Applying sunscreens on earthworms: Molecular response of Eisenia fetida after direct contact with an organic UV filter

Highlights

• Gene expression changes were studied in Eisenia fetida after exposure to a UV filter.

• Whole-body tissue was analyzed after acute (48 h) exposure to 4-OHBP.

• Gene expression of EcR increased, indicating endocrine disruption.

• The filter altered CuZn SOD expression, related to oxidative stress response.

• Gene expression of GAPDH, involved in energy metabolism, decreased.

Abstract

The use of organic Ultraviolet (UV) filters has increased in the last years, either in sunscreens, other cosmetics, or even food packaging. These filters may end up in soil and water since the Wastewater Treatment Plants may not successfully remove them. Among them, benzophenones are known to act as endocrine disruptors. However, most of the studies are directed towards vertebrates and aquatic invertebrates, while there is a lack of information on the molecular mechanisms affected by these compounds on soil dwelling invertebrates. Here, we study the impact of direct acute (48 h) contact of 4-hydroxybenzophenone (4-OHBP) at two sublethal concentrations (0.02 and 0.2 mg/mL) on gene expression of the earthworm Eisenia fetida. Investigated genes were involved in endocrine pathways, stress response, detoxification mechanisms, genotoxicity, energy metabolism and epigenetics. Three of them were identified for the first time in earthworms. Our results suggest that exposure to 4-OHBP affected endocrine pathways, causing an increase in the Ecdysone receptor gene (EcR) expression. Moreover, the UV filter induced changes in the CuZn superoxide dismutase gene (CuZn SOD), indicating an effect in the stress response. Finally, significant changes were detected for glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) expression, indicating that energy metabolism is influenced by the 4-OHBP and highlighting the risks of using GAPDH as an internal reference for Real Time PCR.

Introduction

The emergence of new chemical compounds in the last years has been accompanied with the concern of their effects on humans and environment. Known as xenobiotics, they are rare or nonexistent in the nature, being artificial products synthetized by us (De Bolster, 1997). Among them, endocrine-disrupting chemicals (EDCs) present such a similar chemical structure to that of certain hormones that they can be bound to steroid hormone receptors and meddle with normal regulation of the endocrine system (Sharma et al., 2017). The use of Ultraviolet (UV) filters within cosmetics and sunscreens has increased lately because of the intensification of UV radiation and associated health damages. They are also added to food packaging and other perfumery products in order to avoid their odour and colour degradation (Lewis, 2016; Wypych, 2015). According to their mechanism of action, UV filters can be classified into physical or chemical, and benzophenones are included within the latter. Because of the difficulty and the lack of regulation for eliminating these filters in the Wastewater Treatment Plants, they often end up in water (Harrison et al., 2006). Eventually, they reach the soil ecosystems through fertilization with sewage sludge or irrigation with regenerated water (Camino-Sanchez et al., 2016). Gago-Ferrero et al. (2011) showed the presence of two main products of the benzophenone-3 (BP-3) degradation in the local sludge of 15 treatments plants in Spain: 4,4′-dihydroxibenzophenone and the focus of this study, 4-hydroxibenzophenone (4-OHBP). Benzophenones are known to accumulate in organisms (Gago-Ferrero et al., 2015; Langford et al., 2015; Liao and Kannan, 2019; Tsui et al., 2017) and several studies show that they are endocrine disruptors (Ghazipura et al., 2017; Kim and Choi, 2014), highlighting the necessity for further investigation of the effects of these compounds. Most of the research has been focused on vertebrates, showing that benzophenones can inhibit gamete development (Weisbrod et al., 2007), reduce daily egg production (Kim et al., 2014), induce a decline in hatching and testosterone (Ghazipura et al., 2017) or change gene expression (Bluthgen et al., 2012) of fishes for example. Studies of the effects of UV filters on invertebrates are scarce and mainly involve water ecosystems, because of their relationship with sunscreens through aquatic leisure activities. For example, Schmitt et al. (2008) showed a decrease in reproduction of the freshwater oligochaete Lumbriculus variegatus and the increase in mortality and embryo production of the snail Potamopyrgus antipodarum after their exposure to certain camphors. Kaiser et al. (2012) showed the decrease in reproduction of two snails after exposure to 3 chemical UV filters and Guyon et al. (2018) observed a decrease in egg hatching success of a marine copepod after exposure to benzophenone. Campos et al. (2017b) found a significant decrease in larval growth, developmental delay in females and weight decrease in males of Chironomus riparius after exposures to BP-3. Negative impacts of BP-3 have been also shown in corals such as bleaching and death (Danovaro et al., 2008) or genotoxicity and endocrine disruption (Downs et al., 2016). Casquero et al. (submitted) presented the first results of the effects of organic UV filters on soil dwelling invertebrates, showing that the exposure of the earthworm Eisenia fetida to 4-OHBP resulted in increased mortality and decreased reproduction capacity. However, the harmful concentrations (100 mg/kg or higher) were above those environmentally relevant. Studies on concentrations of UV filters in soils are scarce and there is not much information on the biodegradation or accumulation potential of these compounds in soil. Jeon et al. (2006) showed that concentrations of UV filters in soil ranged from 500 to 18,380 ng/kg in Korea. Casquero et al. (submitted) found that the direct contact of 4-OHBP on the earthworm E. fetida produced significant mortality at 2 mg/mL.

