Acute and chronic effects of environmental realistic concentrations of clofibric acid in Danio rerio: Behaviour, oxidative stress, biotransformation and lipid peroxidation endpoints.

Due to their widespread use, pharmaceuticals can be metabolized, excreted and ultimately discarded in the environment, thereby affecting aquatic organisms. Lipid-regulating drugs are among the most prescribed medications around the world, controlling human cholesterol levels, in more than 20 million patients. Despite this growing use of lipid-regulating drugs, particularly those whose active metabolite is clofibric acid, the potential toxicological effects of these pharmaceuticals in the environment is not fully characterized. This work intended to characterize the toxicity of an acute (120 hours post-fertilization) and chronic (60 days post-fertilization) exposures to clofibric acid in concentrations of 10.35, 20.7, 41.4, 82.8, and 165.6 µg L-1 in zebrafish (Danio rerio). The concentrations which were implemented in both exposures were based on predicted environmental concentrations for Portuguese surface waters. The acute effects were analysed focusing on behavioural endpoints (small and large distance travelled, swimming time and total distance travelled), biomarkers of oxidative stress (activity of the enzymes superoxide dismutase, Cu/Zn- and Mn SOD; catalase, CAT; glutathione peroxidase, Se- and total GPx), biotransformation (activity of glutathione S-transferases, GSTs) and lipid peroxidation (thiobarbituric acid reactive substances, TBARS). Chronically exposed individuals were also histologically analysed for sex determination and gonadal developmental stages. In terms of acute exposure, significant alterations were reported, in terms of behavioural alterations (hypoactivity), followed by an overall increase in all tested biomarkers. Chronically exposed organisms did not show alterations in terms of sex ratio and maturation stages, suggesting that clofibric acid did not act as an endocrine disruptor. Moreover, the metabolism of clofibric acid resulted in increased levels of both forms of SOD activity, especially for animals exposed to higher levels of this drug. An increase of CAT activity was observed in fish exposed to low levels, and a decrease in those exposed to higher amounts of clofibric acid. Both GPx forms had their activities increased. The enzyme of biotransformation GSTs were increased at low levels of clofibric acid but inhibited at higher amounts of this substance. Lipid peroxidation levels were also changed, with an induction of this parameter with increasing amounts of clofibric acid. Changes also occurred in behavioural endpoints and patterns for control organisms and for those exposed to clofibric acid were significantly distinct, for all types (light and darkness) of exposure, and for the two analysed endpoints (small and large distance). Results from this assay allow inferring that clofibric acid can have an ecologically relevant impact in living organisms exposed to this substance, with putative effects on the metabolism of individuals, affecting their behaviour and ultimately their survival.

Metabolism of clofibric acid in zebrafish embryos (Danio rerio) as determined by liquid chromatography-high resolution-mass spectrometry.

The zebrafish embryo (ZFE) is increasingly used in ecotoxicology research but detailed knowledge of its metabolic potential is still limited. This study focuses on the xenobiotic metabolism of ZFE at different life-stages using the pharmaceutical compound clofibric acid as study compound. Liquid chromatography with quadrupole-time-of-flight mass spectrometry (LC-QToF-MS) is used to detect and to identify the transformation products (TPs). In screening experiments, a total of 18 TPs was detected and structure proposals were elaborated for 17 TPs, formed by phase I and phase II metabolism. Biotransformation of clofibric acid by the ZFE involves conjugation with sulfate or glucuronic acid, and, reported here for the first time, with carnitine, taurine, and aminomethanesulfonic acid. Further yet unknown cyclization products were identified using non-target screening that may represent a new detoxification pathway. Sulfate containing TPs occurred already after 3h of exposure (7hpf), and from 48h of exposure (52hpf) onwards, all TPs were detected. The detection of these TPs indicates the activity of phase I and phase II enzymes already at early life-stages. Additionally, the excretion of one TP into the exposure medium was observed. The results of this study outline the high metabolic potential of the ZFE with respect to the transformation of xenobiotics. Similarities but also differences to other test systems were observed. Biotransformation of test chemicals in toxicity testing with ZFE may therefore need further consideration.

