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Chemosphere
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Chemosphere
Single and mixture toxicity evaluation of avobenzone and homosalate to male zebrafish and H295R cells
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Sujin Lee ª, Yujin Kaª, Bomi Lee b, Inhye Lee , Ye Eun Seoª, Hyewon Shine, Younglim Khoe, Kyunghee Ji ª,
a Department of Environmental Health, Graduate School at Yongin University, Yongin, Gyeonggi, 17092, Republic of Korea
b Institute of Natural Science, Yongin University, Yongin, Gyeonggi, 17092, Republic of Korea
Department of Environmental Health Sciences, School of Public Health, Seoul National University, Seoul, 08826, Republic of Korea
d Department of Food Technology & Service, Eulji University, Seongnam, Gyeonggi, 13135, Republic of Korea
e Department of Health, Environment & Safety, Eulji University, Seongnam, Gyeonggi, 13135, Republic of Korea
HIGHLIGHTS
· Homosalate (HS) had estrogenic and anti-androgenic effects in male zebrafish.
· Co-exposure to avobenzone (AVB) and HS enhanced sex hormone disruption.
· Anti-androgenic effect of AVB and HS behaved as synergistically in H295R cell.
ARTICLE INFO
Handling Editor: James Lazorchak
Keywords:
Avobenzone Endocrine disruption Homosalate Mixture Zebrafish
GRAPHICAL ABSTRACT
Avobenzone + Homosalate
Male zebrafish
H295R cell
1200
1.2
TU=0.76
1000
1.0
T conc. (pg/mL)
800
*
T fold-change
0.8
600
*
0.6
*
*
*
400
*
0.4
*
*
200
0.2
0
0.0
SC H(L) H(H) A MI M2
SC x0.2 x0.4x0.8 x1.0 x1.6 x3.2 AVB+HS
Treatment group
ABSTRACT
Avobenzone and homosalate are widely used in sunscreens to provide ultraviolet (UV) protection, either as single compounds or in combination. Some UV filters exhibit estrogenic or anti-androgenic activities, however, studies regarding their interactions and toxicity in mixtures are limited. In this study, the effect of the toxicity of a binary mixture comprising avobenzone (0.72 µg L-1) and homosalate (1.02 and 103 µg L-1) on steroid hormone biosynthesis were investigated using male zebrafish and human adrenocortical carcinoma (H295R) cells. In fish exposed to homosalate, a significant decrease in the gonadosomatic index, testosterone level, and transcription of several genes (e.g, hsd3b2, cyp17a1, and hsd17b1) and a significant increase in the hepatosomatic index, liver steatosis, 17ß-estradiol level, and transcription of vtg gene were observed. These results suggest that estrogenic and anti-androgenic effects of homosalate were mediated by the steroidogenic pathway. The presence of 0.72 µg
* Corresponding author. Department of Occupational and Environmental Health, Yongin University, Yongin, Gyeonggi, 17092, Republic of Korea. E-mail address: kyungheeji@yongin.ac.kr (K. Ji).
https://doi.org/10.1016/j.chemosphere.2023.140271
Received 26 May 2023; Received in revised form 22 September 2023; Accepted 23 September 2023 Available online 25 September 2023
0045-6535/ 2023 Elsevier Ltd. All rights reserved.
L-1 of avobenzone augmented the anti-androgenic responses in male fish. The testosterone level in the H295R cells were significantly decreased after they were exposed to homosalate alone or in combination with avo- benzone, which is consistent with observations in male zebrafish. Further studies need to be conducted to un- derstand the endocrine disrupting properties of long-term exposure to substances typically used in sunscreens.
1. Introduction
Sunscreens contain various ultraviolet (UV) filter components that reflect or obstruct UV light. In general, a higher percentage of UV filters are included in products that absorb UV-A, -B, and -C light (Kunz and Fent, 2006; Uter et al., 2014). Avobenzone provides excellent protection against the broad UV-A spectrum (Ahn et al., 2019), and is often mixed with additional substances such as octocrylene, homosalate, and octi- salate to help filter other UV rays (Afonso et al., 2014). Homosalate is one of the most typically used UV-B filters related to avobenzone, which is used in both the European Union and United States (Ginzburg et al., 2021). In the aquatic environment, contamination of UV filters can occur as a result of recreational activities (Labille et al., 2020) or as result of their incomplete removal by wastewater treatment plant (Cadena-Aizaga et al., 2022).
