Ecotoxicological properties of ketoprofen and the S(+)‐enantiomer (dexketoprofen): Bioassays in freshwater model species and biomarkers in fish PLHC‐1 cell line

The increased use of non-steroidal anti-inflammatory drugs (NSAIDs) has resulted in their ubiquitous presence in the environment. The toxicological properties of these two widely prescribed NSAIDs, namely - racemic ketoprofen (rac-KP) and its enantiomer S(+)-ketoprofen (dexketoprofen, DKP) were evaluated. Firstly, by acute and chronic toxicity tests using three representative model organisms (Vibrio fischeri, Pseudokirchneriella subcapitata and Ceriodaphnia dubia). Secondly, by evaluating the responses of biotransformation systems and multidrug resistance associated proteins (MRP1/MRP2) using the PLHC-1 fish hepatic cell-line. Toxicity data from both acute and chronic DKP exposure indicated higher sensitivity through inhibition of bioluminescence and algal growth and through increased mortality/immobilization compared to rac-KP exposure. The growth inhibition test showed that rac-KP and DKP exhibited different values for EC50 (240.2 µg/L and 65.6 µg/L, respectively). Furthermore, rac-KP and DKP did not exert cytotoxic effects in PLHC-1 cells, and produced compound-, time- and concentration-specific differential effects on CYP1A and GST levels. For CYP1A, the effects of rac-KP and DKP differed at transcriptional and catalytic level. Exposure to rac-KP and DKP modulated MRP1 and MRP2 mRNA levels and these effects were also dependent on compound, exposure time and concentration of the individual drug. The present study revealed for the first time, the interactions between these NSAIDs and key detoxification systems, and different sensitivity to the racemic mixture compared to its enantiomer. This article is protected by copyright. All rights reserved

Novel organ-specific effects of Ketoprofen and its enantiomer, dexketoprofen on toxicological response transcripts and their functional products in salmon

Racemic ketoprofen (RS-KP) and its enantiomer, dexketoprofen (S(+)-KP) are widely used non-steroidal anti-inflammatory drugs (NSAIDs), and commonly detected in the aquatic environment. The present study has evaluated the toxicological effects of RS-KP and S(+)-KP on biotransformation and oxidative stress responses in gills and liver of Atlantic salmon. Fish were exposed for 10 days using different concentrations of RS-KP (1, 10 and 100 μg/L) and S(+)-KP (0.5, 5 and 50 μg/L). Biotransformation and oxidative stress responses were analysed at both transcript and functional levels. In the gills, significant inhibitory effect at transcriptional and enzymatic levels were observed for biotransformation and oxidative stress responses. On the contrary, biotransformation responses were significantly increased at transcriptional and translational levels in the liver, while the associated enzymatic activities did not parallel this trend and were inhibited and further demonstrated by principal component analysis (PCA). Our findings showed that both compounds produced comparable toxicological effects, by producing organ-specific effect differences. RS-KP and S(+)-KP did not bioaccumulate in fish muscle, either due to rapid metabolism or excretion as a result of their hydrophobic properties. Interestingly, the inhibitory effects observed in the gills suggest that these drugs may not undergo first pass metabolism, that might result to downstream differences in toxicological outcomes

Novel organ-specific effects of Ketoprofen and its enantiomer, dexketoprofen on toxicological response transcripts and their functional products in salmon.

Racemic ketoprofen (RS-KP) and its enantiomer, dexketoprofen (S(+)-KP) are widely used non-steroidal anti-inflammatory drugs (NSAIDs), and commonly detected in the aquatic environment. The present study has evaluated the toxicological effects of RS-KP and S(+)-KP on biotransformation and oxidative stress responses in gills and liver of Atlantic salmon. Fish were exposed for 10 days using different concentrations of RS-KP (1, 10 and 100 μg/L) and S(+)-KP (0.5, 5 and 50 μg/L). Biotransformation and oxidative stress responses were analysed at both transcript and functional levels. In the gills, significant inhibitory effect at transcriptional and enzymatic levels were observed for biotransformation and oxidative stress responses. On the contrary, biotransformation responses were significantly increased at transcriptional and translational levels in the liver, while the associated enzymatic activities did not parallel this trend and were inhibited and further demonstrated by principal component analysis (PCA). Our findings showed that both compounds produced comparable toxicological effects, by producing organ-specific effect differences. RS-KP and S(+)-KP did not bioaccumulate in fish muscle, either due to rapid metabolism or excretion as a result of their hydrophobic properties. Interestingly, the inhibitory effects observed in the gills suggest that these drugs may not undergo first pass metabolism, that might result to downstream differences in toxicological outcomes

Regulation of Intestinal UDP-Glucuronosyltransferase 1A1 by the Farnesoid X Receptor Agonist Obeticholic Acid Is Controlled by Constitutive Androstane Receptor through Intestinal Maturation