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.

Effects of the lipid regulating drug clofibric acid on PPARα-regulated gene transcript levels in common carp (Cyprinus carpio) at pharmacological and environmental exposure levels.

In mammals, the peroxisome proliferator-activated receptor α (PPARα) plays a key role in regulating various genes involved in lipid metabolism, bile acid synthesis and cholesterol homeostasis, and is activated by a diverse group of compounds collectively termed peroxisome proliferators (PPs). Specific PPs have been detected in the aquatic environment; however little is known on their pharmacological activity in fish. We investigated the bioavailability and persistence of the human PPARα ligand clofibric acid (CFA) in carp, together with various relevant endpoints, at a concentration similar to therapeutic levels in humans (20mg/L) and for an environmentally relevant concentration (4µg/L). Exposure to pharmacologically-relevant concentrations of CFA resulted in increased transcript levels of a number of known PPARα target genes together with increased acyl-coA oxidase (Acox1) activity, supporting stimulation of lipid metabolism pathways in carp which are known to be similarly activated in mammals. Although Cu,Zn-superoxide dismutase (Sod1) activity was not affected, mRNA levels of several biotransformation genes were also increased, paralleling previous reports in mammals and indicating a potential role in hepatic detoxification for PPARα in carp. Importantly, transcription of some of these genes (and Acox1 activity) were affected at exposure concentrations comparable with those reported in effluent discharges. Collectively, these data suggest that CFA is pharmacologically active in carp and has the potential to invoke PPARα-related responses in fish exposed in the environment, particularly considering that CFA may represent just one of a number of PPAR-active compounds present to which wild fish may be exposed.

Chronic effects of clofibric acid in zebrafish (Danio rerio): a multigenerational study.

Clofibric acid (CA) is an active metabolite of the blood lipid lowering agent clofibrate, a pharmaceutical designed to work as agonist of peroxisome proliferator-activated receptor alpha (PPARa). It is the most commonly reported fibrate in aquatic environments with low degradation rate and potential environmental persistence. Previous fish exposures showed that CA may impact spermatogenesis, growth and the expression of fat binding protein genes. However, there are limited data on the effects of chronic multigenerational CA exposures. Here, we assessed chronic multigenerational effects of CA exposure using zebrafish (Danio rerio) as a teleost model. Zebrafish were exposed through the diet to CA (1 and 10mg/g) during their whole lifetime. Growth, reproduction-related parameters and embryonic development were assessed in the exposed fish (F1 generation) and their offspring (F2 generation), together with muscle triglyceride content and gonad histology. In order to study the potential underlying mechanisms, the transcription levels of genes coding for enzymes involved in lipid metabolism pathways were determined. The results show that chronic life-cycle exposure to CA induced a significant reduction in growth of F1 generation and lowered triglyceride muscle content (10mg/g group). Also, an impact in male gonad development was observed together with a decrease in the fecundity (10mg/g group) and higher frequency of embryo abnormalities in the offspring of fish exposed to the lowest CA dose. The profile of the target genes was sex- and tissue-dependent. In F1 an up-regulation of male hepatic pparaa, pparb and acox transcript levels was observed, suggesting an activation of the fatty acid metabolism (provided that transcript level change indicates also a protein level change). Interestingly, the F2 generation, raised with control diet, displayed a response pattern different from that observed in F1, showing an increase in weight in the descendants of CA exposed fish, in comparison with control animals, which points to a multigenerational effect.

Statistical modeling of the hormetic dose zone and the toxic potency completes the quantitative description of hormetic dose responses.

