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Karl Fent, Sara Zucchi & Verena Christen

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1 Environmental impact of sunscreeens and personal care products in the aquatic environment
Karl Fent, Sara Zucchi & Verena Christen University of Applied Sciences Northwestern Switzerland Muttenz, Switzerland and Swiss Federal Institute of Technology (ETHZ), Zürich

2 UV-filters Physically acting filters: TiO2, ZnO nanoparticles
Scatter and reflect UV rays Organic UV-absorbing chemicals (contain one or more aromatic rings) Absorb energetic solar photons and returning to the ground state by thermally emitting absorbed energy UVA: nm, UVB: nm Currently 28 UV-filters permitted for sunscreens and cosmetics in Europe

3 Organic UV-filters in sunscreens and cosmetics
Shampoo Sunscreens Deo Octocrylene (OC) Ethylhexyl-methoxy cinnamate (EHMC) Benzophenone-3 (BP3) Benzophenone-4 (BP4) OC, EHMC Aftershave Hairspray Nail Polish Lotions EHMC BP1, BP3 BP4 BP4 BP3 2011

4 How do UV-filters enter the aquatic environment?
indirectly directly

5 Many UV-filters are lipophilic and tend to bioaccumulate
in biota and in human milk

6 Glatt Estrogenic UV-filters Polar: BP4 and BP3
May 2007 Estrogenic UV-filters Polar: BP4 and BP3 Lipophilic: 4MBC and EHMC POCIS and fate modelling show that wastewater is a significant source

7 Swiss rivers: BP-4 and EHMC most important
Water: Passive sampling (POCIS) BP-4 (log P = 0.89) up to ng BP-3 (log P = 3.64) up to ng 4-MBC (log P = 4.95) up to ng EHMC (log P = 5.66) up to ng Fish and macroinvertebrates BP-4 not detected BP-3 up to 151 ng/g lipids 4-MBC rarely detected EHMC up to 337 ng/g lipids BP-4 in Spain: up to 850 ng/L in rivers and up to 1480 ng/L in untreated wastewater (Rodil et al., 2008). Zenker et al. 2008, J. Chromatogr. A: 1202: 64-74, Fent et al. (2010) Environ. Pollut. 158: 1817

8 EHMC in all 53 biota, residues higher in cormorants
but no significant biomagnification (BMF max. 1.5) Fent et al. (2010) Environ. Pollut.158:

9 BP4 is most prominent in water and EHMC in biota, others are also present Is there an ecological concern? What is their hormonal activity? What is their mode of action?

10 Effects on zooplankton Daphnia magna
Toxicity increases with lipophilicity UV filter Log Pow LC50 (48 h) (mg/L) Benzophenone-4 (BP-4) 0.89 50 Benzophenone-3 (BP-3) 3.64 1.9 4-Methylbenzylidene camphor (4-MBC) 4.95 0.56 2-Ethyl-hexyl-4-trimethoxycinnamate (EHMC) 5.66 0.29 Chronic (reproduction 21 d) 0.05 Fent et al. 2010, Mar.Env.Res. 69: S4-S6

11 Analysis of endocrine effects
In vitro hormonal activity (YES, YAS) Estrogenicity, antiestrogenicity Androgenicity, antiandrogenicity In vivo estrogenicity in fish: vitellogenin induction BP-1, BP-2, 3-BC, Et-PABA, BP-4, EHMC In vivo reproduction effects: decreased fertility 3-BC, BP-2, EHMC Mixture activity in vitro and in vivo in fish Molecular analysis Gene expression analysis: BP4 Transcriptomics: EHMC

12 Hormonal activity in vitro
19 UV-Filters Estrogenic Antiestrogenic Androgenic Antiandrogenic _______________________________9__________14_____________6___________10_____ Benzophenone Benzophenone 4,4‘-Dihydroxybenzophenone Ethyl-4-aminobenzoate Benzophenone-3 (BP-3) nd Benzophenone-4 (BP-4) nd Benzylsalicylate (BS) nd Phenylsalicylate (PS) nd 3-Benzylidene camphor (3-BC) Ethylhexyl methoxy cinnamate (EHMC = OMC) Octyl salicylate (OSC) 4-Methylbenzylidene camphor (4-MBC) Homosalate (HMS) Isopentyl-4-methylcinnamate (IMC) Octocrylene (OC) nd 4-Aminobenzoate (PABA) nd (N,N')-dimethyl-octyl-4- aminobenzoate (OD-PABA) nd Ethyl 4-aminobenzoate (Peg25 PABA) nd Kunz & Fent (2006), Aquat. Toxicol.79:

