Chemical and toxicological characterisation of anticancer drugs in hospital and municipal wastewaters from Slovenia and Spain

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Derivatives of 2-phenylcinnamic acid as inhibitors of human hydroxysteroid dehydrogenases AKR1C1 and AKR1C3

Hidroksisteroid-dehidrogenaze AKR1C1–AKR1C3 sodijo v naddruţino aldo/keto-reduktaz in so udeleţene v metabolizmu steroidnih hormonov, nevrosteroidov, ksenobiotikov in prostaglandinov. AKR1C3 katalizira redukcijo androstendiona v testosteron in estrona v estradiol, kar posredno lahko privede do povišane proliferacije celic v prostati in dojkah. AKR1C3 katalizira tudi redukcijo prostaglandina PGH2 v PGH2α in PGD2 v 9α,11β-PGF2 ter tako prepreči aktivacijo PPARγ, ki celice usmerja v apoptozo. AKR1C1 je 20-ketosteroid-reduktaza, ki pretvarja aktivni progesteron v manj aktivni 20α-hidroksiprogesteron in močan nevrosteroid 5α-pregnan-3α-ol-20-on v 5α-pregnan-3α,20α-diol. Na ta način AKR1C1 zmanjša zaščitne učinke progesterona v maternici in ektopičnem endometriju in je tako udeleţen pri nastanku raka endometrija, endometrioze in predmenstrualnega sindroma. Zaradi vpliva na metabolizem nevrosteroidov encim AKR1C1 povezujejo tudi z nastankom depresivnih motenj. AKR1C2 katalizira inaktivacijo 5α-dihidrotestosterona v 3α-androstandiol in s tem posledično ščiti celice prostate pred prekomerno proliferacijo. AKR1C1 in AKR1C3 imata pomembne vloge pri različnih patofizioloških stanjih in sta zato pomembni tarči za zdravljenje hormonsko odvisnih in hormonsko neodvisnih rakavih obolenj ter drugih bolezni. Predhodne študije so pokazale, da derivati cimetne kisline in NSAID analogi inhibirajo AKR1C izoencime. Zato smo v tej študiji preverili inhibitorno delovanje 56 strukturno sorodnih spojin iz treh različnih skupin: 1) derivate fumarne kisline, 2) derivate 3-aminobenzojske kisline in 3) derivate 2-fenilcimetne kisline na rekombinantnih encimih AKR1C1, AKR1C2 in AKR1C3. Najprej smo določili % inhibicije reakcije oksidacije 1-acenaftola, ki jo katalizirajo encimi AKR1C1, AKR1C2 in AKR1C3, v prisotnosti 100 μM posamezne spojine in 30 μM, 60 μM oziroma 100 μM substrata. Najboljši inhibitorji so bili derivati 2-fenilcimetne kisline. Spojinam, ki so encime inhibirale za več kot 50 %, smo določili vrednosti IC50. 17 spojin je imelo vrednosti IC50 v nizkem μM območju. Najboljša inhibitorja sta bili spojini 13c z 6,6 μM IC50 vrednostjo za AKR1C1 in spojina 26c z 5,8 μM IC50 vrednostjo za AKR1C3. Našli smo tudi 7 sprecifičnih inhibitorjev AKR1C3, to so bile spojine 26c, 21c, 24c, 10c, 22c, 23c in 1c z vrednostmi IC50 5,8 μM10,8 μM16,8 μM38,7 μM43,1 μM69,8 μM in 121,1 μM. Te spojine predstavljajo dobro izhodišče za razvoj novih specifičnih inhibitorjev AKR1C3 pri zdravljenju raka prostate in dojk ter akutne mieloidne levkemije.Hydroxysteroid dehydrogenases AKR1C1–AKR1C3, members of the aldo-keto reductase superfamily, are involved in metabolism of steroid hormones, neurosteroids, xenobiotics and prostaglandins. AKR1C3 catalyzes reduction of androstenedione to testosterone and estrone to estradiol, and may thus promote the excessive proliferation of prostate and breast cells. AKR1C3 also catalyzes reduction of PGH2 to PGF2α and PGD2 to 9α,11β-PGF2 and thus prevents activation of PPARγ and cell apoptosis. AKR1C1 as 20-ketosteroid reductase inactivates progesterone by producing 20α-hydroxyprogesterone and also converts a potent neurosteroid 5α-pregnane-3α-ol-20-one to 5α-pregnane-3α,20α-diol. In this manner AKR1C1 prevents protective effects of progesterone in uterus and ectopic endometrium and is involved in promotion of endometrial cancer, endometriosis and premenstrual syndrome. Due to its role in neurosteroid metabolism it is also associated with depressive disorders. AKR1C2 catalyzes inactivation of 5α-DHT to 3α-androstanediol and thus protects prostate cells from excessive proliferation. AKR1C1 and AKR1C3 have important roles in different pathophysiological conditions so they represent important drug targets in hormone-dependent and hormone-independent cancers and other diseases. Previous studies showed that cinnamic acid derivatives and NSAID analogs inhibit AKR1C isoenzymes. Therefore, the aim of this study was to evaluate 56 compounds with similar structural characteristics from three different groups of compounds: 1) derivatives of fumaric acid, 2) derivatives of 3-aminobenzoic acid and 3) derivatives of 2-phenylcinnamic acid for their inhibition of the recombinant AKR1C1, AKR1C2 and AKR1C3 enzymes. First, we determined the percentages of inhibition of 1-acenaphthenol oxidation in the presence of 100 μM of individual compound and 30 μM, 60 μM and 100 μM concentration of substrate, respectively. The best inhibitors were derivatives of 2-phenylcinnamic acid. For compounds that showed at least 50 % inhibition of AKR1C1-AKR1C3 the IC50 values were determined. 17 compounds showed low μM IC50 values. The best inhibitor of AKR1C1 and AKR1C3 were compounds 13c and 26c with 6.6 μM and 5.8 μM IC50 values, respectively. We found 7 AKR1C3-specific inhibitors, compounds 26c, 21c, 24c, 10c, 22c, 23c and 1c, showing 5.8 μM10.8 μM16.8 μM38,7 μM43.1 μM69.8 μM and 121.1 μM IC50 values. These compounds represent good starting points for development of new AKR1C3-specific agents for treatment of acute myeloid leukemia and prostate and breast cancer