Avobenzone and homosalate typically occur in freshwater, as well as in marine water, sediments, and biota worldwide. In rivers and lakes, avobenzone has been detected at concentrations of 1.37-145.24 ng L-1 (Tsui et al., 2019), 2.3 ± 0.5 ng L-1 (Yang et al., 2020), 18-721 ng L-1 (Tsui et al., 2014), and 2431 ± 220 ng L-1 (Rodil et al., 2009). In freshwater, homosalate has been detected at 3.1 ± 1.6 ng L-1 (Yang et al., 2020), 3.6-41.84 ng L-1 (Tsui et al., 2019), 15.5-187.9 ng L-1 (He et al., 2019), 460 ± 100 ng L-1 (Celeiro et al., 2020), and 29-2812 ng L-1 (Tsui et al., 2014). Meanwhile, 33.48 ng g-1 dry weight of avo- benzone were detected in sediments (Sun et al., 2021), whereas up to 74.2 and 158.3 ng g-1 dry weight of homosalate were detected in sed- iments and oyster tissue, respectively (He et al., 2019). In red swamp crayfish (Procambarus clarkii), homosalate persisted through a 14 d elimination period, even after chemical exposure was terminated (He et al., 2021).
Recently, an increasing number of studies have demonstrated the reproductive toxicity, developmental toxicity, neurotoxicity, and endo- crine disrupting properties caused by avobenzone and homosalate (de Paula et al., 2022; Ka and Ji, 2022; Klopcic and Dolenc, 2017; Liu et al., 2022). Avobenzone showed potent anti-androgenic activity in human breast cancer (MDA-kb2) cells (Klopcic and Dolenc, 2017) but no es- trogenic activity in human breast cancer (MCF-7) cells (Schlumpf et al., 2001). In the F1 generation of Daphnia magna exposed to 4.4 µg L-1 of avobenzone, reproduction was delayed, and the reproduction rate decreased (de Paula et al., 2022). Avobenzone affected the development of the nervous and retinal systems in zebrafish, inhibited locomotor behavior (Liu et al., 2022), and induced a significant decrease in thyroxine (T4) levels (Ka and Ji, 2022).
Previous studies involving in vitro cell bioassays suggest that homo- salate possesses anti-androgenic (median effective concentration (EC50) 0.69-1.46 mg L-1; Jiménez-Díaz et al., 2013; Ma et al., 2003; Schlumpf et al., 2001) or estrogenic activities (EC50 0.40-2.62 mg L-1; Alamer and Darbre, 2018; Jiménez-Díaz et al., 2013; Schlumpf et al., 2001). Homosalate adversely affected the proliferation of human trophoblast cells (Yang et al., 2018) and the levels of luteinizing hormone and estradiol before birth, during lactation, and during the early postnatal period in female Wistar Hannover rats (Erol et al., 2017). In response to these toxicity reports, recent research has applied in silico techniques to find proper alternative UV-filters with similar photoprotective proper- ties but less toxicity (Zambrano et al., 2023). Additionally, some re- searchers have tested whether alternative substances are safer for aquatic organisms than classic UV-filters using in vivo zebrafish embryos (Damiani et al., 2023). Most of the previous studies have focused on the toxic effects of individual UV filters, while few have investigated the
adverse effects of UV filter mixtures. In a screening assay for a recom- binant yeast that transports the human estrogen receptor alpha, a set of eight UV filters induced an elevation in estrogenic activity (Kunz and Fent, 2006). Since UV filters are present as mixtures in products and environmental media, the toxic effects of various UV filters under mixed exposure scenarios must be investigated.
In the present study, the endocrine disrupting effects of mixture exposure to avobenzone and homosalate, which are commonly used together in sunscreen, were investigated using an in vivo male zebrafish and an in vitro human adrenal cortex-derived (H295R) cells. Zebrafish can be used to examine the expression of various genes in the hypothalamus-pituitary-gonad axis and as sex hormones (Jang and Ji, 2015); therefore, it is often used for in vivo evaluations of various UV filters including benzophenone-3 (Tao et al., 2023), ethylhexyl methoxy cinnamate (Zhou et al., 2019), 4-methyl benzylidene camphor (Xian et al., 2023), and octocrylene (Zhang et al., 2016). The steroidogenic assay using H295R cells is a method validated by the Organization for Economic Co-operation and Development (OECD) and is used in the ToxCast program of the US Environmental Protection Agency (EPA) as an in vitro technique to evaluate potential endocrine disruptors (Haggard et al., 2019). The results of this study highlight changes in toxicity of substances that are frequently mixed in products and provide informa- tion on the toxicity of avobenzone and homosalate in combination.