Quantifying the characteristics of hormesis provides valuable insights into this low-dose phenomenon and helps to display and capture its variability. A prerequisite to do so is a statistical procedure allowing quantification of general hormetic features, namely the maximum stimulatory response, the dose range of hormesis, and the distance from the maximum stimulation to the dose where hormesis disappears. Applying extensions of a hormetic dose-response model that is well-established in plant biology provides a direct estimation of several quantities, except the hormetic dose range. Another dose range that is difficult to model directly is the distance between the dose where hormesis disappears and the dose giving 50% inhibition, known as toxic potency. The present study presents 2 further model extensions allowing for a direct quantification of the hormetic dose range and the toxic potency. Based on this, a 4-step mathematical modeling approach is demonstrated to quantify various dose-response quantities, to compare these quantities among treatments, and to interrelate hormesis features. Practical challenges are exemplified, and possible remedies are identified. The software code to perform the analysis is provided as Supplemental Data to simplify adoption of the modeling procedure. Because numerous patterns of hormesis are observed in various sciences, it is clear that the proposed approach cannot cope with all patterns; however, it should be possible to analyze a great range of hormesis patterns.

Toxicological effects of clofibric acid and diclofenac on plasma thyroid hormones of an Indian major carp, Cirrhinus mrigala during short and long-term exposures.

In the present investigation, the toxicity of most commonly detected pharmaceuticals in the aquatic environment namely clofibric acid (CA) and diclofenac (DCF) was investigated in an Indian major carp Cirrhinus mrigala. Fingerlings of C. mrigala were exposed to different concentrations (1, 10 and 100µgL(-1)) of CA and DCF for a period of 96h (short term) and 35 days (long term). The toxic effects of CA and DCF on thyroid hormones (THs) such as thyroid stimulating hormone (TSH), thyroxine (T4) and triiodothyronine (T3) levels were evaluated. During the short and long-term exposure period TSH level was found to be decreased at all concentrations of CA (except at the end of 14(th) day in 1 and 10µgL(-l) and 21(st) day in 1µgL(-l)) whereas in DCF exposed fish TSH level was found to be increased when compared to control groups. T4 level was found to be decreased at 1 and 100µgL(-l) of CA exposure at the end of 96h. However, T4 level was decreased at all concentrations of CA and DCF during long-term (35 days) exposure period. Fish exposed to all concentrations of CA and DCF had lower level of T3 in both the treatments. These results suggest that both CA and DCF drugs induced significant changes (P<0.01 and P<0.05) on thyroid hormonal levels of C. mrigala. The alterations of these hormonal levels can be used as potential biomarkers in monitoring of pharmaceutical drugs in aquatic organisms.

Effects of clofibric acid alone and in combination with 17ß-estradiol on mRNA abundance in primary hepatocytes isolated from rainbow trout.

Clofibric acid (CA) is the active substance of lipid lowering drugs. It is resistant to degradation, polar in nature, and has been found ubiquitously in the aquatic environment. Though CA is classified as a peroxisomal proliferator in rodents and is considered as a potential endocrine disruptor, little information exists on the effects of CA in aquatic organisms, such as fish. In the present study, we examined the mRNA levels of peroxisome proliferator- and estrogen-sensitive genes on the exposure of primary rainbow trout (Oncorhynchus mykiss) hepatocytes to CA alone and in combination with the natural female sex hormone, 17ß-estradiol (E2). Our results demonstrate that rainbow trout hepatocytes are relatively refractory to the effects of CA on the PPAR signaling pathway and lipid metabolism. Moreover, CA did not show recognizable estrogenic activity, but after the induction of vitellogenesis by E2, CA significantly reduced vitellogenin (VTG) mRNA abundance. Apparently, the indirect repression of VTG transcription, independent of estrogen receptors, occurred. The mechanism is not yet clearly understood but may involve disruption of the stabilization of VTG mRNA known to be induced by E2.

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.

Short and long-term effects of clofibric acid and diclofenac on certain biochemical and ionoregulatory responses in an Indian major carp, Cirrhinus mrigala.