13 Estrogenic activity in vitro – in vivo
Kunz et al. (2006). Toxicological Sciences 93:

14 Activity of UV-filter mixtures in vitro
Four and eigth UV filters show marked activity at NOEC They act synergistically in vitro Recombinant yeast YES Kunz & Fent (2006) Tox. Appl Pharmacol 217: 86-99

15 Mixture activity of 3 estrogenic UV-filters
BP1, BP2, 3BC in fish in vivo UV filters show concentation addition for estrogenicity in mixtures Tendency to antagonism at low concentrations Kunz and Fent, Toxicol.Appl.Pharmacol. 234: 77-88, 2009

16 Benzophenone 2 Estrogenic activity (vitellogenin induction) and effects on fertility and reproduction in fathead minnows

17 Effects of BP-2 on fertility in fathead minnows: egg production reduced at 1.2 mg/L and higher

18 Secondary sexual characteristics in males
Dose-dependent feminization and effects on fertility (decrease in egg production): LOEC for 3BC: 3 ug/L, for BP-2: 1.2 mg/L 3-Benzylidene camphor Benzophenone-2 Kunz et al. (2006) Tox Sci.; Weisbrod et al. (2007), Toxicol. Appl. Pharmacol..

19 Effects of benzophenone-4: gene expression analysis with focus on hormonal pathways in zebrafish
Zebrafish: embryo study 5 days exposure in 24-well microtiter plates, 6 replicates per dose 30 µg/L (worst case environmental level) and 3 mg/L after 120 hpf, 15 larvae were pooled for qRT-PCR Zebrafish: male adults 14 d exposure to 3 concentrations, 5 replicates each 3 concentrations: 30 µg/L, 300 µg/L and 3 mg/L after 14 d dissection of brain, liver and testis, RNA extraction qRT-PCR Targeted gene expression approach Zucchi et al. (2011), Tox. Appl. Pharmacol. 250:

20

21 Expression of genes involved in 4 different hormonal pathways investigated by qRT-PCR
Estrogenic, androgenic, steroidogenesis, thyroid hormone system develop. Embryo: 10 gene transcripts Vtg1 Vtg3 Estrogen receptor alpha (esr1) Estrogen receptor beta1 (esr2b) Androgen receptor 17 ß-HSD (hydroxsteroid dehydr.) P450aromA (CYP19A) P450aromB (CYP19B) paired box gene 8 (pax8) hematopoietically expressed homeobox (hhex) Adult males: 11 transcripts Vtg1 Vtg3 Estrogen receptor alpha (esr1) Estrogen receptor beta1 (esr2b) igfbp1a (liver) Androgen receptor 17 ß-HSD (hydroxsteroid dehydr.) P450aromA (CYP19A) (testis) P450aromB (CYP19B) (brain) StAR (testis) 3 ß–HSD (testis)

22 Effects on genes involved in steroidogenesis
P450aromB (cyp19b) P450aromA (cyp19a) Hydroxysteroid 17-ß dehydrogenase-3 (hsd17ß3)

23 Brain Liver Estrogen responsive genes (Hoffman et al., 2006) Testis
P450aromB (cyp19b) Brain *** Liver Insulin-like growth factor binding protein 1 (igfbp1) *** ** * Testis ** * P450aromA (cyp19a)

24 BP-4: Summary BP4 leads to alteration of gene expression in adult fish and embryos. Effects in adults occur already at 30 µg/L, in embryos mainly at 3 mg/L. Expressional changes differ between different tissues, and also between embryos and adults. Embryos: Estrogen-like activity Up-regulation of vtg1 and vtg3 at 3 mg/L, esr1, esr2b (at 30 µg/L ) Embryos: Upregulation of genes involved in steroidogenesis aromatases cyp19a, cyp19b and hsd17ß3 Adults: Liver: Antiestrogen-like activity by down-regulation of vtg, esr, but up-regulation of igfbp1 Brain and testis: Induction of vtg > Consequence: Interference with hormonal pathways in embryos and adults BP-4 has low activity: alterations occur mainly at 3 mg/L Effects on physiology, steroid levels, sex determination and fertility? Zucchi et al. 2011, TAAP 250:

25 Effects of EHMC on gene expression, vitellogenin induction and histology of gonads in fathead minnows Christen et al. (2011)

26 Down-regulation of androgen receptor in liver
after 14 d of exposure of fathead minnows E: 5.4 g/L EHMC L: 37.5 g/L EHMC M: mg/L EHMC H: 394 mg/L EHMC