2. Materials and methods
2.1. Analysis of chemical mixtures
Avobenzone (Chemical Abstracts Service (CAS) no. 70356-09-1) and homosalate (CAS no. 118-56-9) were purchased from Merck (Merck KGaA, Darmstadt, Germany). Stock solutions were prepared by dis- solving the test chemicals in dimethyl sulfoxide (DMSO) at 0.01% (zebrafish test) or 0.1% (H295R bioassay) (Junsei Chemical Co., Tokyo, Japan). Test solution of the chosen concentrations were prepared by serially dilution of a stock solution.
The concentrations of avobenzone and homosalate in the media exposed to fish were measured by obtaining samples at the start of exposure and 24 h thereafter. The exposure medium was collected 4 times during exposure period. Briefly, 1 ml of water samples were filtered through 0.2 um regenerated cellulose filters (Sartorius, Nur- emberg, Germany). All chemicals were analyzed using a Nexera X2 ultra high-performance liquid chromatography (Shimadzu, Kyoto, Japan) coupled with a Triple Quad 4500 mass spectrometry system (SCIEX, Framingham, MA, USA). The injection volume of the sample was 3 µL and the flow rate was 0.2 mL min-1. Additionally, 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B) were used as the mobile phases. Homosalate was separated in isocratic mode using a Kinetex C18 column (3 pm x 150 um, 2.6 um: Phenomenex, Torrance, CA, USA). Avobenzone was separated using an Acquity UPLC BEH C18 column (2.1 µm × 100 um, 1.7 um; Waters, Milford, MA, USA) in gradient mode. The analytical conditions for UPLC-MS/MS are shown in Tables S1 and S2. The limits of detections were 0.13 and 0.17 µg L-1 for avobenzone and homosalate, respectively.
2.2. Chemicals and mixture exposure in fish
Zebrafish (AB strain, 3-4 mo, approximate wet weight male 0.32-0.48 g, female 0.52-0.78 g) were cultured in a flow-through sys- tem (ZebTEC, Buguggiate, Italy) under a constant water temperature (26
± 1 °℃) and photoperiod (14 h light:10 h dark). Ten male fish per rep- licates (a total of 30 per concentration group) were exposed to the control, solvent control (DMSO 0.01%), 1 µg L-1 of avobenzone (measured concentration: 0.72 µg L-1), 1 µg L-1 of homosalate (measured concentration: 1.02 µg L-1), 100 µg L-1 of homosalate (measured concentration: 103 µg L-1), mixture 1 (0.72 µg L-1 avo- benzone + 1.02 µg L-1 homosalate), and mixture 2 (0.72 µg L-1 avo- benzone + 103 µg L-1 homosalate) for 21 d. The test concentration of avobenzone (1 µg L-1) was selected as a concentration that could be detected in the water environment without affecting the E2 and T levels of male zebrafish through a preliminary experiment (Fig. S1). The high concentration of homosalate (100 µg L-1) was selected as a concentra- tion that can affect the E2 and T levels of male zebrafish through a preliminary experiment (Fig. S1), and the low concentration was selected as a concentration that can be detected in the water environ- ment (Fig. S2). During the exposure period, the fish were fed with Artemia nauplii twice daily. The exposure medium was changed daily, and the pH, water temperature, conductivity, and dissolved oxygen were measured (Table S3).
At the end of the 21 d exposure period, the fish were euthanized by submersion in ice water for at least 10 min. The body weight and organ (liver and testis) weight of each fish were measured using an electronic balance. The hepatosomatic index (HSI) and gonadosomatic index (GSI) were calculated as the organ weight/body weight x 100. Brain, liver, and testis samples were obtained for the transcriptional analysis of genes related to the HPG axis and vitellogenin. Plasma samples were collected for sex hormone analyses.
