Delayed clearance of the pro-carcinogen benzo[a]pyrene in PLHC-1 cells when co-exposed to the antifungal drug clotrimazole and effects on the CYP1A biomarker.

Cytochrome P450 1 A (CYP1A) is a key enzyme in the metabolism of the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) in animals, and a biomarker for environmental PAH exposure. The common antimycotic imidazole drug clotrimazole (CLO) has been detected in the aquatic environment and likely co-exists with BaP. Like BaP, CLO can bind to CYP1A enzymes and can act as a CYP1A inhibitor. Co-exposure of BaP with CLO significantly delayed BaP elimination in a fish liver cell line (PLHC-1). Intracellular BaP concentration was 2.4 times higher after 6 h in co-exposed cells, compared to cells exposed to BaP alone. Higher BaP concentrations in cells co-exposed to CLO positively correlated with CLO dose, indicating CLO-mediated delays in BaP clearance. After 24 h, BaP was undetectable irrespective of CLO co-exposure. In contrast, intracellular CLO concentrations remained constant over the 72 h experimental period. Co-exposure of BaP with CLO caused synergistic and time-dependent increases on the CYP1A biomarker both on CYP1A mRNA levels and on CYP1A enzyme activity, in accordance with an apparent delayed BaP elimination in the presence of CLO. These results indicate a toxicokinetic interaction between BaP and CLO on the CYP1A enzyme that delays metabolic clearance of BaP.

Evaluation of parental and transgenerational effects of clotrimazole in Daphnia magna - A multi-parametric approach.

Azole antifungals inhibit the cytochrome P450 complex, decreasing the production of ergosterol in fungi, and compromising the biosynthesis of ecdysteroids in crustaceans, which are hormones regulating reproduction and ecdysis. The azole antifungal clotrimazole (CLO) raises environmental concerns due its toxicity. This work evaluated the effects on the number of moults, feeding rate, growth, reproduction, transgenerational reproductive effects on two different generations (F0, parental generation; and F1, organisms born from F0), and energetic balance in Daphnia magna. Neonates (<24 h) were exposed to sublethal concentrations (0, 2.7, and 3.4 mg/L) of CLO, to assess its effects on the moulting process. Neonates were also exposed to environmentally realistic concentrations of CLO (0, 30, 150, 750, and 3750 ng/L) for 24 and 96 h, to assess adverse effects on their feeding behaviour. Effects on energy reserves (fatty acids, glycogen, and protein levels) were also measured in animals exposed to CLO. A reproduction test was carried out to evaluate the amount and size of neonates from F0 and F1 generations. CLO exposure decreased the number of moults, and the size of organisms, but did not alter the feeding pattern of 5 days old individuals. However, neonates (<24 h) exposed to CLO had a significant decrease in their feeding pattern. CLO decreased the fatty acids content in exposed animals, but did not change glycogen and protein. CLO also decreased the size of adult daphnids from the third brood, born from animals exposed in F0; in F1 animals, the size of neonates from the third brood was decreased. This study evidenced the toxic effects caused by CLO on growth, feeding and reproduction of D. magna. Nevertheless, it is not possible to conclude whether the effects are due to the inhibition of cytochrome P450 enzymes, or to unspecific effects caused by general toxic stress and decreased nutrition.

Imidazole antifungal drug clotrimazole alters the behavior, brain acetylcholinesterase and oxidative stress biomarkers in African catfish Clarias gariepinus (Burchell 1822).