27 Down-regulation of 3-beta hydroxysteroid dehydrogenase
Conversion of pregnenolone > progesterone E: 5.4 g/L EHMC L: 37.5 g/L EHMC M: mg/L EHMC H: 394 mg/L EHMC

28 Induction of plasma vitellogenin in males
E: 5.4 g/L EHMC L: 37.5 g/L EHMC M: mg/L EHMC H: 394 mg/L EHMC

29 Histology of gonads: fathead minnows
Testis A: water B: solvent C: 5.4 g/L EHMC D: 37.5 g/L EHMC E: mg/L EHMC F: 394 mg/L EHMC Sg: spermatogonia Sc: spermatocytes M: spermatides Christen et al. (2011)

30 Histological effects on gonads
Testes Ovaries E: 5.4 g/L EHMC L: 37.5 g/L EHMC M: mg/L EHMC H: 394 mg/L EHMC Increase in vitellogenic (and in atretic) oocytes Decreased number of spermatocytes increase in spermatides (sperms)

31 What is the mode of action of EHMC
What is the mode of action of EHMC? What toxicological pathways are affected? In vitro study showed antiestrogenic and antiandrogenic activity

32 Analysis of molecular effects and modes of action
of EHMC in zebrafish: Transcriptomics (Agilent 4×44 K Zebrafish) Quantitative real time PCR Microarrays Aim: Determine molecular effects and mode of action in whole body of fish after 14 d exposure to 2.2 (environmental) and 890 µg/L (toxicological relevant) (measured concentrations). Whole body (RNA extraction) for microarray analysis 3 tissue collected for qRT-PCR validation analysis (liver, brain and testis).

33 Venn Diagramm: Whole body transcription profile
3000 µg/L (ANOVA; p<0.05, fold change ≥ 2, TukeyHSD as Posthoc Test, FDR = 0.05) At 2.2 µg/L: 1096 genes At 890 µg/L: 1137 genes Environmental Concentration (ANOVA; p<0.05, und fold change ≥ 2, All genes 2.2 µg/L 890 µg/L 594 (54.2%) ↓ 699 (61.48%) ↓ 502 (45.8%) ↑ 438 (38.52 %) ↑ 708 genes altered in common GeneSpring GX11 4 replicates for each exposure groups Zucchi et al. (2011) Env. Poll. 159: 3086 33

34 Selected transcripts among altered pathways
GO process Investigated transcripts (qPCR) Lipid metabolism/ Response to estrogen stimulus vitellogenin 1 (vtg1) ↓ growth arrest and DNA-damage-inducible beta (gadd45b) ↑ Apoptosis/cell differentiation Response to stress Lipid Biosynthesis and regulation prostaglandin D2 synthase (ptgds) ↑ vitamin A metabolic process/ retinol metabolic process retinol binding protein 2 (rbp2) ↓ regulation of cell growth insulin receptor signaling pathway tissue regeneration Insulin-like growth factor binding protein 1 (igfbp1) ↑ MetaCore software (FDR< 0.05) 34

35 After confirmation of microarray data with qRT-PCR:
Whole body vs. liver brain and testis qRT-PCR validation prostaglandin D2 synthase Whole body Liver Brain Testis Whole body Liver Brain Testis Zucchi et al. (2011) Env. Poll. 159: 3086 35

36 EHMC: Summary EHMC leads to differential gene expression in liver, gonads and brain. Changes differ between tissues, and between males and females of zebrafish Expressional changes point to multiple hormonal activities Estrogen-like Induction of vitellogenin protein in plasma Down-regulation of ar in liver Interference with genes involved in steroidogenesis Down-regulation of hsd3 Histological effects: lower spermatocytes and altered oocyte maturation (ovary) in fathead minnows (fhm) Expressional changes at 2.2 and 890 g/L (zebrafish). Histology: g/L, VTG induction: 245 g/L (fhm). Highest level in raw wastewater 19, in surface water 3 g/L Christen et al. 2011, Aquat. Toxicol. 102:

37 Measured environ conc.(MEC)
Environmental risk assessment for UV filters see also Fent et al. 2008, Chimia 62: for 3BC, BP1, BP2, Et-PABA Measured environ conc.(MEC) (Langford 2007; Rodil 2008,2009) (own data) BP4: ug/L EHMC: 0.238, 3 ug/L ng/L 4MBC: 1.14 ug/L ng/L BP3: ug/L ng/L 3BC: det. BP1, BP2: n.d. Et-PABA: n.d. Effect concentration (PNEC) Ref: fish: literature, own data, Daphnia: own data BP4: 0.03 ug/L (fish), 50 ug/L (Daphnia) EHMC: ug/L (fish), 0.29 ug/L (Daphnia) 4MBC: 0.74 ug/L (fish), 0.56 ug/L (Daphnia) BP3: 0.62 ug/L VTG, decreased fecundity in fish 3BC: 30 ng/L (Kunz et al. 2006) (SF 100) Safety factor (SF) 1000 (acute tox., gene expr.) MEC / PNEC Environmental risk for BP4, EHMC, 4MBC. 3BC and BP2: Risk probable, however not yet detected in environment Mixture activity must be considered: additive action of EHMC, BP3, OC Fent et al. 2010, Mar.Env.Res. 69: S3-S6

38 Conclusions Chemicals in cosmetics and personal care products lead to chronic exposure of humans and environment UV-filters 3BC, Et-PABA, BP2, BP4 and EHMC affect hormonal system of fish, although some at higher than environmental concentrations BP4, EHMC: Targeted gene expression and transcriptomics reveals molecular effects and modes of action: More pathways than hormonal signalling are affected Gene expression analysis is more sensitive than physiological or histological effects Transcriptional changes are complex, tissue-dependent and different in males and females EHMC shows multiple hormonal activities and affects many different cellular, biochemical and physiological pathways

39 Cosmetics containing phthalates, disinfectans (triclosan)
Phthalates (DEP) Disinfectants Shampoos Tooth paste Hair styling After shave Shaving foam Fragrances Lotions Cleansing Deo Conditioner Anti ageing products Shaving foam

40 Some phthalates have anti-androgenic activity in vitro (MDA-kb2 cells) benzylbutyl phthalate, dibutyl phthalate, diethyl phthalate Christen et al. Chemosphere 2010

41 Binary mixtures of phthalates BBP, DBP and DEP show
concentration addition

42 Binary mixtures show concentration addition with tendency to
antagonism at low and synergism at high levels (toxic unit approach) EC50 mixture EC25 mixture no lines EC10 mixture

43 Antimicrobials/disinfectants triclosan and triclocarban
Triclocarban (TCC) used in PCPs since 1957 Triclosan (TCS) widespread use Widespread in environment. Found in surface waters: TCC: ng/L, median 95 ng/L TCS: ng/L, median 48 ng/L Algae have high sensitivity to TCS through disruption of lipid synthesis, membrane destabilization, uncoupling of oxidative phosphorylation Acute ffects in invertebrates and fish in the hundreds of ug/L through unspecific narcosis Hormonal effect of TCS in fish (Raut & Angus, Env.Tox.Chem. 2010, 29:1287) Vitellogenin mRNA expression significantly elevated at ug/L (350 nM) TCS. Sperm counts significantly decreased in western mosquitofish, Gambusia affinis)

44 Enhancement of androgenic activity by biocides
triclosan (TCS) and triclocarban (TCC) LOEC of TCS 10 nM=2.9 ug/L Christen et al. 2010, Chemosphere 81:

45 Financial support

46

47 Endocrine activity shown mammals…

48 Number of positive samples (%)
UV filters accumulate in human milk 54 samples analysed between UV filter Number of positive samples (%) Mean level (ng/g) lipids Ethylhexyl-methoxy cinnamate (EHMC) 42 (78%) 27.5 Octocrylene (OC) 36 (67%) 30.2 4-Methylbenzylidene camphor (4MBC) 11 (20%) 22.1 Benzophenone-3 (BP3) 7 (13%) 52.2 Homosalate 3 (6%) 29.4 Octyldimethyl-PABA 1 (2%) 49.0 Schlumpf et al. 2010, Chemosphere 81:

49 Mixtures of 2 UV filters show mainly synergistic activity in vitro
Recombinant yeast YES Kunz & Fent (2006) Tox. Appl Pharmacol 217: 86-99