2.3. Measurement of sex hormones in fish
A total of 10-20 µL of blood were obtained from each fish using a glass capillary tube. The blood samples were centrifuged at 2000xg for 10 min, and the supernatant was transferred meticulously to a clean tube. The final plasma samples were reconstituted in 120 uL of assay buffer. The E2 (Cat No. 582251; Cayman Chemical Company, Ann Arbor, MI, USA) and T (Cat No. 582701; Cayman Chemical Company) levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions.
2.4. Determination of gene transcription using real-time PCR
Twenty-two genes related to the HPG axis and vitellogenin induction were analyzed in the brain, gonad, and liver samples (n = four per group) using quantitative real-time polymerase chain reaction (qRT- PCR), as previously described (Lee et al., 2022). The total ribonucleic acid (RNA) was extracted using a RNeasy mini kit (QIAGEN, Hilden, Germany), and complementary deoxyribonucleic acid (cDNA) was synthesized using an iScript™M cDNA synthesis kit (BIORAD, Hercules, CA, USA). qRT-PCR was performed using SYBR Green® PCR Master Mix kits on an Applied Biosystems™ 7500 real-time PCR system. Genes related to the gonadotropin releasing hormone (gnrh2, gnrh3, gnrhr1, gnrhr2, and gnrhr4), gonadotropin hormones (fshb and lhb), aromatase (cyp19a1b), estrogen receptor (esr1 and esr2b), and androgen receptor (ar) were analyzed in the brain samples. In the testis, genes related to the gonadotropin hormone receptor (fshr and lhcgr), steroidogenic acute regulatory protein (star), hydroxymethylglutaryl CoA reductase (hmgcra and hmgcrb), steroidogenic cytochrome P450 enzymes (cyp11a1.1, cyp17a1, and cyp19a1a), and hydroxysteroid dehydrogenase (hsd3b2 and hsd17b1) were analyzed. In the liver, gene related to the vitellogenin (vtg) was analyzed. The primer sequences are presented in Table S4. Thermal cycling was performed as follows: 1) 50 ℃ for 1 min, 2) 95 ℃ for 10 min, 3) 40 cycles at 95 ℃ for 15 s and 60 ℃ for 1 min. The threshold cycle (Ct) of the analyzed gene was normalized to the average of the Ct of two housekeeping genes, i.e., beta-actin (actb1) and ribo- somal protein L8 (rpl8), and then expression levels were compared using
the 2-AACt method (Livak and Schmittgen, 2001).
2.5. Histological analysis
To observe histological changes, five males from each treatment group were additionally exposed to the same concentration and expo- sure time. All fish were euthanized and are placed in an individual container of Davidson’s fixative for 24 h. Tissue samples were decal- cified and embedded in paraffin. Samples were transversely sectioned using a microtome (RM2245, Leica Biosystems, Deer Park, IL, USA) and stained with hematoxylin and eosin. Histological changes (nuclear displacement and cytoplasm vacuolization) to define liver steatosis were evaluated under a light microscope and a semiquantitative severity score was assigned: 0-not observed; 1-mild, 2-moderate, 3-severe.
2.6. Measurement of individual toxicity using H295R cells
H295R cells (CRL-2128) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM/F12 (Gibco 11320033) containing an ITS + premix (Corning 354352), 2.5% Nu-serum (Corning 355100), and 1% penicillin/streptomycin. Cells were seeded at a density of 3.0 x 105 cells mL-1 in 24-well plates, and the medium was renewed every other day. Based on the #456 test guideline of the OECD (OECD, 2022), the cells were exposed to several concentrations (0, solvent control (DMSO 0.1%), avobenzone (10, 30, 100, 300, and 1000 nM), and homosalate (1, 10, 30, 90, 200, 330, and 1000 nM)) for 48 h. Since the ability of cells to produce E2 changes with increasing passages, cells were used for testing within the cell passages recommended in the test guideline. Each concentration group was tested with three replicates on two different days, resulting in a total of six replicates. Concentrations that were not cytotoxic in the MTT assay (i.e., greater than 85% viability compared with the solvent control) were specified as the exposure concentrations for assaying sex hormones. To validate the experiment, two positive controls were used: forskolin (CAS no. 66575-29-9; 1 µM), a strong inducer of hormone production, and prochloraz (CAS no. 67747-09-5; 1 µM), a strong inhibitor of androgen production (Haggard et al., 2018).