This study was carried out to determine the effect of clotrimazole (CTZ), an imidazole fungicide on behavior, brain acetylcholinesterase, lipid peroxidation and oxidative stress parameters in Clarias gariepinus juveniles. Fish were acutely exposed to five nominal concentrations of CTZ and control to assess the behavioral effects on fish. To determine the effects on brain acetylcholinesterase, lipid peroxidation and oxidative stress parameters, fish were exposed to three sub-lethal concentrations vis 7.76, 3.89 and 1.94 mg/L which corresponds to 20 10 and 5% of 96 h LC50 value of CTZ respectively and a control for 21 days and allowed to recover for 7 days. The brain, liver and gills were sampled weekly. Fish exposed to different concentrations of the drug displayed behavioral responses such as reduced swimming rate, mucus secretion, decrease in both the feeding frequency and rate. A duration dependent increase in the levels of brain acetylcholinesterase was observed among the exposed groups. The result of the sub-lethal exposure revealed concentration and duration significant increase in lipid peroxidation (LPO), catalase activity (CAT), superoxide dismutase (SOD), reduced glutathione (GSH), glutathione reductase (GR) and glutathione peroxide (GPx) in both the liver and gill tissues of the exposed groups compared to the control. The effects of the drug on many of the observed parameters did not wane after the 7-day withdrawal period. This study revealed that CTZ has a negative impact on the observed parameters, thus providing additional evidence of its toxic effect on non-target aquatic species, especially fish.

Human-relevant concentrations of the antifungal drug clotrimazole disrupt maternal and fetal steroid hormone profiles in rats.

Clotrimazole is a non-prescription and broad-spectrum antifungal drug sold under brand names such as Canesten® and Lotrimin®. It is used to treat different types of fungal infections, from oral thrush to athlete's foot and vaginal mycosis. The level of exposure to clotrimazole is uncertain, as the exact usage amongst self-medicating patients is unclear. Recent studies have raised potential concern about the unsupervised use of clotrimazole during pregnancy, especially since it is a potent inhibitor of CYP enzymes of the steroidogenesis pathway. To address some of these concerns, we have assessed the effects of intrauterine exposure to clotrimazole on developing rat fetuses. By exposing pregnant rats to clotrimazole 25 or 75 mg/kg bw/day during gestation days 7-21, we obtained internal fetal concentrations close to those observed in humans. These in vivo data are in strong agreement with our physiologically-based pharmacokinetic (PBK)-modelled levels. At these doses, we observed no obvious morphological changes to the reproductive system, nor shorter male anogenital distance; a well-established morphometric marker for anti-androgenic effects in male offspring. However, steroid hormone profiles were significantly affected in both maternal and fetal plasma, in particular pronounced suppression of estrogens was seen. In fetal testes, marked up-concentration of hydroxyprogesterone was observed, which indicates a specific action on steroidogenesis. Since systemic clotrimazole is rapidly metabolized in humans, relevant exposure levels may not in itself cause adverse changes to the reproductive systems. Its capacity to significantly alter steroid hormone concentrations, however, suggests that clotrimazole should be used with caution during pregnancy.

Human ES and iPS cells display less drug resistance than differentiated cells, and naïve-state induction further decreases drug resistance.

Pluripotent stem cells (PSCs) possess unique characteristics that distinguish them from other cell types. Human embryonic stem (ES) cells are recently gaining attention as a powerful tool for human toxicity assessment without the use of experimental animals, and an embryonic stem cell test (EST) was introduced for this purpose. However, human PSCs have not been thoroughly investigated in terms of drug resistance or compared with other cell types or cell states, such as naïve state, to date. Aiming to close this gap in research knowledge, we assessed and compared several human PSC lines for their resistance to drug exposure. Firstly, we report that RIKEN-2A human induced pluripotent stem (iPS) cells possessed approximately the same sensitivity to selected drugs as KhES-3 human ES cells. Secondly, both ES and iPS cells were several times less resistant to drug exposure than other non-pluripotent cell types. Finally, we showed that iPS cells subjected to naïve-state induction procedures exhibited a sharp increase in drug sensitivity. Upon passage of these naïve-like cells in non-naïve PSC culture medium, their sensitivity to drug exposure decreased. We thus revealed differences in sensitivity to drug exposure among different types or states of PSCs and, importantly, indicated that naïve-state induction could increase this sensitivity.

Ecotoxicological effects of the azole antifungal agent clotrimazole on the macrophyte species Lemna minor and Lemna gibba.

