Acute exposure to bis(2-ethylhexyl)phthalate disrupts calcium homeostasis, energy metabolism and induces oxidative stress in the testis of Danio rerio

Highlights

• BEHP disrupts testicular Ca²⁺ homeostasis by increasing Ca²⁺ influx.

• BEHP increases the Ca²⁺ influx mainly through L-VDCC and reverse-mode NCX.

• activation.

• BEHP decreases testicular lactate content and LDH activity.

• BEHP increases ROS production, lipid peroxidation and GGT activity in the testis.

• BEHP increases hepatic GPT activity, but not GOT neither GGT.

Abstract

Bis(2-ethylhexyl)phthalate (BEHP) negatively affects testicular functions in different animal species, disturbing reproductive physiology and male fertility. The present study investigated the in vitro acute effect of BEHP on the mechanism of action of ionic calcium (Ca²⁺) homeostasis and energy metabolism. In addition, the effect of BEHP on oxidative stress was studied in vitro and in vivo in the testis of Danio rerio (D. rerio). Testes were treated in vitro for 30 min with 1 μM BEHP for ⁴⁵Ca²⁺ influx measurements. Testes were also incubated with 1 μM BEHP for 1 h (in vitro) or 12 h (in vivo) for the measurements of lactate content, ¹⁴C-deoxy-d-glucose uptake, lactate dehydrogenase (LDH) and gamma-glutamyl transpeptidase (GGT) activity, total reactive oxygen species (ROS) production and lipid peroxidation. In addition, the effect of BEHP (1 μM) on GGT, glutamic oxaloacetic transferase (GOT) and glutamic pyruvic transferase (GPT) activity in the liver was evaluated after in vivo treatment for 12 h. BEHP disturbs the Ca²⁺ balance in the testis when given acutely in vitro. BEHP stimulated Ca²⁺ influx occurs through L-type voltage-dependent Ca²⁺ channels (L-VDCC), transitory receptor potential vaniloid (TRPV1) channels, reverse-mode Na⁺/Ca²⁺ exchanger (NCX) activation and inhibition of sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). BEHP affected energy metabolism in the testis by decreasing the lactate content and LDH activity. In vitro and in vivo acute effects of BEHP promoted oxidative stress by increasing ROS production, lipid peroxidation and GGT activity in the testis. Additionally, BEHP caused liver damage by increasing GPT activity.

Introduction

Fish are exposed to numerous chemicals that are considered to be endocrine disruptor compounds (EDC). These synthetic or natural chemicals, principally produced by the plastic chemical industry, include phthalates, an important class of EDC that are rapidly metabolized [[1], [2], [3]]. The most abundantly manufactured phthalate is bis(2-ethylhexyl)phthalate (BEHP), of which more than two million tons are produced annually worldwide to increase the flexibility and elasticity of plastic products [4,5]. As phthalates are not covalently incorporated into the plastic matrix, they are released to the environment under certain conditions, leading to generalized human exposure through ingestion, inhalation and dermal contamination [6,7]. Furthermore, BEHP is the major phthalate detected in high concentrations in surface water, seawater, rivers, lakes, sediments and sewage water, consequently affecting aquatic ecosystems [[8], [9], [10]]. Additionally, the detection of BEHP in different aquatic animals, mainly fish [11], emphasizes the importance of studies in models of these animal. The Danio rerio (D. rerio) fish, known as zebrafish, is a suitable animal model for studying the impact of EDC on testicular functions and male fertility [[12], [13], [14]], since it completes its spermatogenesis wave every 6 days, instead of the 45 days required in rodents [15,16].

The effects of phthalates on males have been reported in different animals, but especially in mammalian models. BEHP inhibits steroidogenesis in human testicular cells [17], in the offspring of male rats [18] and inhibits spermatogenesis progression in D. rerio [19]. Additionally, BEHP induces the death of spermatocytes, Leydig and Sertoli cells, testicular atrophy and increases the production of reactive oxygen species (ROS) in rat testis [[20], [21], [22]].

Calcium (Ca²⁺) is one of the most important and versatile intracellular messenger and is crucial for triggering cellular processes, such as testicular Sertoli cell secretion [23,24]. Ca²⁺ signaling plays a fundamental role on sperm motility and in the fertilization process, being important for cellular differentiation, maturation, motility and the acrosome reaction of spermatozoa [[25], [26], [27], [28]], as well as cellular secretion [28,29]. However, high concentrations of intracellular Ca²⁺ and mitochondrial Ca²⁺ overload may lead to apoptosis [23,30], emphasizing the importance of Ca²⁺ homeostasis for cell functions. The involvement of ionic channels activity, osmolality and ionic concentrations associated with sperm motility can be studied in the male reproductive system using a pharmacological approach [31,32]. Phthalates may increase Ca²⁺ influx and intracellular Ca²⁺ in germ cells [33], ovary cells and renal cells [34], mast cells [29], red blood cells [35] and pancreatic cells [36], but the mechanism is still not clearly defined.

Although glucose is the main source of energy in the testis, low glucose concentration is present in the seminiferous tubules [37]. Therefore, the attachment of germ cells to the Sertoli cells is necessary, since germ cell ATP is derived from the degradation of lactate, synthesized by the lactate dehydrogenase (LDH) enzyme of the Sertoli cells [38,39]. Chronic exposure to a high dose of the phthalate, BEHP, reportedly induces severe testicular injury, reduces LDH activity and increases lipid peroxidation, culminating in impaired spermatogenesis, in rats [40]. Additionally, there is a close relationship between intracellular Ca²⁺ overload, increases in ROS production, oxidative stress and lipid peroxidation, leading to Sertoli cell apoptosis [[41], [42], [43], [44], [45]] and, consequently, an impaired testicular function in males [46,47].

In addition to the testes, the liver is another organ that is very affected by the phthalates, where these two organs are known to be the major targets of phthalate toxicity [48]. The enzymes, gamma-glutamyltranspeptidase (GGT), glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT), are recognized markers of hepatic injury after acute and chronic exposure to chemicals, including BEHP [[49], [50], [51]]. Some studies indicate a close correlation between high levels of GPT and hepatic diseases and others consider that GOT is mostly associated with mitochondrial injury [49,[51], [52], [53], [54]]. In addition to its recognition as a marker of hepatic injury [50], GGT is also considered a marker of Sertoli cell function [55,56], since it is present at the plasma membrane of these cells and is involved in cell secretion and transferrin synthesis [57,58]. In addition, GGT catalyses the transfer of gamma-glutamyl peptides either to other peptides or to l-amino acids, contributing to the synthesis and secretion of specific proteins secreted by the testis [59,60]. GGT is also involved in oxidative stress processes, participating in the maintenance of GSH redox homeostasis and in oxyreduction-dependent processes [61,62]. The present work investigated the in vitro acute effects of BEHP on mechanisms of Ca²⁺ homeostasis and energetic metabolism. In addition, the in vitro and in vivo effect of BEHP on oxidative stress was observed in the testis of D. rerio.