The levels of E2 and T were measured in H295R cells using the ELISA (Cayman Chemical; E2 (Cat No. 582251) and T (Cat No. 582701)). Briefly, media samples (500 µL) of exposed H295R cells were extracted twice using 2.5 mL of diethyl ether and then evaporated using a nitrogen concentrator. The residue was dissolved in 300 µL of the ELISA buffer and then diluted at ratios of 1:1 and 1:75 to measure the E2 and T levels, respectively. Hormonal change (y) was converted to a ratio between 0 and 1 as (y-minimum)/(maximum-minimum). Four-parameter log- logistic curves were fitted and median effect concentration (EC50) values were calculated (Nielsen et al., 2015).
2.7. Measurement of binary mixture toxicity using H295R cells
The toxicity of avobenzone and homosalate mixtures was deter- mined using the respective EC50 values and exposure concentrations for each sex hormone, referring to the method presented in previous studies (Chen and Lu, 2002; Roberts and Costello, 2003). Synergistic, additive, and antagonistic effects were determined by performing exposures at concentrations corresponding to 0.2, 0.4, 0.8, 1.0, 1.6, and 3.2 times the EC50 values for sex hormones of the individual substances (Ding et al., 2017). To quantify the toxicity of mixture, the toxic units (TUs) were calculated using the following equation (Son et al., 2016; Tian et al., 2012):
TU = CA /EC50A + CB/EC50B (1)
where CA and CB are the concentrations of chemicals A and B in the mixtures, respectively. EC50A and EC50B represent the concentrations that exert a median effect on maximal hormone production when
exposed to chemicals A and B, respectively. If the TU value was between 0.8 and 1.2, then a simple addition was assumed. Meanwhile, a TU value greater than 1.2 or less than 0.8 indicate the antagonistic and synergistic effect, respectively (Long et al., 2016; Wang et al., 2016).
2.8. Statistical analyses
One-way analysis of variance (ANOVA) with post-hoc Dunnett’s test was performed using SPSS (IBM Corp., New York, NY, USA) to analyze the statistical significance of the treatment groups compared with the solvent controls. Before running ANOVA, the Kolmogorov-Smirnov test and Levene’s test were performed to check the normality and homoge- neity of data variance, respectively. Spearman correlation analysis from SAS (version 9.2; SAS Institute, Cary, NA, USA) was used to investigate whether there was a correlation between gene transcripts. Principal component analysis (PCA) in SAS was used to convert a number of possibly associated genetic variables into principal components (PCs) and determine their effects on sex hormones in male zebrafish. Regres- sion analysis was used to evaluate the effects of the first and second principal components on sex hormone concentrations. In the statistical analysis, a p-value of less than 0.05 was considered statistically significant.
3. Results
3.1. Chemical analysis
The mean concentrations measured at the beginning of exposure ranged from 108% to 119% of the nominal concentrations of avo- benzone and homosalate (Table S5). The concentrations of homosalate collected after the 24 h exposure was also ranged from 86 to 92% of the nominal concentration (Table S5). However, the concentrations of avobenzone in the test water obtained after 24 h of exposure decreased
to 29-31% (Table S5). The estimated half-life of avobenzone is known to be 7.1 h at pH 7.41 (Mbah, 2007). Subsequent analyses of biological effects were based on average measured concentrations; nominal con- centrations of 1 µg L-1 of avobenzone were expressed as 0.72 µg L-1, 1 µg L-1 of homosalate as 1.02 µg L-1, and 100 µg L-1 of homosalate as 103 µg L-1.
3.2. Changes in relative organ weight and histology in male zebrafish
The GSI was significantly reduced after exposure to 103 µg L-1 of homosalate, mixture 1 (0.72 µg L-1 avobenzone + 1.02 µg L-1 homo- salate), and mixture 2 (0.72 µg L-1 avobenzone + 103 µg L-1 homo- salate) treatment groups (Fig. 1A). The HSI was significantly increased following the exposure to 1.02 µg L-1 and 103 µg L-1 of homosalate and mixture 2 (0.72 µg L-1 avobenzone + 103 µg L-1 homosalate) (Fig. 1A). Histological changes were observed in gonad and liver following 21 d exposure to avobenzone and homosalate (Fig. 1A and Fig. S3). Compared to solvent control, liver steatosis defined by cellular vacuo- lization (partial or entire removal of hepatocyte cytoplasm in stained tissue sections) and degenerated hepatocytes was significantly increased in fish exposed to homosalate or co-exposed to avobenzone.