Extensive use of pharmaceuticals in human and veterinary medicine and aquaculture practices pose a serious threat to aquatic organisms. In the present investigation, Cirrhinus mrigala an Indian major carp was exposed to different concentrations of clofibric acid (CA) and diclofenac (DCF) and certain biochemical and ionoregulatory responses were assessed under short and long term exposures. During short-term (96h) exposure period, plasma glucose and sodium (Na(+)) levels were increased at all concentrations (1, 10 and 100µgL(-1)) of CA and DCF treated fish. Plasma protein and chloride (Cl(-)) levels were found to be decreased at all concentrations of CA and DCF exposed fish comparatively to control groups. Meanwhile an increase in plasma potassium (K(+)) level was noted in fish exposed to CA treatments alone and in DCF treatments it was decreased. In long-term exposure (35d), plasma Na(+) and Cl(-) levels were found to be significantly increased at all concentration of CA and DCF. However, a biphasic trend was observed in plasma glucose, protein and K(+) levels. In both the treatments, a significant (P<0.01 and P<0.05) changes were observed in all parameters measured in fish exposed to different concentrations of CA and DCF. The results of the present investigation indicate that both the drugs caused significant changes in biochemical and ionoregulatory responses of fish at all concentrations. The alterations of these parameters can be useful in monitoring of pharmaceutical residues present in aquatic environment.

Alteration in certain enzymological parameters of an Indian major carp, Cirrhinus mrigala exposed to short- and long-term exposure of clofibric acid and diclofenac.

The extensive use of pharmaceuticals in human and veterinary medicine may enter the aquatic environment and pose a serious threat to non-target aquatic organisms like fish. In this study, Indian major carp Cirrhinus mrigala was exposed to different concentrations (1, 10 and 100 µg L⁻¹) of most commonly used pharmaceutical drugs clofibric acid (CA) and diclofenac (DCF) to evaluate its impacts on certain enzymological parameters during short- and long-term exposures. During short-term (96 h) exposure period, plasma glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and gill Na⁺/K⁺-ATPase activity were significantly altered at all concentrations of both the CA- and DCF-treated fish. In long-term exposure (35 days), gill Na⁺/K⁺-ATPase activity was found to be significantly increased at all concentration of CA and DCF exposures throughout the study period (except at the end of 7th day in 10 and 100 µg L⁻¹) . However, a biphasic trend was observed in plasma GOT and GPT activity when compared to the control groups. In both short- and long-term exposure, a significant (P < 0.01 and P < 0.05) changes were observed in all enzymological parameters of fish C. mrigala exposed to different concentrations of CA and DCF. The alterations of these enzymological parameters can be effectively used as potential biomarkers in monitoring of pharmaceutical toxicity in aquatic environment and organisms.

Distribution and temporal evolution of pharmaceutically active compounds alongside sewage sludge treatment. Risk assessment of sludge application onto soils.

In this work, the distribution and the ecotoxicological risk of sixteen pharmaceutically active compounds belonging to seven different therapeutic groups (five anti-inflammatory drugs, two antibiotics, an anti-epileptic drug, a ß-blocker, a nervous stimulant, four estrogens and two lipid regulators) have been studied in sewage sludge from wastewater treatment plants. Only three of the sixteen pharmaceutical compounds were never detected in sludge while eleven of the studied pharmaceuticals were still detected in compost. Mean concentration levels of the pharmaceutically active compounds ranged between 24.9 and 4105 µg/kg dm, 14.5-944 µg/kg dm, 3.29-636 µg/kg dm and 9.19-974 µg/kg dm in primary, secondary, digested sludge and compost, respectively. An increase in the concentration levels of most of the pharmaceuticals was observed from summer to winter (mean values in primary and secondary sludge were 304 and 85.1 µg/kg dm in summer and 435 and 175 µg/kg dm in winter, respectively) probably due to an increase of their consumption during the coldest season and a reduction of the microbial activity under colder temperatures. The highest ecotoxicological risk, in digested sludge and compost, was due to the estrogenic compound 17ß-estradiol. The ecotoxicological risk significantly decreased after the application of digested sludge or compost to the soils (risk quotient values ranged between 0.04 and 252 in digested sludge and 0.002-37.8 in compost and decreased to 8·10(-4)-1.92 in digested sludge-amended soil and 1·10(-4)-0.23 in compost-amended soil).

Ecotoxicological impacts of clofibric acid and diclofenac in common carp (Cyprinus carpio) fingerlings: hematological, biochemical, ionoregulatory and enzymological responses.