UDP-glucuronosyltransferase (UGT) 1A1 is the only transferase capable of conjugating serum bilirubin. However, temporal delay in the development of the UGT1A1 gene leads to an accumulation of serum bilirubin in newborn children. Neonatal humanized UGT1 (hUGT1) mice, which accumulate severe levels of total serum bilirubin (TSB), were treated by oral gavage with obeticholic acid (OCA), a potent FXR agonist. OCA treatment led to dramatic reduction in TSB levels. Analysis of UGT1A1 expression confirmed that OCA induced intestinal and not hepatic UGT1A1. Interestingly, Cyp2b10, a target gene of the nuclear receptor CAR, was also induced by OCA in intestinal tissue. In neonatal hUGT1/Car(-/-) mice, OCA was unable to induce CYP2B10 and UGT1A1, confirming that CAR and not FXR is involved in the induction of intestinal UGT1A1. However, OCA did induce FXR target genes, such as Shp, in both intestines and liver with induction of Fgf15 in intestinal tissue. Circulating FGF15 activates hepatic FXR and, together with hepatic Shp, blocks Cyp7a1 and Cyp7b1 gene expression, key enzymes in bile acid metabolism. Importantly, the administration of OCA in neonatal hUGT1 mice accelerates intestinal epithelial cell maturation, which directly impacts on induction of the UGT1A1 gene and the reduction in TSB levels. Accelerated intestinal maturation is directly controlled by CAR, since induction of enterocyte marker genes sucrase-isomaitase, alkaline phosphatase 3, and keratin 20 by OCA does not occur in hUGT1/Car(-/-) mice. Thus, new findings link an important role for CAR in intestinal UGT1A1 induction and its role in the intestinal maturation pathway. SIGNIFICANCE STATEMENT Obeticholic acid (OCA) activates FXR target genes in both liver and intestinal tissues while inducing intestinal UGT1A1, which leads to the elimination of serum bilirubin in humanized UGT1 mice. However, the induction of intestinal UGT1A1 and the elimination of bilirubin by OCA is driven entirely by activation of intestinal CAR and not FXR. The elimination of serum bilirubin is based on a CARdependent mechanism that facilitates the acceleration of intestinal epithelium cell differentiation, an event that underlies the induction of intestinal UGT1A1

Studio di un popolamento profondo di Corallium rubrum in un'area del Mar Tirreno.

Corallium rubrum (Cnidaria, Anthozoa, Ottocorallia, Gorgonacea L.1758) è un organismo endemico del Mediterraneo che fa parte delle comunità sciafile di substrato duro. Il forte interesse commerciale, legato al suo scheletro carbonatico rosso brillante, fa sì che questa specie sia pescata da oltre 2000 anni. Metodi di pesca non selettivi hanno portato ad un sovrasfruttamento di interi popolamenti di corallo con la loro conseguente riduzione o scomparsa. In seguito a ciò, sarebbe opportuno intervenire con l'attuazione di una politica di gestione adeguata che garantisca un prelievo sostenibile della risorsa. Questo può essere reso possibile dallo studio demografico, che fornisce informazioni utili nella conservazione della specie. Le analisi dei parametri demografici e riproduttivi permettono di conoscere lo stato attuale dei popolamenti e di fare proiezioni della loro struttura nel tempo. Le conoscenze sui popolamenti superficiali (10- 50 metri) di corallo rosso sono abbastanza estese; al contrario i popolamenti profondi (50-300 metri, noti anche come mesofotici), di importanza commerciale maggiore, sono ancora poco conosciuti. Questa tesi si basa sui dati raccolti durante la "Campagna oceanografica per lo studio del corallo rosso profondo", finanziata dal Ministero dell'Ambiente. Questo studio coinvolge diversi ricercatori e istituzioni che collaborano attivamente nel "Gruppo di ricerca italiana sul corallo rosso". La raccolta dei dati è stata condotta utilizzando la nave oceanografica "Astrea" (ISPRA) nel Luglio 2010. Durante questo studio è stata esplorata un'area del Tirreno Settentrionale appartenente all' Arcipelago Toscano, l'Isola d' Elba. Questa ricerca è stata realizzata con l'utilizzo: della sonda "Multibeam" per ottenere la cartografia batimetrica tridimensionale del fondo marino, del ROV (Remotely Operated Vehicle) per ottenere il video campionamento dell'area e dei sommozzatori professionisti per la raccolta dei campioni. Sono stati misurati i parametri morfometrici delle colonie (diametro, altezza, peso) su un campione totale di 199 colonie. E' stato, anche, identificato il sesso ed i principali parametri riproduttivi del popolamento. La maggior parte delle colonie era riproduttiva (89,9%) con una fecondità media di 0,82 oociti o planule per polipo e la sex ratio della popolazione non differiva significativamente da 1:1 (χ2=0,27; P>0,05). Le colonie sono state suddivise in classi di taglia in base al diametro e per ciascuna classe è stato calcolato l'output riproduttivo. La determinazione dell'età dei campioni di un popolamento è di fondamentale importanza per gli studi demografici. Per la prima volta è stata determinata l'età delle colonie di un popolamento profondo, utilizzando il metodo delle sezioni sottili messo a punto da Marschal et al. nel 2004. Questa tecnica permette di visualizzare la presenza di anelli concentrici di matrice organica depositati ogni anno lungo lo scheletro assiale. Le colonie sono state sezionate alla base, da questo primo taglio si è ricavata una sezione sottile spessa 50 μm. Successivamente, le sezioni sono state sottoposte a decalcificazione della matrice carbonatica e la componente organica è stata evidenziata mediante colorazione con blu di Toluidina. Una volta evidenziati gli anelli è stato possibile fotografare le sezioni delle colonie al microscopio. L'età delle colonie è stata conteggiata da tre diversi osservatori. Attraverso l'analisi della varianza (ANOVA) si è dimostrato che tra le stime d'età degli osservatori non vi erano differenze significative. E' stata ottenuta, per la prima volta, una relazione tra il diametro medio delle colonie profonde e l'età (y=1,33x^0,49; R2=0,59; P<0,0001). Il valore del tasso di accrescimento medio della colonia è di 0,26 mm/anno (maggiore rispetto ai popolamenti superficiali, 0,24 mm/anno). I valori dei parametri stimati, durante questo studio, forniscono una prima descrizione della struttura demografica dei popolamenti profondi di Corallium rubrum