3.3. Hormonal changes in male zebrafish
Although the concentration of E2 in plasma collected from male zebrafish was significantly increased in the 103 µg L-1 homosalate treatment group, the result obtained from the combined treatment with homosalate and avobenzone did not differ significantly from that of the solvent control (Fig. 1B). The plasma concentration of T decreased significantly in the 1.02 µg L-1 and 103 µg L-1 of homosalate, and mixture 2 (0.72 µg L-1 avobenzone + 103 µg L-1 homosalate) treatment groups (Fig. 1B). The elevation of the E2/T ratio was significantly increased after individual exposure to homosalate as well as combined
(A)
2.5
2.5
6
*
Gonadosomatic index
2.0
Hepatos omatic index
2.0
*
*
Liver steatosis score
5
*
4
*
1.5
1.5
*
3
1.0
*
*
*
1.0
2
*
0.5
0.5
1
0.0
0.0
0
SC
H(L) H(H)
A
M1
M2
SC
CH(L) H(H)
A
M1
M2
SC
H(L) H(H)
A
M1
M2
Treatment group
Treatment group
Treatment group
(B)
1200
1200
5
E2 conc. (pg/mL)
1000
1000
4
*
800
T conc. (pg/mL)
*
800
*
E2/T ratio
3
600
600
*
*
400
400
2
*
*
200
200
1
0
I
0
I
0
I
SC H(L) H(H)
H) A M1 M2
SC H(L) H(H)
A
M1
M2
SC H(L) H(H)
A
M1
M2
Treatment group
Treatment group
Treatment group
exposure to mixture 2 (0.72 µg L-1 avobenzone + 103 µg L-1 homo- salate) (Fig. 1B).
3.4. Regulation of genes in male zebrafish
Transcriptional changes in the analyzed genes were more evident in the mixed treatment group than in the individual exposure groups (Fig. 2, Table S6). In fish exposed to homosalate, the transcriptions of cyp19a1a, cyp19a1b, esr1, esr2b, gnrh3, and vtg genes were significantly upregulated, whereas the transcriptions of ar, cyp11a1.1, cyp17a1, gnrh2, gnrhr2, fshb, fshr, hmgcra, hmgcrb, hsd3b2, hsd17b1, lhb, lhcgr, and star genes were significantly downregulated. Following exposure to avobenzone alone, significant up-regulation was observed in ar, cyp19a1b, esr2b, and gnrh3 genes, whereas down-regulation of cyp17a1, fshb, fshr, hsd17b1, and star genes were observed. After exposure to both avobenzone and homosalate, cyp19a1a, cyp19a1b, esr1, esr2b, and gnrh3 genes were significantly upregulated, whereas ar, cyp11a1.1, cyp17a1, gnrh2, gnrhr2, fshb, fshr, hmgcra, hmgcrb, hsd3b2, hsd17b1, lhb, lhcgr, and star genes were significantly downregulated.
Because most of the 21 genes analyzed showed highly correlated expression (Table S7), we performed PCA to reduce the number of in- dependent variables to PCs. PC1 and PC2 constituted 51.3% and 11.1% of total variance, respectively (Table S8). The first PC was affected significantly by genes fshb, hmgcrb, hsd17b1, lhb, and lhcgr, whereas the second PC was affected significantly by genes ar, cyp19a1b, esr2b, gnrh2, hmgcra, and hsd3b2. In the male zebrafish, PC1 ($ = 0.259, p < 0.001) was significantly correlated with the concentration of T, but not with that of E2.
3.5. Hormonal changes in H295R cells
The EC50 values of avobenzone and homosalate in the H295R cells are shown in Fig. 3A and B. The EC50 values of avobenzone were 103 nM for E2 and 610 nM for T, whereas those of the homosalate were 126 nM for E2 and 195 nM for T.
The binary mixture toxicity was determined using a mixture of avobenzone and homosalate at concentrations proportional to 0.2 to 3.2 times their respective EC50 values (Fig. 3C). Although the EC50 of the two substances was significantly increased compared to the solvent control in the samples mixed with 0.2-fold and 0.4-fold EC50 of the two substances, the TU could not be calculated because no concentration-
dependent increase was observed. The T hormone inhibition rate of the binary mixtures of avobenzone and homosalate increased with the mixture concentration. The obtained EC50-TU value for T was <0.8 for the binary mixture comprising avobenzone and homosalate.