Investigation on the toxic effects of pharmaceutical drugs namely clofibric acid (CA) and diclofenac (DCF) were studied in a common carp Cyprinus carpio at different concentrations such as 1, 10 and 100 µg L(-1) for a short-term period of 96 h under static bioassay method. At all concentrations, red blood cell (RBC), plasma sodium (Na(+)), potassium (K(+)), and glutamate oxaloacetate transaminase (GOT) levels were decreased in fish treated with CA and DCF. Contrastingly, white blood cell (WBC), plasma glucose, protein, lactate dehydrogenase (LDH) and gill Na(+)/K(+)-ATPase level were increased. However, a mixed trend was observed in hemoglobin (Hb), hematocrit (Hct), plasma chloride (Cl(-)), mean cellular volume (MCV), mean cellular hemoglobin (MCH), mean cellular hemoglobin concentration (MCHC) and glutamate pyruvate transaminase (GPT) levels. There was a significant (P<0.01 and P<0.05) change in all parameters measured in fish exposed to different concentrations of CA and DCF. In summary, the alterations in hematological, biochemical, ionoregulatory and enzymological parameters can be used as biomarkers in monitoring the toxicity of CA and DCF in aquatic environment. However, more detailed studies on using of specific biomarkers to monitor the human pharmaceuticals are needed.

Can pharmaceuticals interfere with the synthesis of active androgens in male fish? An in vitro study.

The in vitro interference of fibrate (gemfibrozil, clofibrate, clofibric acid), anti-inflammatory (ibuprofen, diclofenac), and anti-depressive (fluoxetine, fluvoxamine) drugs with key enzymatic activities-C17,20-lyase and CYP11ß-involved in the synthesis of active androgens in gonads of male carp have been investigated. Among the tested compounds, fluvoxamine and fluoxetine were the strongest inhibitors of C17,20-lyase and CYP11ß enzymes, with IC50s in the range of 321-335 µM and 244-550 µM, respectively. To our knowledge this is the first report on the interaction of pharmaceutical compounds with enzymatic systems involved in the synthesis of oxy-androgens. As oxy-androgens are known to influence spermatogenesis and stimulate reproductive behavior and secondary sexual characteristics in male fish, this work highlights the need for further investigating these endpoints when designing specific in vivo studies to assess the endocrine disruptive effect of pharmaceuticals in fish.

The value of repeating studies and multiple controls: replicated 28-day growth studies of rainbow trout exposed to clofibric acid.

Two studies to examine the effect of waterborne clofibric acid (CA) on growth-rate and condition of rainbow trout were conducted using accepted regulatory tests (Organisation for Economic Co-operation and Development [OECD] 215). The first study (in 2005) showed significant reductions after 21 d of exposure (21-d growth lowest-observed-effect concentration [LOEC] = 0.1 µg/L, 21-d condition LOEC = 0.1 µg/L) that continued to 28 d. Growth rate was reduced by approximately 50% (from 5.27 to 2.67% per day), while the condition of the fish reduced in a concentration-dependant manner. Additionally, in a concentration-dependent manner, significant changes in relative liver size were observed, such that increasing concentrations of CA resulted in smaller livers after 28-d exposure. A no-observed-effect concentration (NOEC) was not achieved in the 2005 study. An expanded second study (in 2006) that included a robust bridge to the 2005 study, with four replicate tanks of eight individual fish per concentration, did not repeat the 2005 findings. In the 2006 study, no significant effect on growth rate, condition, or liver biometry was observed after 21 or 28 d (28-d growth NOEC = 10 µg/L, 28-d condition NOEC = 10 µg/L), contrary to the 2005 findings. We do not dismiss either of these findings and suggest both are relevant and stand for comparison. However, the larger 2006 study carries more statistical power and multiple-tank replication, so probably produced the more robust findings. Despite sufficient statistical power in each study, interpretation of these and similar studies should be conducted with caution, because much significance is placed on the role of limited numbers of individual and tank replicates and the influence of control animals.

Application of the combination index (CI)-isobologram equation to study the toxicological interactions of lipid regulators in two aquatic bioluminescent organisms.