Pharmaceuticals are a large and diverse group of compounds used to treat, prevent and diagnose disease. Among these, a group that has been recently detected in the aquatic environment is that of the azole compounds, commonly used as antifungals. Clotrimazole (CLO) is a nonbiodegradable persistent azole compound, with broad-spectrum antifungal activity for which virtually no toxicological data are available, especially towards aquatic plants. The few existent data point to a documented interference with cytochrome P450 system of exposed organisms. Therefore, the aim of this paper was to evaluate the ecotoxicological effects of the fungicide CLO on two aquatic macrophyte species, namely, Lemna minor and Lemna gibba. To attain this purpose, an acute assay (96 h) was performed with both species being exposed to CLO, in a concentration range of 0 to 5 µg L-1. The analyzed endpoints were levels of chlorophyll a and b, total, carotenoids, catalase (CAT) and glutathione -s-transferases activities (GSTs). In general, CLO exposure caused some minor alterations in L. minor and L. gibba pigment contents. Antioxidant enzymes exhibited a different pattern in both species, since the highest concentrations of CLO caused an increase on CAT activity, and a decrease on GSTs activity in L. minor, and the opposite in L. gibba, reflected by a decrease on CAT activity and an increase on GSTs activity in all tested concentrations. These results demonstrate that CLO exposure resulted in potential deleterious effects on macrophytes, namely with the involvement of the antioxidant defense mechanisms that were likely deployed to cope with pro-oxidative conditions established by CLO.

Morphological, haematological and biochemical changes in African catfish Clarias gariepinus (Burchell 1822) juveniles exposed to clotrimazole.

Clotrimazole (CLO) is an imidazole fungicide used in human and veterinary medicine for treating fungal infection. This study evaluated the changes in morphological, haematological and biochemical indices in Clarias gariepinus juveniles exposed to CLO. After the acute exposure, the 96 h LC50 value of CLO determined by probit analysis was 38.79 mgl-1. Based on this value, fish were exposed to sublethal concentrations of 7.76, 3.89, 1.94 and 0.00 mgl-1 (control) of CLO for 21 days and were allowed to recover for 7 days. The result revealed no significant effect on the hepatosomatic index and condition factor of the exposed fish. There were concentration and time-dependent significant decreases in red blood cell (RBC), haemoglobin (Hb), packed cell volume (PCV) and mean corpuscular volume (MCV) with significant increase in the white blood cell (WBC), mean corpuscular haemoglobin concentration (MCHC), and mean corpuscular haemoglobin (MCH) in the exposed group when compared with the control. A mixed trend was observed in the levels of neutrophils, lymphocytes, monocytes and eosinophils. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and glucose values significantly increased, while protein levels were reduced (p < 0.05) throughout the 21-day exposure and the 7-day recovery period. The present research indicated that CLO may have potential toxic effect on non-target organisms especially fish and, therefore, should be monitored in the aquatic ecosystem.

Acetoacetate enhances oxidative metabolism and response to toxicants of cultured kidney cells.

Cultured kidney cells maintained in conventional growth media with high glucose levels exhibit increased glycolytic activity compared to the cells in vivo. In contrast, renal proximal tubules utilize substrates such as ketone bodies and rely on mitochondrial oxidative phosphorylation. LLC-PK1 cells maintain many features of the proximal tubule but are exposed to glucose concentrations ranging from 17 to 25 mM. This may impact their reliability in predicting mitochondrial toxicity. This study is designed to test the impact of the ketone body acetoacetate on metabolic characteristics of LLC-PK1 cells. Basal respiration, maximal respiration, spare respiratory capacity and ATP-linked respiration were significantly increased in cells grown in growth medium supplemented with 5 mM acetoacetate. In contrast, glycolytic capacity, as well as glycolytic reserve were significantly reduced in the acetoacetate group. There was an increased expression in biomarkers of mitochondrial biogenesis, and an increase in mitochondrial protein expression. Cells grown in medium complemented with acetoacetate displayed a significantly lower LC50 when treated with clotrimazole and diclofenac. There was a marked increase in uncoupled respiration in the presence of diclofenac, while clotrimazole and ciprofibrate significantly decreased respiration in the acetoacetate. The results indicate that acetoacetate complemented media can alter cellular metabolism and increase sensitization to toxicants.

