on critical physiological processes, such as somatic growth, male secondary sexual characteristics expression, and gonad maturation. Juvenile fish (60 days post-hatch) were exposed to 20 and 200 ng/L of KT and DHT for 45 days. Exposure to both androgens significantly stimulated somatic growth in both males (20 and 200 ng/L) and females (200 ng/L). Nuptial tubercle formation was induced by both KT and DHT, but only the latter, at 200 ng/L, caused the appearance of dorsal fin spot in 92% of males and 75% of females. Circulating plasma T concentrations showed a sex-specific response; a significant increase was recorded in exposed males and a decrease in females. Both androgens induced a significant advancement of the spermatogenic processes in males at 200 ng/L. In contrast, only DHT caused a severe disruption of ovarian physiology and morphology in females, inducing the development of spermatogenic tissue (intersex). These results show that in fathead minnow juveniles, DHT had in vivo androgenic potency comparable to KT in males, and higher than KT in females, suggesting a potential involvement of DHT in the mediation of fathead minnow androgenic responses.

Internal exposure dynamics drive the Adverse Outcome Pathways of synthetic glucocorticoids in fish.

The Adverse Outcome Pathway (AOP) framework represents a valuable conceptual tool to systematically integrate existing toxicological knowledge from a mechanistic perspective to facilitate predictions of chemical-induced effects across species. However, its application for decision-making requires the transition from qualitative to quantitative AOP (qAOP). Here we used a fish model and the synthetic glucocorticoid beclomethasone dipropionate (BDP) to investigate the role of chemical-specific properties, pharmacokinetics, and internal exposure dynamics in the development of qAOPs. We generated a qAOP network based on drug plasma concentrations and focused on immunodepression, skin androgenisation, disruption of gluconeogenesis and reproductive performance. We showed that internal exposure dynamics and chemical-specific properties influence the development of qAOPs and their predictive power. Comparing the effects of two different glucocorticoids, we highlight how relatively similar in vitro hazard-based indicators can lead to different in vivo risk. This discrepancy can be predicted by their different uptake potential, pharmacokinetic (PK) and pharmacodynamic (PD) profiles. We recommend that the development phase of qAOPs should include the application of species-species uptake and physiologically-based PK/PD models. This integration will significantly enhance the predictive power, enabling a more accurate assessment of the risk and the reliable transferability of qAOPs across chemicals.

Identification and quantification of 5α-dihydrotestosterone in the teleost fathead minnow (Pimephales promelas) by gas chromatography-tandem mass spectrometry.

The steroid hormone 5α-dihydrotestosterone (DHT) is one of the most physiologically important androgens in male vertebrates, with the exception of teleost fish, in which it is generally assumed that DHT does not play any major physiological role. However, this assumption is challenged by the fact that all the components involved in DHT biosynthesis and action are present and evolutionary conserved in teleost fish. In fact, testosterone (T) is converted into DHT by two isoforms of the enzyme steroid-5-alpha-reductase (5αR), and both 5αRs gene expression and enzymatic activity have been detected in several tissues of different teleost species, which also have an androgen receptor with high binding affinity to DHT. This body of evidence strongly suggest that DHT is synthesised by teleost fish. We investigated this hypothesis using the cyprinid fathead minnow (Pimephales promelas) as the experimental model. The study of the evolutionary and functional conservation of 5αRs in teleost fish was used to support the experimental approach, based on an ultrasensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) method to identify and measure simultaneously T and DHT in fathead minnow biological fluids and tissues. The analyses were performed using plasma samples collected from both male and female adult fish and samples of testicular tissue collected from sexually mature males. Both T and DHT were identified and quantified in all the samples analysed, and in particular, the high concentrations of DHT quantified in the testes suggested that these organs are a likely site of synthesis of DHT in the teleost fathead minnow, as they are in mammals. These results may represent the basis for future studies aimed at elucidating the physiological role, if any, of DHT in teleost fish.

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.

Preliminary data on the influence of rearing temperature on the growth and reproductive status of fathead minnows Pimephales promelas.

An investigation into the influence of temperature on the growth and reproductive status of the fathead minnow Pimephales promelas revealed that, while there was no clear effect of treatment on sex differentiation, ovarian tissue from female fish reared under the highest temperature regime contained large amounts of undefined tissue containing no germ cells. Furthermore, both male and female fish exhibited differences in length mass, condition and somatic indices, and in the expression of secondary sexual characteristics. The patterns observed are discussed in the context of climate change.

A New Role for Carbonic Anhydrase 2 in the Response of Fish to Copper and Osmotic Stress: Implications for Multi-Stressor Studies

The majority of ecotoxicological studies are performed under stable and optimal conditions, whereas in reality the complexity of the natural environment faces organisms with multiple stressors of different type and origin, which can activate pathways of response often difficult to interpret. In particular, aquatic organisms living in estuarine zones already impacted by metal contamination can be exposed to more severe salinity variations under a forecasted scenario of global change. In this context, the present study aimed to investigate the effect of copper exposure on the response of fish to osmotic stress by mimicking in laboratory conditions the salinity changes occurring in natural estuaries. We hypothesized that copper-exposed individuals are more sensitive to osmotic stresses, as copper affects their osmoregulatory system by acting on a number of osmotic effector proteins, among which the isoform two of the enzyme carbonic anhydrase (CA2) was identified as a novel factor linking the physiological responses to both copper and osmotic stress. To test this hypothesis, two in vivo studies were performed using the euryhaline fish sheepshead minnow (Cyprinodon variegatus) as test species and applying different rates of salinity transition as a controlled way of dosing osmotic stress. Measured endpoints included plasma ions concentrations and gene expression of CA2 and the α1a-subunit of the enzyme Na+/K+ ATPase. Results showed that plasma ions concentrations changed after the salinity transition, but notably the magnitude of change was greater in the copper-exposed groups, suggesting a sensitizing effect of copper on the responses to osmotic stress. Gene expression results demonstrated that CA2 is affected by copper at the transcriptional level and that this enzyme might play a role in the observed combined effects of copper and osmotic stress on ion homeostasis.

Adverse outcome pathway networks II: Network analytics

Toxicological responses to stressors are more complex than the simple one-biological-perturbation to one-adverse-outcome model portrayed by individual adverse outcome pathways (AOPs). Consequently, the AOP framework was designed to facilitate de facto development of AOP networks that can aid in the understanding and prediction of pleiotropic and interactive effects more common to environmentally realistic, complex exposure scenarios. The present study introduces nascent concepts related to the qualitative analysis of AOP networks. First, graph theory-based approaches for identifying important topological features are illustrated using 2 example AOP networks derived from existing AOP descriptions. Second, considerations for identifying the most significant path(s) through an AOP network from either a biological or risk assessment perspective are described. Finally, approaches for identifying interactions among AOPs that may result in additive, synergistic, or antagonistic responses (or previously undefined emergent patterns of response) are introduced. Along with a companion article (part I), these concepts set the stage for the development of tools and case studies that will facilitate more rigorous analysis of AOP networks, and the utility of AOP network-based predictions, for use in research and regulatory decision-making. The present study addresses one of the major themes identified through a Society of Environmental Toxicology and Chemistry Horizon Scanning effort focused on advancing the AOP framework. Environ Toxicol Chem 2018;37:1734-1748.