Impatto ambientale di farmaci veterinari

Sono state valutate le proprietà tossicologiche di ketoprofene (miscela recemica) e del suo enantiomero S (+) (dexketoprofene) in diversi modelli sperimentali. Inizialmente, sono stati condotti saggi di tossicità acuta e cronica con tre organismi modello appartenenti a diversi livelli della catena trofica (Vibrio fischeri, Pseudokirchneriella subcapitata e Ceriodaphnia dubia). Successivamente, sono state valutate diverse risposte metaboliche in modelli cellulari in vitro. La linea cellulare di epatociti di pesce PLHC-1 (modello non target) è stata utilizzata per saggiare le risposte dei sistemi di biotrasformazione e le proteine di resistenza multi-farmaco (MRP1/MRP2). Invece, la linea cellulare di epatociti di ratto H4IIE (modello target) è stata utilizzata per saggiare le risposte di biotrasformazione e dei sistemi antiossidanti. In ultima analisi, utilizzando il modello in vivo con giovanili di salmone, sono stati valutati biomarkers di biotrasformazione e di stress ossidativo in campioni di fegato e branchie. I dati di tossicità, a seguito di esposizione sia acuta che cronica negli organismi modello, hanno indicato una maggiore sensibilità per dexketoprofene rispetto a ketoprofene in tutti gli endpoints valutati (inibizione della bioluminescenza, inibizione della crescita algale e mortalità/immobilizzazione dei crostacei). Inoltre, il test di inibizione della crescita algale ha mostrato rispettivamente per ketoprofene e dexketoprofene diversi valori di concentrazione al quale non si è manifestato nessun effetto (NOEC: 7.81- e 3.9 µg/l) e di concentrazione più bassa al quale è stato osservato un effetto (LOEC:15.63 e 7.81 µg/l). Entrambe le molecole non hanno esercitato effetti citotossici nelle cellule PLHC-1, sebbene siano state osservate risposte differenti per CYP1A e GST. Per CYP1A, ketoprofene e dexketoprofene hanno prodotto effetti diversi sia a livello trascrizionale che catalitico. L’esposizione a questi farmaci ha modulato i livelli di mRNA di MRP1 e MRP2, risultati essere di tipo farmaco, tempo e dose-dipendenti. Nessun effetto citotossico è stato osservato anche in cellule H4IIE esposte a farmaci scelti. In questo modello ketoprofene è stato metabolizzato dal CYP1A a 48 h, mentre dexketoprofene dopo 72 h e solo alla concentrazione massima testata. Le risposte allo stress ossidativo sono state diverse: ketoprofene ha evidenziato un’inibizione delle attività enzimatiche, mentre dexketoprofene ha mostrato un aumento di queste attività alla concentrazione più alta. L’esposizione in vivo ha evidenziato differenze tra fegato e branchie. Nelle branchie sia ketoprofene che dexketoprofene hanno provocato un’inibizione di tutti i biomarkers testati sia a livello trascrizionale che enzimatico. Diversamente, l’espressione genica delle difese antiossidanti (CAT, GR e GPX) e del sistema di disintossicazione da xenobiotici (CYP1A1 e CYP3A) è risultata essere up-regolata nei campioni di fegato. In conclusione, il presente studio ha rivelato per la prima volta le interazioni tra questi composti e i sistemi di disintossicazione chiave nonché delle difese antiossidanti, mostrando una diversa sensibilità tra la miscela racemica ketoprofene e il suo enantiomero destrogiro dexketoprofene

