Toxic effects of octocrylene on zebrafish larvae and liver cell line (ZFL)

Highlights

• The toxic effects of octocrylene (OC) on zebrafish larvae and ZFL were studied.

• OC had no obvious phenotypic toxicity to larvae, but it was very toxic to ZFL.

• OC upregulated CYP1A, CYP3A65, ERα, ERβ1, CYP19A, and DMRT1 genes but downregulated 11β-HSD.

• OC upregulated glutathione transferases.

• OC is not a ligand of AhR1B and AhR2, but it can activate ERα and GPER.

Abstract

Octocrylene (OC) is a broad-spectrum ultraviolet-absorbing chemical used in sunscreen and other personal care products. Its health effects are a concern because it has been detected in water, fish, humans, and food chains. In vivo and in vitro investigations were performed in zebrafish (Danio rerio) larvae and a zebrafish liver cell line (ZFL), respectively, to understand the potential risks and molecular mechanisms of OC toxicity. The 96-h median lethal concentration (LC₅₀) of OC was determined to be 251.8 μM in larvae and 5.5 μM in ZFL cells. Quantitative real-time PCR (qRT-PCR) showed that OC induced the expression of genes for CYPs (CYP1A, CYP3A65), estrogen receptors (ERα, ERβ1, GPER), vitellogenin (VTG1), and sex determination (BRCA2, CYP19A, DMRT1, SOX9A), both in vitro and in vivo. A whole-transcriptome sequencing method was used to evaluate the gene expression profile of larvae exposed to OC. OC was found to mediate the biosynthesis of estrogens (such as estriol) and affect the antioxidant pathway (glutathione transferases and peroxisome). These findings clarify the toxic effects and molecular mechanisms of OC and support banning its use in cosmetics.

Introduction

High-energy UV rays have adverse effects and cause cellular damage to human skin exposed to sunlight for prolonged periods. Long-wave UV (UVA; 320–400 nm) is strongly penetrating and can act directly on the dermis, resulting in destruction of elastic fibers and collagen fibers (Bernerd and Asselineau 1998). Medium-wave UV (UVB; 280–320 nm) has weaker penetration ability than UVA, but it also causes skin tanning, redness, peeling, and other harmful effects (Adil et al., 2010). Sunscreen chemicals are thus used to protect our skin from harm from UV light and decrease the possibility of skin cancer (Latha et al., 2013). However, as the amount and variety of sunscreen chemicals being used increases, the effects of sunscreen chemicals on the natural environment and human public health become increasingly serious (Kim and Choi 2014).

Octocrylene (OC) is a type of chemical sunscreen (the structure as shown in Fig. 1), which absorbs UVA and UVB (Manová et al., 2014) and is usually used with other sun-screening agents to increase a sunscreen's sun protection factor (SPF) (Avenel-Audran et al., 2010). The use of OC in sunscreen is permitted in many European and other Western countries. Due to its good UV-absorbing ability, OC often appears on the list of high-sales sunscreen ingredients (Avenel-Audran et al., 2010). However, OC can liberate oxygen radicals under exposure to UV radiation to capture an electron from other substances and form their own stable substance (Gago-Ferrero et al., 2013b; Yan et al., 2020). Excessive production of oxygen radicals in organisms may cause breaks or mutations in DNA, thus affecting the transcription of genetic information; it may also damage proteins, leading to cell aging and death (Breimer 1990; Sugden et al., 1992).

The toxicity profile of OC has been poorly studied in comparison with other sunscreen chemicals (Samantha et al., 2018), but it appears to act as a photoallergen because OC can cause contact dermatitis when exposed to sunlight (de Groot and Roberts 2014). OC was recently reported to increase the expression level of plasma sex-steroid hormones (estradiol (E2) and 11-ketotestosterone (11-KT)) and VTG, inhibit the synthesis of spermatozoa, and promote the maturation of oocytes in Japanese medaka (Oryzias latipes) (Yan et al., 2020). Previous studies demonstrated that OC can bioaccumulate, with OC concentrations increasing in organisms in the higher trophic levels (Balmer et al., 2005; Blüthgen et al., 2014; Gago-Ferrero et al., 2013b; Pawlowski et al., 2019). In addition, the concentrations of OC extracted from exfoliated human cuticle samples indicated that OC can penetrate to the epidermis (Treffel and Gabard 1996; Walters and Roberts 2002) and that the OC concentrations in humans may become significant over time due to continual exposure.

At present, water bodies in many regions worldwide are contaminated with OC (Amine et al., 2012). For example, in the Nueces River in Texas, USA, the input of OC to surface water was found to be up to 318 kg/year (Sharifan et al., 2016). The concentration of OC detected in the seawater of Folly Beach, South Carolina was 1409 ng/L (Bratkovics and Sapozhnikova 2011), and the concentration of OC in the seawater of Hong Kong reached 6812 ng/L (Tsui et al., 2015). OC has also been found in many aquatic animals. For example, the concentration of OC was found to be 7112 μg/kg in marine mussels from French coastal regions (Bachelotet al. 2012), 89–782 ng/g in marine mammal livers in Brazil (Gago-Ferrero et al., 2013a; Gago-Ferrero et al., 2013b), and 25–11,875 ng/g in cod livers in Norway (Langford et al., 2015). Due to this pervasiveness of OC in the environment, it is imperative to study the potential risks of this chemical because it is possible for biotic concentrations of OC to exceed concentrations regarded by regulatory bodies as safe in products intended for human consumption via bioaccumulation.

Zebrafish (Danio rerio) is chosen as the model organism for this study because its embryos are transparent and it is therefore easy to observe embryonic development and directly view the effect of added chemicals on the phenotype (Scholz et al., 2008; Spitsbergen and Kent 2003). The zebrafish liver cell line (ZFL) is a good in vitro system to simulate the metabolic process of toxins entering the liver (Cheuk et al., 2008; Meng et al., 2020; Yang et al., 2016; Zhou et al., 2017) and complements the data obtained in vivo (Chen et al., 2011; Chen and Chan 2018; Chen et al., 2014). Furthermore, examination of the genome of zebrafish showed that it possessed many orthologues of human genes (Hill et al., 2005), which allows us to use zebrafish as a model to study ligand-receptor pathway interactions that are similar to those in humans and make reasonable extrapolations to form preliminary hypotheses on human disease mechanisms (Kwok et al., 2020). Thus far, studies on the toxicity and potential molecular mechanisms of OC have been limited, but the risk of OC to humans is increasing. That is, because humans are at the highest trophic level, the problem of bioaccumulation of organic contaminants such as OC is a serious consideration that requires further inquiry.

In this study, we elucidated the molecular mechanism by which OC regulates CYPs and ER gene expression at the transcriptional level and compared the toxicity of OC in vivo and in vitro. High-throughput whole-transcriptome shotgun sequencing (RNA-Seq) was used to reveal the comprehensive effects of OC on whole-body gene expression in zebrafish larvae. The results of this study will help to assess the safety of OC, predict its potential threats to aquatic organisms and the environment, and establish a risk-assessment model for OC in personal care products.