4. Discussion
The results of our study showed that exposing male zebrafish to homosalate alone affected the expression of various genes in the HPG axis, which altered the sex hormone levels and relative organ weights. Measuring endpoints at the organ level to obtain the GSI and HSI has been proposed to comprehensively consider endocrine disruption (Car- nevali et al., 2018). We observed a significant decrease in the testes of male zebrafish exposed to 103 µg L-1 of homosalate, which indicates that homosalate may affect sexual maturity and fecundity. Previous studies have shown that reduced gonad weight in males due to chemical exposure is associated with decreased egg production as well as T con- centration. For example, egg production, GSI, and T production in males were decreased in zebrafish exposed to bisphenol SIP (Lee et al., 2018) or ibuprofen (Ji et al., 2013). Inhibition of relative gonadal weight has been previously reported in fish exposed to UV filter benzophenone-2, which supports our results (Weisbrod et al., 2007).
The HSI is an indicator that reflects the state of energy reserves in fish, and its value is determined by the feeding capacity and physio- logical status of the liver (Lin et al., 2023). Our finding, i.e., exposure to homosalate increased the HSI, liver steatosis, and vtg gene transcription, suggests that homosalate may stimulate the swelling of liver and pro- motes the participation of the liver in yolk formation. Especially, liver steatosis, which is defined by the abnormal accumulation of fat in liver hepatocytes (Goh et al., 2019), agrees well with the results of HSI. The results of a recent study (Carvalhais et al., 2021) that found simulta- neous exposure to organic (oxybenzone) and inorganic (titanium diox- ide nanoparticles) UV filters targeted the liver, exacerbated oxidative stress, and altered metabolic function, together with the findings of our study, suggesting that UV filters can cause severe liver damage. The increase in HSI in male fish exposed to homosalate may be related to vitellogenin, an egg yolk protein produced by the liver. Previous studies have also frequently confirmed an association between increased HSI and plasma vitellogenin concentrations, e.g., male rare minnow exposed to ethynylestradiol and nonylphenol (Zha et al., 2006) as well as Chilean flounders (Leonardi et al., 2012).
Brain
Hypothalamus
SC |H(L) H(H) A M1 M2
gnrh2
gnrh3
T
cyp 19afb
1.25 2.0 4.0
Pituitary
0.8 0.5 0.25
esr1
gnrhr1
gnrhr2
gnrhr4
p<0.05
E2
esr2b
fshb
thb
Blood
ar
FSH
LH
FSH, LH
Gonad
cyp17at
FSH
fshr
LH
thegi
HDL LDL
Progesterone - 17-hydroxyprogesterone
hsd3b2
syp1701
hmgcra
Prognenolone
Aldostenedione
hmgcrb
star
cyp11a1.1
hsd17b1
Cholesterol
Cholesterol
Testosterone (T)
T
Outer mitochondrial membrane
Inner mitochondrial membrane
cyp19a1a
Estradiol (E2)
E2
(A)
20
EC50 = 103 nM
20
(C)
3
EC50 = 126 nM
TU = NA
18
18
E2 fold-change
5
E2 fold-change
5
E2 fold-change
2
4
4
*
*
3
3
T
T
T
2
2
1
T
1
1
0
0
0
SC
0
1
2
3
SC
0
1
2
3
SC x0.2 x0.4x0.8x1.0x1.6x3.2
AVB conc. (log nM)
HS conc. (log nM)
AVB + HS
(B)
5.0
EC50 = 610 nM
5.0
1.2
4.0
EC50 = 195 nM
TU = 0.76
1.0
T
4.0
2.0
T fold-change
T fold-change
T fold-change
3.0
0.8
1.5
0.6
*
2.0
1.0
*
*
0.4
*
*
1.0
0.5
0.2
0.0
0.0
0.0
SC
0
1
2
3
SC
0
1
2
3
SC x0.2 x0.4 x0.8x1.0x1.6x3.2
AVB conc. (log nM)
HS conc. (log nM)
AVB + HS
Sex hormone homeostasis is essential for the regular operation of the endocrine system; additionally, increases or decreases in sex hormone concentrations are typically used to assess endocrine-disrupting effects. Several studies reported that UV filters can disturb the balance of sex hormones (Xian et al., 2023; Yan et al., 2020; Zhou et al., 2019). For example, exposure to 1 µg L-1 octinoxate for 120 day decreased levels of E2 and VTG in zebrafish (Zhou et al., 2019). In addition, plasma con- centrations of 11-ketotestosterone, E2, and VTG were significantly increased in Japanese medaka after 28 days of exposure to octocrylene (Yan et al., 2020). A decrease in T in the male zebrafish exposed to homosalate alone indicated the suppression of endogenous hormone secretion. The anti-androgenic effect of homosalate was also observed in previous cell bioassays (Jiménez-Díaz et al., 2013; Ma et al., 2003), but the EC50 range (0.69-1.46 mg L-1) was much higher than our test concentration. In the presence of avobenzone, the homosalate-induced anti-androgen responses enhanced in the male zebrafish. Taken together, further studies are needed for investigating the anti-androgenic effects and increased E2/T ratio in fish exposed to homosalate.