The classic azole antifungal drugs are highly potent endocrine disruptors in vitro inhibiting steroidogenic CYP enzymes at concentrations lower than therapeutic Cmax.

Azole antifungal drugs are used worldwide to treat a variety of fungal infections such as vulvovaginal candidiasis, particularly in pregnant women who are at increased risk. The aim of this study was to mechanistically investigate the endocrine disrupting potential of four commonly used azole antifungal drugs; clotrimazole, miconazole, ketoconazole and fluconazole in vitro using the H295R cell assay and two recombinant, CYP17A1 and CYP19A1 (aromatase), assays. Steroids were quantified using LC-MS/MS. In both recombinant assays, all four azoles inhibited the CYP enzymes investigated, at therapeutically relevant concentrations. However, responses were much more complex in the H295R cell line. Clotrimazole inhibited steroid production in a dose-dependent manner with IC50 values for CYP17A1 and CYP19A1 in the range 0.017-0.184 µM. Miconazole and ketoconazole increased all steroids on the hydroxylase axis (IC50 MIC: 0.042-0.082 µM, KET: 0.041-1.2 µM), leading to accumulation of progestagens and corticosteroids and suppression of androgens and estrogens, indicating inhibition of CYP17A1, in particular lyase activity. However, ketoconazole suppressed all steroids at higher concentrations, resulting in bell-shaped curves for all steroids on the hydroxylase axis. Fluconazole was found to inhibit CYP17A1-lyase activity, causing suppression of androgens (IC50 = 114-209 µM) and estrogens (IC50 = 28 µM). The results indicate that these four azole drugs are highly potent in vitro and, based on plasma Cmax values, may exert endocrine disrupting effects at therapeutically relevant concentrations. This raises concern for endocrine related effects in patients using azole antifungal drugs, particularly when taken during sensitive periods like pregnancy.

Biomarker-based assessment of the toxicity of the antifungal clotrimazol to the microcrustacean Daphnia magna.

Among the vast list of xenobiotics that may promote harmful effects in aquatic ecosystems, pharmaceuticals are currently a prominent class due to their ability to persist in these environments and also due to the lack of information regarding their effects on the different components of the aquatic biota. Antifungals in particular, despite their massive use, are not extensively studied in environbmental terms. The main objective of this study was to characterize the toxicity of the antifungal clotrimazole to the aquatic organism Daphnia magna. To attain this purpose, the effects of this compound were measured, focusing on the determination of acute lethality, and quantification of biomarkers, such as neurotoxicity (soluble cholinesterases, ChEs); and oxidative stress and metabolism (such as catalase, CAT; and glutathione-S-transferases, GSTs). The toxicity assessment with biomarkers was based on animals exposed to concentrations similar to those already found in surface waters in order to increase the ecological relevance of the obtained data. The results showed that exposure to clotrimazole was able to induce significant increases in both CAT amd GSTs activities. ChE activity was not significantly altered after clotrimazol exposure. In view of the above, it is concluded that the drug studied caused adverse effects in terms of oxidative stress, at an ecological relevant levels, showing that the presence of clotrimazol in the wild is not innocuous.

Sub-lethal effects and bioconcentration of the human pharmaceutical clotrimazole in rainbow trout (Oncorhynchus mykiss).

The aim of this study was to characterize biomarker responses, haematological profiles, structural changes and uptake in juvenile rainbow trout exposed to clotrimazole (CLO) at three concentrations (0.01 - [lowest environmentally relevant concentration], 1.0 [highest environmentally relevant concentration] and 10 µg L(-1)) in a semi-static system over a period of 42 days. Antioxidant defence enzymes, which responded to CLO exposure, changed the oxidative stress status of cells, but no differences were observed in lipid peroxidation. Clotrimazole triggered a biphasic response of CYP3A-like activity in liver microsomes, which may indicate a detoxification process in the liver. Histopathological alterations were most pronounced in kidneys and testes in the group exposed to 10 µg L(-1). Structural changes in the kidney included tubulonephrosis and hyaline droplet degeneration in the tubular epithelial cells. The relative proportions of germ cells in testes were changed: The number of spermatozoa was reduced, and the spermatogonia and spermatocytes were increased. The highest CLO concentration was detected in fish liver (3710 ng per gram wet tissue) and kidney (4280 ng per gram wet tissue). Depuration half-life was estimated to be 72, 159, and 682 h in liver, muscle, and kidney, respectively. Taken together, these results provide valuable toxicological data on the effects of CLO on aquatic non-target organisms, which could be useful for further understanding of the potential risks in the real aquatic environment.