Evaluation of acute and chronic ecotoxicity of cyclophosphamide, ifosfamide, their metabolites/transformation products and UV treated samples

Cyclophosphamide (CP) and Ifosfamide (IF) are two nitrogen mustard drugs widely prescribed in cancer therapy. They are continuously released via excreta into hospital and urban wastewaters reaching wastewater treatment plants. Although CP and IF, their metabolites and transformation products (TPs) residues have been found in the aquatic environment from few ng L−1 to tens of μg L−1, their environmental toxic effects are still not well known. The present study aimed to investigate the acute and chronic ecotoxicity of CP and IF and their commercially available human metabolites/TPs, i.e. carboxy-CP, Keto-CP and N-dechloroethyl-CP on different organisms of the aquatic trophic chain. The experiments were performed using the green alga Pseudokirchneriella subcapitata, the rotifer Brachionus calyciflorus and the crustaceans Thamnocephalus platyurus and Ceriodaphnia dubia. Moreover, to assess the treatment conditions in regards to parent compound removal and formation of new TPs, CP and IF were UV- irradiated for 6 h, 12 h, 24 h, 36 h and 48 h, followed by toxicity evaluation of treated samples by algae, rotifers and crustaceans. Between the parent compounds, IF resulted as more toxic drug under tested conditions, exerting both acute and chronic effects especially on C. dubia (LC50:196.4 mg L−1, EC50:15.84 mg L−1). Among the tested metabolites/TPs, only carboxy-CP inhibited the reproduction in the rotifer. However, LOEC and NOEC values were calculated for CP and IF for all organisms. In addition, despite a low degradation of CP (28%) and IF (36%) after 48 h UV-irradiation, statistically significant effect differences (p < 0.05) from not-irradiated and irradiated samples were observed in both acute and chronic assays, starting from 6 h UV-irradiation. Our results suggest that the toxic effects found in the aquatic organisms may be attributable to interactions between the parent compounds and their metabolites/TPs. The interactions among CP, IF and their metabolites/TPs may cause environmental toxic effects on aquatic organisms