Earthworms are ideal candidates for evaluating the effects of contaminants (Novo et al., 2018) because of their essential role for soil structure and fertility and their basal position in the food chain, representing the main animal biomass in the soil (Lavelle et al., 2006). Standardized tests are available for the earthworm species E. fetida (OECD, 1984, test No. 207; OECD, 2004, test No. 222), which has been widely used for soil toxicity assessments. The typical endpoints are survival, growth, and reproduction, providing eco-toxicologically relevant information. Nevertheless, the insights provided by molecular studies can aid unravelling the mechanisms involved in UV filter effects in invertebrates, which are poorly understood. Direct contact tests (OECD, 1984 test No. 207) are ideal for the study of the molecular effects exerted by a toxicant since the acute stimulus can clearly unveil the genetic mechanisms involved in the response. Some studies showed that exposure to benzophenones altered endocrine, stress and detoxification pathways of the aquatic midge Chironomus riparius; upregulating the Ecdysone Receptor (EcR, Ozaez et al., 2014; Ozaez et al., 2016b), Heat Shock proteins (HSP70, Ozaez et al., 2014) or cognates (HSC70, Martin-Folgar et al., 2018) and glutathione-peroxidases (GSTs, Martinez-Guitarte, 2018). Moreover, they have shown to have genotoxic effects in corals (Downs et al., 2016). No data on molecular effects of organic UV filters on soil-dwelling animals is available at the moment.

The aim of this work is to assess gene expression changes in the earthworm E. fetida, after acute exposures (48 h) to different concentrations of the chemical UV filter 4-OHBP by direct contact tests. Genes involved in different pathways that are potentially interesting in the evaluation of the effects of endocrine disruptors and toxicants are analyzed. Some of them were already described by Novo et al. (2018) and three more have been identified here for the first time in earthworms. Selected genes were involved in endocrine pathways (Ecdysone Receptor [EcR], Membrane Associated Progesterone Receptor [MAPR], Adiponectin Receptor [AdipoR], and Estrogen Receptor [ER]), stress response (heat shock protein cognate 70-4 [HSC70 4], and CuZn superoxide dismutase [CuZn SOD]), detoxification mechanisms (Metallothionein, and glutathione-peroxidase Pi [GST Pi]), genotoxicity (poly [ADP-ribose] polymerase 1 [PARP1], and X-ray repair cross-complementing protein 1 [XRCC1]), energy metabolism (Lumbricine kinase, and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and epigenetics (DNA methylation = DNA (cytosine-5)-methyltransferases 1 [DNMT1] and 3 beta [DNMT3b]; RNA interference = Piwi2).

This study constitutes the first attempt to understand the molecular mechanisms of a soil-dwelling animal affected by an organic UV filter. Moreover, some of the biomarkers used to evaluate these mechanisms in earthworms are presented here for the first time.