Individual and mixture effects of selected pharmaceuticals on larval development of the estuarine shrimp Palaemon longirostris.

Few ecotoxicological studies incorporate within the experimental design environmental variability and mixture effects when assessing the impact of pollutants on organisms. We have studied the combined effects of selected pharmaceutical compounds and environmental variability in terms of salinity and temperature on survival, development and body mass of larvae of the estuarine shrimp Palaemon longirostris. Drug residues found in coastal waters occur as mixture, and the evaluation of combined effects of simultaneously occurring compounds is indispensable for their environmental risk assessment. All larval stages of P. longirostris were exposed to the nonsteroidal anti-inflammatory drug (NSAID) diclofenac sodium (DS: 40 and 750 µg L(-1)), the lipid regulator clofibric acid (CA: 17 and 361 µg L(-1)) and the fungicide clotrimazole (CLZ: 0.14 and 4 µg L(-1)). We observed no effect on larval survival of P. longirostris with the tested pharmaceuticals. However, and in contrast to previous studies on larvae of the related marine species Palaemon serratus, CA affected development through an increase in intermoult duration and reduced growth without affecting larval body mass. These developmental effects in P. longirostris larvae were similar to those observed in the mixture of DS and CA confirming the toxic effects of CA. In the case of CLZ, its effects were similar to those observed previously in P. serratus: high doses affected development altering intermoult duration, tended to reduce the number of larval instars and decreased significantly the growth rate. This study suggests that an inter-specific life histories approach should be taken into account to assess the effect of emergent compounds in coastal waters.

Uptake of azoles by lamb's lettuce (Valerianella locusta L.) grown in hydroponic conditions.

An uptake and translocation study of azole compounds was performed in lamb's lettuce (Valerianella locusta L.) grown in nutrient solution fortified with different azoles. Three azoles, (clotrimazole, fluconazole and propiconazole), which have different physico-chemical properties and are ubiquitous in the aquatic environment, were the compounds selected. An analytical method, based on matrix solid phase dispersion (MSPD) followed by LC-MS/MS determination, was developed to quantify these compounds in aqueous solution and in roots and leaves. The physicochemical properties of azoles are the main factors governing the uptake and plant accumulation. These azoles were detected in leaves indicating their transport within lamb's lettuce. Translocation from nutrient solution to the aerial part of lamb's lettuce was found to be highly dependent on the hydrophobicity of the azole. Clotrimazole accumulates in roots causing necrosis in roots and leaves, whereas fluconazole was the azole with the highest concentration in leaves without causing apparent phytotoxicity symptoms. The assessment of the levels of these azoles in leaves indicates that the risk for human health is negligible.

Toxicidad de agentes antiparasitarios, antimicrobianos e insecticidas sobre larvas del camarón salino Artemia franciscana (Crustacea: Artemiidae) / Toxicity of antiparasitic, antimicrobial agents and insecticides on larvae of brine shrimp Artemia franciscana (crustacea: artemiidae)