Exposure to homosalate resulted in anti-androgenic effects via interference with the expression of transcripts involved in steroidogenic pathways in the male zebrafish, which was similarly supported by the PCA results. The primary contributors to the first PC (hsd17b1) and second PC (hsd3b2) were positively correlated with the concentrations of T. Significant down-regulation of ar at 1.02 µg L-1 of homosalate is similar to changes in genes exhibiting anti-androgenic activity in a previous in vitro study (Kunz and Fent, 2006). An efficient conversion of progesterone to T requires the activities of cyp17a1 and hsd17b1. The down-regulation of cyp17a1 and hsd17b1 may have decreased the T in the fish plasma. This is consistent with the experimental results reported
by Bluthgen et al. (2012) and Kim et al. (2014), who discovered that exposure to UV filter benzophenone-3 reduced the hsd17b1 gene in male zebrafish.
The H295R cell bioassay has been used by the U.S. EPA Endocrine Disruptor Screening Program and OECD to rapidly screen for endocrine disrupting chemicals associated with steroidogenesis (Haggard et al., 2018). Our findings that homosalate alone as well as combined exposure to avobenzone and homosalate can reduce T levels in H295R cells sug- gest that they correlate well with the results observed in male zebrafish. Changes in T concentrations have been used as a representative indi- cator of hormone levels, which may result in reproductive failure (Huda Bhuiyan et al., 2019). Although the results of the H295R cell experi- ments cannot directly support the findings based on fish, the observation that simultaneous exposure to avobenzone and homosalate can reduce T hormone supports the finding of decreased T and increased E2/T ratio in male fish.
In conclusion, we demonstrated that homosalate can activate the anti-androgenic activity and exposure to avobenzone may enhance anti- androgenic responses in male zebrafish. In addition, the method for determining mixture toxicity using the hormone EC50 of the two sub- stances proposed here can be used to confirm the combined toxicity using biomarkers, where 100% is not determined. Because sunscreen contains a mixture of various active ingredients, users of sunscreen as well as the ecosystem will be affected. Our results showed that the tested compound combinations may interfere with the normal endocrine sys- tem and should not be dismissed. Taken together, our study indicates the importance of considering the toxicity of mixtures as well as individual substances when assessing the risk of UV-filters, and provides guidance for the design of future experiments investigating mixture toxicity.
CRediT authorship contribution statement
· Sujin Lee: Investigation, Formal analysis, Data curation, Visualiza- tion, Writing-original draft
· Yujin Ka: Investigation, Formal analysis, Visualization, Writing- original draft
· Bomi Lee: Investigation, Formal analysis, Visualization, Writing- original draft
· Inhye Lee: Investigation, Formal analysis, Visualization, Writing- original draft
· Ye Eun Seo: Investigation, Formal analysis, Writing-original draft
· Hyewon Shin: Investigation, Formal analysis, Writing-original draft
. Younglim Kho: Conceptualization, Writing-review & editing, Supervision
· Kyunghee Ji: Conceptualization, Methodology, Writing-review & editing, Supervision, Funding acquisition
Ethics approval statement
This study was approved by the Institutional Animal Care and Use Committee of Yongin University, Korea (YUIACUC-2022-01).
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgement
This study was carried out with the support of the Korea Environ- ment Industry & Technology Institute (KEITI) through “the Technology Development Project for Safety Management of Household Chemical Products”, funded by Korea Ministry of Environment (MOE) (grant number 288 2020002960006; 1485017189).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi. org/10.1016/j.chemosphere.2023.140271.
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