First inter-laboratory comparison exercise for the determination of anticancer drugs in aqueous samples

The results of an inter-laboratory comparison exercise to determine cytostatic anticancer drug residues in surface water, hospital wastewater and wastewater treatment plant effluent are reported. To obtain a critical number of participants, an invitation was sent out to potential laboratories identified to have the necessary knowledge and instrumentation. Nine laboratories worldwide confirmed their participation in the exercise. The compounds selected (based on the extent of use and laboratories capabilities) included cyclophosphamide, ifosfamide, 5-fluorouracil, gemcitabine, etoposide, methotrexate and cisplatinum. Samples of spiked waste (hospital and wastewater treatment plant effluent) and surface water, and additional non-spiked hospital wastewater, were prepared by the organising laboratory (Jožef Stefan Institute) and sent out to each participant partner for analysis. All analytical methods included solid phase extraction (SPE) and the use of surrogate/internal standards for quantification. Chemical analysis was performed using either liquid or gas chromatography mass (MS) or tandem mass (MS/MS) spectrometry. Cisplatinum was determined using inductively coupled plasma mass spectrometry (ICP-MS). A required minimum contribution of five laboratories meant that only cyclophosphamide, ifosfamide, methotrexate and etoposide could be included in the statistical evaluation. z-score and Q test revealed 3 and 4 outliers using classical and robust approach, respectively. The smallest absolute differences between the spiked values and the measured values were observed in the surface water matrix. The highest within-laboratory repeatability was observed for methotrexate in all three matrices (CV ≤ 12 %). Overall, inter-laboratory reproducibility was poor for all compounds and matrices (CV 27–143 %) with the only exception being methotrexate measured in the spiked hospital wastewater (CV = 8 %). Random and total errors were identified by means of Youden plots

Human metabolites and transformation products cyclophosphamide and ifosfamide: analysis, occurrence and formation during abiotic treatments

This study describes a gas chromatography-mass spectrometry analytical method for the analysis of cytostatic cyclophosphamide (CP), ifosfamide (IF) and their selected metabolites/transformation products (TPs): carboxy-cyclophosphamide (carboxy-CP), keto-cyclophosphamide (keto-CP) and 3-dechloroethyl-ifosfamide/N-dechloroethyl-cyclophosphamide (N-decl-CP) in wastewater (WW). Keto-cyclophosphamide, CP and IF were extracted with Oasis HLB and N-decl-CP and carboxy-CP with Isolute ENV+ cartridges. Analyte derivatization was performed by silylation (metabolites/TPs) and acetylation (CP and IF). The recoveries and LOQs of the developed method were 58, 87 and 103 % and 77.7, 43.7 and 6.7 ng L−1 for carboxy-CP, keto-CP and N-decl-CP, respectively. After validation, the analytical method was applied to hospital WW and influent and effluent samples of a receiving WW treatment plant. In hospital WW, levels up to 2690, 47.0, 13,200, 2100 and 178 ng L−1 were detected for CP, IF, carboxy-CP, N-decl-CP and keto-CP, respectively, while in influent and effluent samples concentrations were below LOQs. The formation of TPs during abiotic treatments was also studied. Liquid chromatography-high-resolution mass spectrometry was used to identify CP and IF TPs in ultrapure water, treated with UV and UV/H2O2. UV treatment produced four CP TPs and four IF TPs, while UV/H2O2 resulted in five CPs and four IF TPs. Besides already known TPs, three novel TPs (CP-TP138a, imino-ifosfamide and IF-TP138) have been tentatively identified. In hospital WW treated by UV/O3/H2O2, none of the target metabolites/TPs resulted above LOQs