Artemia franciscana "camarón salino", es un crustáceo sensible a un amplio rango de compuestos químicos, de fácil manejo en el laboratorio, y con un cultivo relativamente sencillo y barato. El objetivo del presente trabajo fue evaluar la toxicidad de agentes antiparasitarios, antimicrobianos e insecticidas sobre A. franciscana para establecer la concentración prevista que no causa efectos (PNEC) sobre los organismos marinos y obtener los niveles guía para la protección de la vida acuática. Con los nauplios II de A. franciscana, dentro de las 24 h de eclosión, se procedió a realizar los bioensayos de toxicidad calculando la Concentración letal media (CL50) a 24 h y 48 h de exposición. Se observó la siguiente secuencia de mayor a menor toxicidad a 48 h de exposición para tres agentes antiparasitarios comerciales: Mebendazol >Albendazol >Metronidazol. Con relación al efecto tóxico letal de seis agentes antimicrobianos comerciales se vio la siguiente secuencia de mayor a menor toxicidad a 48 h de exposición: Triclosan >Clotrimazol >Itraconazol >Ketoconazol >Oxitetraciclina >Mimosa. El camarón salino mostró efectos de mortalidad por acción de cinco sustancias con propiedades insecticidas, encontrándose el siguiente orden de mayor a menor mortalidad a 48 h de exposición: Cipermetrina >Rotenona >Carbaryl >Canela >Malation. Las tres sustancias químicas calificadas como muy tóxicas y que presentaron los niveles guía más bajos para la protección de la vida acuática fueron Triclosan (0,72 ug·L-1), Cipermetrina (0,84 ug·L-1) y Clotrimazol (0,97 ug·L-1). Se observó que diez (71,42%) de las sustancias químicas mostraron fuerte actividad citotóxica (AU)

Artemia franciscana "brine shrimp", is sensitive to a wide range of chemical structures, and easy handling in the laboratory and with a relatively simple and inexpensive crustacean culture. The aim of this study was to evaluate the toxicity of parasiticides agent, antimicrobials agent and insecticides on A. franciscana to establish Predicted No-Effect Concentration (PNEC) on marine organisms and obtain guidance levels for the protection of aquatic life. With A. franciscana nauplii II, within 24 h of hatching, we proceeded to perform toxicity bioassays calculating the average lethal concentration (LC50) at 24 h and 48 h of exposure. The following sequence of high to low toxicity to 48 h of exposure to three commercial antiparasitic agents were observed: Mebendazole> Albendazole> Metronidazole. Regarding the lethal toxic effect of six commercial antimicrobial agents about A. franciscana, the following sequence of toxicity at 48 h of exposure was observed: Triclosan> Clotrimazole> Itraconazole> Ketoconazole> oxytetracycline> Mimosa. The brine shrimp mortality showed effects on five substances with insecticidal properties, meeting the following order from highest to lowest mortality at 48 h of exposure Cipermethrin >Rotenone >Carbaryl >Cinnamon >Malathion. The three chemicals were classified as very toxic and presented lower levels guidance for the protection of aquatic life were Triclosan (0,72 ug·L-1), Cipermetrina (0,84 ug·L-1) y Clotrimazol (0,97 ug·L-1). Ten of chemicals (71.42%) showed strong cytotoxic activity (AU)

Climate change and pollution speed declines in zebrafish populations.

Endocrine disrupting chemicals (EDCs) are potent environmental contaminants, and their effects on wildlife populations could be exacerbated by climate change, especially in species with environmental sex determination. Endangered species may be particularly at risk because inbreeding depression and stochastic fluctuations in male and female numbers are often observed in the small populations that typify these taxa. Here, we assessed the interactive effects of water temperature and EDC exposure on sexual development and population viability of inbred and outbred zebrafish (Danio rerio). Water temperatures adopted were 28 °C (current ambient mean spawning temperature) and 33 °C (projected for the year 2100). The EDC selected was clotrimazole (at 2 µg/L and 10 µg/L), a widely used antifungal chemical that inhibits a key steroidogenic enzyme [cytochrome P450(CYP19) aromatase] required for estrogen synthesis in vertebrates. Elevated water temperature and clotrimazole exposure independently induced male-skewed sex ratios, and the effects of clotrimazole were greater at the higher temperature. Male sex ratio skews also occurred for the lower clotrimazole exposure concentration at the higher water temperature in inbred fish but not in outbred fish. Population viability analysis showed that population growth rates declined sharply in response to male skews and declines for inbred populations occurred at lower male skews than for outbred populations. These results indicate that elevated temperature associated with climate change can amplify the effects of EDCs and these effects are likely to be most acute in small, inbred populations exhibiting environmental sex determination and/or differentiation.