Biotransformation and oxidative stress responses in rat hepatic cell-line (H4IIE) exposed to racemic ketoprofen (RS-KP) and its enantiomer, dexketoprofen (S(+)-KP)

Pharmaceuticals such as racemate ketoprofen (RS-KP) and its enantiomer, dexketoprofen (S(+)-KP) are highly detectable non-steroidal anti-inflammatory drugs (NSAIDs) in the aquatic environment and therefore are designated as one of the most emerging groups of pollutants that can affect environmental and human health. The potential impact of these pharmaceuticals was assessed for the first time in vitro using a rat hepatocellular carcinoma cell line (H4IIE). Cells were exposed to low and high concentrations of these drugs. Cytotoxicity was determined by MTT reduction assay; CYP1A1 transcriptional and enzymatic levels together with canonical oxidative stress responsive markers (GPx, GR, GST and CAT) were also investigated. Cells exposed to RS-KP and S(+)-KP did not show cytotoxicity effect at the concentrations tested. However, this study highlighted differences between RS-KP and S(+)-KP in most of the evaluated markers, showing compound-, concentration- and time-specific effect patterns which suggest a potential stereo-selective toxicity of these drugs

Assessing the effects of Awba dam sediment (Nigeria) on the steroidogenesis of H295R cells using different extraction methods

In the present study, H295R human cells were used to investigate the endocrine disruptor potential of three different sediments extracts taken from a Nigerian tropical freshwater dam (Awba Dam), using three extraction methods that allowed a selective consideration of contaminants based on their binding affinity, which is mainly driven by polarity, to sediment particles. After exposure to different concentration of each extract, H295R cells were evaluated for the expression profiles of 10 steroidogenic enzyme genes and estradiol (E2) and testosterone (T) levels. Our results showed a comparable concentrated-related increase in the expression of 17β-hsd1, 3β-hsd2 and cyp21 in cells treated with the polar and non-polar extracts. The star, hmgr, cyp11b2 and 17β-hsd4 were slightly decreased, in an apparent concentration-specific manner, after treatment with the polar extract and decreased in the non-polar treatment. The cyp11a and cyp17 showed an opposite trend in the polar and non-polar treatments. E2 was significantly higher in cell treated with the non-polar extract. Elutriate exposure produced less pronounced variation in mRNA and hormones levels. Overall the extract with non-polar compounds produced the most severe effects in H295R cells. Thus, direct ingestion of detritus and mud from fishes and other benthonic organisms represent possible transfer of contaminants in the trophic web, and mainly account for alteration of the endocrine system previously observed in fish from the same study site

NCoR1 Protects Mice From Dextran Sodium Sulfate Induced Colitis by Guarding Colonic Crypt Cells From Luminal Insult

BACKGROUND & AIMS: Colonic stem cells are essential for producing the mucosal lining, which in turn protects stem cells from insult by luminal factors. Discovery of genetic and biochemical events that control stem cell proliferation and differentiation can be leveraged to decipher the causal factors of ulcerative colitis and aid the development of more effective therapy. METHODS: We performed in vivo and in vitro studies from control (nuclear receptor corepressor 1 [NCoR1(F/F)]) and intestinal epithelial cell-specific NCoR1-deficient mice (NCoR1(Delta IEC)). Mice were challenged with dextran sodium sulfate to induce experimental ulcerative colitis, followed by colitis examination, barrier permeability analysis, cell proliferation immunostaining assays, and RNA sequencing analysis. By using crypt cultures, the organoid-forming efficiency, cell proliferation, apoptosis, and histone acetylation were analyzed after butyrate and/or tumor necrosis factor alpha treatments. RESULTS: NCoR1(Delta)(IEC) mice showed a dramatic increase in disease severity in this colitis model, with suppression of proliferative cells at the crypt base as an early event and a concomitant increase in barrier permeability. Genome expression patterns showed an important role for NCoR1 in colonic stem cell proliferation and secretory cell differentiation. Colonic organoids cultured from NCoR1(Delta)(IEC) mice were more sensitive to butyrate-induced cell growth inhibition and apoptosis, which were exaggerated further by tumor necrosis factor a cotreatment, which was accompanied by increased histone acetylation. CONCLUSIONS: NCoR1 regulates colonic stem cell proliferation and secretory cell differentiation. When NCoR1 is disrupted, barrier protection is weakened, allowing luminal products such as butyrate to penetrate and synergistically damage the colonic crypt cells. Transcript profiling: RNA sequencing data have been deposited in the GEO database, accession number: GSE136153