Pharmaceuticals in the aquatic environment do not appear singly and usually occur as complex mixtures, whose combined effect may exhibit toxicity to the aquatic biota. We report an environmental application of the combination index (CI)-isobologram equation, a method widely used in pharmacology to study drug interactions, to determine the nature of toxicological interactions of three fibrates toward two aquatic bioluminescent organisms, Vibrio fischeri and the self-luminescent cyanobacterial recombinant strain Anabaena CPB4337. The combination index-isobologram equation method allows computerized quantitation of synergism, additive effect and antagonism. In the Vibrio test, the fibrate combinations showed antagonism at low effect levels that turned into an additive effect or synergism at higher effect levels; by contrast, in the Anabaena test, the fibrate combinations showed a strong synergism at the lowest effect levels and a very strong antagonism at high effect levels. We also evaluated the nature of the interactions of the three fibrates with a real wastewater sample in the cyanobacterial test. We propose that the combination index-isobologram equation method can serve as a useful tool in ecotoxicological assessment.

Effects of lipid-lowering pharmaceuticals bezafibrate and clofibric acid on lipid metabolism in fathead minnow (Pimephales promelas).

The lipid-lowering agents bezafibrate and clofibric acid, which occur at concentrations up to 3.1 and 1.6 microg/L, respectively, are among the most frequently found human pharmaceuticals in the aquatic environment. In contrast to knowledge about their environmental occurrence, little is known about their effects in the environment. The aim of the present study was to analyze effects of these lipid-lowering agents in fish by focusing on their modes of action, lipid metabolism. Fathead minnows were exposed in aquaria to measured concentrations of 0.1, 1.27, 10.18, 101.56, and 106.7 mg/L bezafibrate and to 1.07, 10.75, and 108.91 mg/L clofibric acid for 14 and 21 d, respectively. After exposure, fish liver was analyzed for expression of peroxisome proliferator-activated receptor alpha (PPARalpha) by quantitative polymerase chain reaction (PCR), and the PPAR-regulated enzyme fatty acyl-coenzyme-A oxidase (FAO) involved in fatty acid oxidation. Bezafibrate had no effect, either on PPARalpha expression or on FAO activity, at all concentrations. In contrast, clofibric acid induced FAO activity in male fathead minnows at 108.91 mg/L. No increase in expression of PPARalpha messenger ribonucleic acid was observed. Egg production was apparently decreased after 21 d of exposure to 108.91 mg/L clofibric acid. The present study demonstrates that bezafibrate has very little or no effect on PPARalpha expression and FAO activity, but clofibric acid affects FAO activity.

Single and combined toxicity of pharmaceuticals and personal care products (PPCPs) on the rainbow trout liver cell line RTL-W1.

The toxicological implications of the presence of pharmaceuticals and personal care products (PPCPs) in the aquatic environment remain largely unknown. Acute toxicity tests have generally failed to detect the subtle action elicited by those compounds at environmentally relevant concentrations and they have often overlooked the fact that toxicity can be influenced by additive and synergistic effects. The aim of this study was to further assess the cytotoxicity of different pharmaceuticals and synthetic musks as well as their mixtures on the rainbow trout liver cell line RTL-W1. Eleven pharmaceuticals from different therapeutic classes (anti-inflammatory drugs, serotonin re-uptake inhibitors and lipid regulators) and five synthetic musks from the two major groups (nitro- and polycyclic musks) were selected for the study. Two fluorescent dyes were used to monitor cell viability. Among the tested compounds, estimated EC50s (effective concentration causing 50% decline of cell viability) denoted that polycyclic musks (7-25 microM) followed by anti-depressives (7-50 microM) showed the highest potential to induce cytotoxicity, whereas lipid regulators (20-380 microM), anti-inflammatory drugs (160-260 microM) and nitromusks (100-240 microM) had the lowest toxicity. Within a given therapeutic class, combined toxicity of mixtures was additive, following in most cases the concentration addition concept. However, the combined toxicity was higher than additive for those mixtures that included one compound from each class (i.e. dissimilar mixtures). Overall, this study shows that in the aquatic environment, toxicity of PPCPs on non-target organisms may occur at concentrations lower than expected due to synergistic effects between the different toxicants.