50 Effects on fecundity and reproduction: 3BC and BP2
3BC (3-Benzylidene camphor) Kunz et al. (2006). Toxicol.Sci. 90 (2): BP2 (Benzophenone-2) Christine Weisbrod investigated the UV fiter Benzophenone-2 and I looked at possible adverse effects of 3BC on fish reproduction. So, you may ask yourself why these to compounds were chosen, and not 4-MBC for example, which is known to be estrogenic in rats?? We found that both compounds, BP2 as well as 3BC, were estrogenic in vitro in yeast reporter gene systems. Which were carrying either a human estrogen receptor or a rainbowtrout estrogen receptor. ABB yeast: Here you see the plotted estrogenic activity of these compounds, whereas on the X-axes you see the concentration and on the Y-axes the effect elicited by the compound, as a percentage of the maximal effect of E2, which was used as a standard. The dotted lines represent our findings for the hER, whereas the solid lines represent the estrogenicity found towards the rtER. As you probably already noticed, BP2 and 3BC show different estrogenic activites. Interestingly BP2 possesses maximal activity towards both ERs, whereas 3BC only shows submaximal activity towards both ERs. Furthermore BP-2 is more active towards the hER whereas 3BC on the other hand is more active towards the rtER. If you look at the sensitivities of the two different ER to E2 your see that the hER is more sensitive to E2 than the rtER, and this leads when looking at the potencies of BP2 and 3BC to a generally higher potency of these UV filters toward the rtER. So we asked ourselves, is the yeast assay carrying the rtER better in order to predict in vivo activity of 3BC and BP2 in fish?? In order to see if this is the case we investigated these two UV filters in juvenile fish for vitellogenin induction during a 14-d exposure. VTG is a biomarker for estrogenic compounds and can be only measured in fish when exposed to estrogens or xenoestrogens. This is what we found ABB fish: BP2 and 3BC were estrogenic in juvenile fish, but 3BC, which possessed weaker estrogenic activity in yeast was the more estrognic compound in fish. BP2 was approx. 10-times weaker than 3BC. TAB und FRAGE: Hence BP2 as well as 3BC were estrogenic in vitro and in juvenile fathead minnows IN VIVIO. So we were wondering if……. After we saw Yeast in vitro Fish in vivo BP2 estrogenic (full agonist) estrogenic (VTG induction) 3BC estrogenic (partial agonist) estrogenic (VTG inductionI Kunz & Fent (2006). Aquat. Toxicol. 79:

51 Histology of gonads Ovary A: water B: solvent C: 5.4 g/L EHMC
D: 37.5 g/L EHMC E: mg/L EHMC F: 394 mg/L EHMC 1: primary oocyte 2: previtellogenic oocyte 3: vitellogenic oocyte

52 Expression of estrogen receptor
Males Females E: 5.4 g/L EHMC L: 37.5 g/L EHMC M: mg/L EHMC H: 394 mg/L EHMC

53 Expression of vitellogenin (qPCR)
Males Females E: 5.4 g/L EHMC L: 37.5 g/L EHMC M: mg/L EHMC H: 394 mg/L EHMC

54 cyp19b fold change is 2.4 Whole body Liver Brain Testis Whole body

55 Scan and quantification of the raw data
Exposure of male zebrafish to 3 µg/L and 3000 µg/L 14 d exposure: whole body (RNA extraction) for microarray analysis For transcriptome analysis, 16 microarrays (Agilent 4 × 44 K) were analysed 3 tissue collected for qRT-PCR validation analysis (liver, brain and testis) Fish exposure mRNA extracted from whole body Cy3 labeled Cy3 cDNA RT Hybridisation Microarray Wash Danio rerio Scan and quantification of the raw data GeneSpring GX GO analysis

56 Hormonal activity in vitro of phthalates and disinfectants in personal care products
Aims Determination of antiandrogenic activity of single compounds in personal care products Method: in vitro activity in MDA-kb2 cells - Human AR/luciferase cell line from mammary gland - express androgen responsive firefly luciferase reporter gene

57 Aim 2-ethyl-hexyl-4-trimethoxycinnamate (EHMC)
untreated municipal wastewater up to 19 μg/L EHMC detected in human milk (Hany and Nagel,1995) and tap water; found in all biota in Swiss rivers, EHMC tend to bioaccumulate in the food chain (Fent et al., 2010) (logP=5.66). estrogenic activity in male medaka at high concentrations (Inui et al., 2003). Aim Determine the modes of action of this wide use UV filter compound in zebrafish (Danio rerio) adult males by analyzing the global gene expression pattern (microarray) and by investigating mRNA expression (qRT-PCR) of more classical genes involved in hormonal pathways .

58 Estrogenic, androgenic, steroidogenesis
Expression of target genes involved in 3 different hormonal pathways in liver, gonads, brain after exposure for 14 days and comparison with plasma vitellogenin and gonad histology Estrogenic, androgenic, steroidogenesis Adult males and females: 8 transcripts Estrogen receptor alpha (esr1) Vitellogenin (vtg1) Prostaglandin D2 synthase (ptgds): induced by estradiol gluthation S-transferase alpha (gst): induced by estradiol, phase II enzyme Androgen receptor (ar) 3 ß–hydroxysteroid dehydrogenase (converts pregnelolone > progesterone) P450aromA (cyp19a1a) P450aromB (cyp19a1b)


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