Bioavailability of the imidazole antifungal agent clotrimazole and its effects on key biotransformation genes in the common carp (Cyprinus carpio).

Clotrimazole (CTZ) is a persistent imidazole antifungal agent which is frequently detected in the aquatic environment and predicted to bio-concentrate in fish. Common carp (Cyprinus carpio) were exposed to mean measured concentrations of either 1.02 or 14.63µgl(-1) CTZ for 4 and 10 days, followed by a depuration period of 4 days in a further group of animals. Following each exposure regimen, plasma and liver CTZ concentrations were measured. Mean measured plasma concentrations of CTZ in animals exposed to the lower concentration of CTZ were 30 and 44µgl(-1) on days 4 and 10, respectively, and in the higher concentration were 318 and 336µgl(-1). Mean measured liver levels in the same animals were 514, 1725, 2111 and 7017µgl(-1) suggesting progressive hepatic accumulation. Measurement of CTZ in plasma after depuration suggested efficient elimination within 4 days, but appreciable levels of CTZ remained in the liver after depuration suggesting a degree of persistence in this tissue. In addition we measured responses of a number of key hepatic detoxification gene targets in the liver associated with the transcription factor pregnane X receptor (PXR); namely cyp450s 2k and 3a, glutathione-S-transferases a and p (gsta and p), and drug transporters multidrug resistance protein1 (mdr1), and MDR-related protein2 (mrp2). CTZ is a potent ligand of the PXR in humans and there is some evidence of PXR activation following exposure to CTZ in fish. The highest concentration of CTZ was adopted to explore the potential for alterations to detoxification gene expression in fish at a pharmacologically relevant dose level, and the lower concentration is within the range reported in effluents from waste water treatment works (WWTW). The genes for all biotransformation enzymes were up-regulated after exposure to the higher concentration of CTZ for 10 days, and alterations in expression occurred for the drug transporter genes mdr1 and mrp2 following exposure to the lower concentration of 1.02µgl(-1) CTZ (mean measured concentration). These data support the potential for CTZ to induce alterations in biotransformation and drug transporter genes associated with PXR in fish at concentrations measured in some WWTW effluents.

A multiple stressor approach to study the toxicity and sub-lethal effects of pharmaceutical compounds on the larval development of a marine invertebrate.

We studied the effects of three common pharmaceutical compounds on growth, development and body mass of larval stages of the marine shrimp Palaemon serratus at different temperatures and salinities. The pharmaceuticals compounds tested were the anti-inflammatory and analgesic diclofenac sodium, the lipid regulator clofibric acid and the fungicide clotrimazole. Neither diclofenac nor clofibric acid had any effect on growth, development or survival, although the maximum concentrations tested were 40 times higher than those observed in European coastal waters. Clotrimazole had significant effects at the higher concentration (2.78 µg L(-1)) when larvae were reared in full salinity sea water (32 PSU) and at the lower concentration (0.14 µg L(-1)) when larvae were reared at 20PSU. Changes in body mass at larval stage resulted from effects of these compounds on growth and developmental rates, specifically the changes in intermoult duration and in the number of larval instars required to reach the juvenile stage. The results demonstrate that the effects of emergent compounds on growth and development may be stronger when organisms are under some additional stress.

QSAR model for human pregnane X receptor (PXR) binding: screening of environmental chemicals and correlations with genotoxicity, endocrine disruption and teratogenicity.

The pregnane X receptor (PXR) has a key role in regulating the metabolism and transport of structurally diverse endogenous and exogenous compounds. Activation of PXR has the potential to initiate adverse effects, causing drug-drug interactions, and perturbing normal physiological functions. Therefore, identification of PXR ligands would be valuable information for pharmaceutical and toxicological research. In the present study, we developed a quantitative structure-activity relationship (QSAR) model for the identification of PXR ligands using data based on a human PXR binding assay. A total of 631 molecules, representing a variety of chemical structures, constituted the training set of the model. Cross-validation of the model showed a sensitivity of 82%, a specificity of 85%, and a concordance of 84%. The developed model provided knowledge about molecular descriptors that may influence the binding of molecules to PXR. The model was used to screen a large inventory of environmental chemicals, of which 47% was found to be within domain of the model. Approximately 35% of the chemicals within domain were predicted to be PXR ligands. The predicted PXR ligands were found to be overrepresented among chemicals predicted to cause adverse effects, such as genotoxicity, teratogenicity, estrogen receptor activation and androgen receptor antagonism compared to chemicals not causing these effects. The developed model may be useful as a tool for predicting potential PXR ligands and for providing mechanistic information of toxic effects of chemicals.