In vitro genotoxic assessment of xenobiotic diacylglycerols in an in vitro micronucleus assay.

Xenobiotic diacylglycerols (DG) may induce pathological disorders by causing abnormal chromosomal segregation, which could be aneuploid. In this study, seven xenobiotic-diacylglycerols (four of drug origin and three of pesticide origin) were evaluated for their ability to induce aneuploidy in mammalian cultures using in vitro cytokinesis blocked micronucleus (CBMN) assay coupled with kinetochore labeling and interphase fluorescent in situ hybridization. Out of seven xeno-DGs, two (ibuprofen-DG and fenbufen-DG) induced statistically significant (P < 0.001) and dose-dependent increase in micronucleus induction, but this apparent micronucleus induction was very weak in case of fenbufen-DG. These MN were produced predominantly by aneugenic and clastogenic mechanisms, respectively, confirmed by immunofluorescent labeling of kinetochores. Fluorescent in situ hybridization analysis revealed that ibuprofen-DG induced significantly higher nondisjunction for chromosomes 10, 17, and 18. Other xenobiotic diacylglycerols (indomethacin-DG, salicylic acid-DG, 4-(2-methyl-4-chlorophenoxy) butanoic acid-DG (MCPB-DG), 2-(2-methyl-4-chlorophenoxy) propanoic acid-DG (MCPP-DG) and 2-(4-dichlorophenoxy)-butanoic acid-DG (2,4 DB-DG) did not induce micronuclei, but the concentrations tested did not reach levels that caused the marked growth suppression typically required for testing for regulatory testing purposes. However, the levels of growth suppression achieved were similar to that seen with ibuprofen-DG, which was positive. This study shows that xeno-DGs, which have been neglected in the past for their possible link to any pathological disorders, need serious assessment of their mutagenic potential.

Ciprofibrate, clofibric acid and respective glycinate derivatives. Effects of a four-week treatment on male lean and obese Zucker rats.

Fibrates are widely prescribed in hyperlpidemic patients to prevent atherosclerosis. Their therapeutic use, however, can be associated with adverse effects like gastrointestinal disorders, myalgia, myositis and hepatotoxicity. In rodents large doses can even cause hepatocellular carcinoma. Additionally, interactions with the biotransformation of other compounds at the cytochrome P450 (CYP) system have been observed. Thus, the discovery of new substances or derivatives with less side effects is of great interest. In the present study the influence of a four-week daily oral administration of 2 mg/kg body weight ciprofibrate (CAS 52214-84-3) or of 100 mg/kg body weight clofibric acid (CAS 882-09-7) was compared to that of the respective doses of their newly synthesized glycine conjugates in adult male lean and obese Zucker rats. Although obese rats displayed distinctly higher serum lipid concentrations, after fibrate treatment values were significantly lowered in lean animals only. Livers of obese rats were significantly enlarged, histologically showing a fine-droplet like fatty degeneration and an increase in glycogen content, but no signs of inflammation. After fibrate administration histologically a hypertrophy, an eosinophilia, a reduced glycogen content and also hepatocyteapoptosis were observed. Livers of obese rats displayed higher CYP1A1 andCYP2E1 expression, but lower immunostaining for CYP2B1 and CYP3A2. No differences between the two groups of rats were seen with respect to CYP4A1 expression. Due to fibrate treatment especially CYP2E1 and CYP4A1, but also CYP1A1, 2B1 and 3A2 were induced. Resulting CYP mediated monooxygenase activities were also elevated in most cases. In general, effects of clofibric acid and clofibric acid glycinate (CAS 4896-55-3) were less distinct than those of ciprofibrate and its glycinate (CAS 640772-36-7). With no parameterinvestigated major differences were seen between the parent fibrates and their glycine conjugates. Thus, the present investigations revealed no noticeable advantages of the glycinates over ciprofibrate or clofibric acid.