A critical role of follicle-stimulating hormone (Fsh) in mediating the effect of clotrimazole on testicular steroidogenesis in adult zebrafish.

Clotrimazole is a pharmaceutical fungicide known to inhibit several cytochrome P450 enzyme activities, including several steroidogenic enzymes. This study aimed to assess short-term in vivo effects of clotrimazole exposure on blood 11-ketotestosterone (11-KT) levels and on the transcriptional activity of genes in pituitary and testis tissue that are functionally relevant for androgen production with the view to further characterize the mode of action of clotrimazole on the hypothalamus-pituitary-gonad axis in zebrafish, a model vertebrate in toxicology. Adult male zebrafish were exposed to measured concentrations in water of 71, 159 and 258µg/L of clotrimazole for 7 days. Expression of pituitary gonadotropins ß subunit (lhb, fshb), testicular gonadotropins receptors (lhcgr, fshr) and testicular steroidogenesis-related genes (e.g., star, cyp17a1, cyp11c1) were assessed. Blood concentrations of 11-KT were measured. Short-term exposure to clotrimazole induced a concentration-dependent increase of star, cyp17a1, and cyp11c1 gene expression and Cyp17a1 and Cy11c1 protein synthesis in Leydig cells, but androgen levels in blood remained unchanged. fshb, but not lhb mRNA levels in the pituitary tended to increase in clotrimazole-exposed zebrafish. Testicular expression of the Fsh receptor gene was significantly up-regulated following exposure, when expression of this receptor was significantly correlated to the expression of steroidogenesis-related genes. Moreover, the Fsh-regulated insulin-like growth factor 3 (igf3) gene, a fish-specific Igf peptide expressed in Sertoli cells, was induced in testes. By using a network of genes functioning in pituitary and testis tissue, our study demonstrated that clotrimazole induced a cascade of molecular and cellular events which are in agreement with a role for Fsh (1) in stimulating Leydig cell steroidogenesis to compensate the inhibitory action of clotrimazole on 11-KT synthesis and (2) in inducing the expression of Fsh-regulated igf3 in Sertoli cells.

Clotrimazole nanoemulsion for malaria chemotherapy. Part II: stability assessment, in vivo pharmacodynamic evaluations and toxicological studies.

The aim of present investigation was to evaluate the potential of clotrimazole as antimalarial drug. Due to poor aqueous solubility and high lipophilicity, it was previously formulated in a nanoemulsion based system. The intrinsic effects of nanoemulsion on improvement of antimalarial activity of clotrimazole were assessed in mice infected with Plasmodium berghei and compared to its suspension formulation. In four-day suppressive test, mice treated with 10mg/kg clotrimazole nanoemulsion showed the highest suppression of parasitemia and; parasitemia was significantly lower than that of 10mg/kg clotrimazole suspension. In onset of activity and recrudescence test, percent reduction of parasitemia was significantly higher in 10 and 15 mg/kg clotrimazole nanoemulsion groups compared to 15 mg/kg suspension group. In both murine models, survival of mice treated with nanoemulsion was significantly prolonged compared to suspension at equivalent doses. The inhibition of parasite growth by clotrimazole in the nanoemulsion was dose dependent as determined by test for linear trend. In repeated dose oral toxicity, levels of serum liver enzymes and biomarkers of hepatotoxicity did not vary significantly from control. Six-month stability testing of the clotrimazole nanoemulsion exhibited no changes in various physiochemical attributes of drug product compared to initial analysis.