Testosterone disruptor effect and gut microbiome perturbation in mice: Early life exposure to doxycycline
Graphical abstract
Introduction
In recent years it has become clear that the increase in the frequency of male reproductive disorders such as cryptorchidism, hypospadias, testicular cancer, as well as decreased quality of semen (Vega et al., 2012), are related to early developmental environmental exposures, and particularly endocrine disrupting chemicals (EDCs) (Rahman et al., 2017). A major cause of these disorders is believed to be due to a deficiency in testosterone production during a critical period of early life (van den Driesche et al., 2015).
In testicular Leydig cells, luteinizing hormone (LH) and human chorionic gonadotropin (hCG) interact with their cognate receptors and initiate activation of the adenylate cyclase/cAMP/PKA pathway, which regulates the expression of steroidogenic proteins and enzymes (Payne and Youngblood, 1995), including steroidogenic acute regulatory protein (StAR); cytochrome P450s such as P450 side chain cleavage enzyme (P450scc; Cyp11a1), P450 hydroxylase/17,20 lyase (P450c17; Cyp17a1) and P450 aromatase (P450arom); and hydroxysteroid dehydrogenases (HSDs) such as 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) (Stocco and Clark, 1996). The mitochondria is a vital organelle in steroid hormone synthesis. StAR transfers free cholesterol from the cytoplasm into the inner membrane of mitochondria, whereupon Cyp11a1 catalyzes the conversion of cholesterol to pregnenolone, the first step in the steroidogenesis biosynthetic pathway (Clark et al., 1994). EDCs have long been known to exhibit estrogenic properties and the ability to compete with endogenous steroid hormones for receptor binding sites, thus altering male reproductive health (Jeng, 2014). Recent studies provide new insights that certain EDCs can induce mitochondrial dysfunction involved in steroidogenesis, which leads to the reduction of hormone synthesis (Desdoits-Lethimonier et al., 2012; Datta et al., 2017).
Various classes of antibiotics are used widely in human therapy, livestock agriculture, and aquaculture treatments (Sarmah et al., 2006; Bártíková et al., 2016). The extensive use of antibiotics results in environmental contamination and have recently been considered as emerging organic pollutants that lead to serious ecological issues and human health risks (Li et al., 2011). When 1064 school students in eastern China were assessed for exposure due to residues in drinking water and food at least one antibiotic type was detected in urine of 50% of students, while two or more antibiotic types were detected in urine of more than 20% of students (Wang et al., 2015). Some animal antibiotics, such as chlortetracycline, enrofloxacin and tylosin, had the highest frequency of detection in children in China (Wang et al., 2015, 2016). The US Environmental Protection Agency (EPA) implemented the Endocrine Disruptor Screening Program (EDSP) for identification and characterization of endocrine disrupting activity (EPA, 2014). However, research on antibiotics as EDCs and their human health risk implication is still lacking.
Tetracyclines, which are available over the counter in most countries, are the most common antibiotic used to control disease as well as being an additive in livestock feed. The human consumption of tetracyclines is increasing as is their detection frequency in surface water (Wei et al., 2011), drinking water (Liu and Wong, 2013), soil, vegetables (Li et al., 2011), meat, milk, and egg products (Li et al., 2017). They can subsequently bioaccumulate throughout food chains and humans are therefore exposed. Experimental investigations in mice indicated that tetracyclines could induce testicular damage (Farombi et al., 2008; Elzeinova et al., 2013). On the other hand, some studies have documented the anti-oxidant, anti-apoptotic, and anti-fibrotic effects of docycycline in various tissues besides the testis (Yeh et al., 2007; Yoon et al., 2015). Consequently, it remains unclear whether these chemicals could prevent or reverse testicular toxicity. Previous observations have demonstrated that tetracyclines can cause mitochondria dysfunction in vitro (Moullan et al., 2015). However, little attention has been given to the effects that drugs and environmental chemicals have on mitochondria function and testosterone biosynthesis in Leydig cells.
Several studies have shown that the early life stage is a critical window of time for metabolic development, and that antibiotic exposure during this window affects the course of growth and development by altering the gut microbial population (Cox et al., 2014; Bokulich et al., 2016). Long-term exposure to low-dose antibiotic is associated with an increased risk of asthma and child obesity (Korpela et al., 2016; Wang et al., 2016). But it is unclear whether antibiotics affect hormone levels in male children when exposed to low levels of these compounds over an extended period of time.
For the present study, we hypothesized that long-term exposure to low concentrations of doxycycline might disrupt testosterone production in Leydig cells by altering steroidogenesis via mitochondrial dysfunction. We evaluated the in vitro effects of doxycycline as a representative tetracycline compound on mitochondrial function and steroidogenesis in mouse Leydig tumor cell lines. Further, we determined whether the in vivo effects of exposure to low and therapeutic doses of doxycycline would result in a reduction in testosterone production during early life periods, and to also identify potential targets in the steroidogenic pathway that may responsible for the reduced testosterone production. Finally, we examined the hypothesis that gut microbiota disruption in early life could affect long-term body weight gain in adulthood.
Section snippets
Cell culture and doxycycline treatment
Mouse Leydig tumor cell lines (MLTC-1) from Shanghai Cell Bank (Shanghai, China) were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) under a humid atmosphere at 37 °C with 5% CO2. When MLTC-1 cells were approximately 80% confluent they were cultured in fresh media with no FBS for 24 h. Then various concentrations of doxycycline in RPMI 1640 medium with no FBS were overlaid on the cells and incubation proceeded for an additional 24 or 48 h.
CCK-8 assay and determination of progesterone levels
The CCK-8 assay was
Doxycycline inhibits progesterone production by mitochondria in vitro
To assess impaired mitochondrial metabolism, we used the MLTC-1 cell line that maintains the consistent gonadotropin-response compared with the primary Leydig cell before progesterone synthesis (Han et al., 2012). We analyzed the effect of doxycycline on MLTC-1 cell viability using the CCK-8 assay. As shown in Fig. 1A, cell viabilities were not significantly (P > 0.05) changed after 24 or 48 h incubation at concentrations ≤5 μM, and cell apoptosis was increased after 24 h at 15 μM or 48 h at 10
Discussion
EDCs are synthetic chemicals that interfere with hormone biosynthesis and catabolism, thereby affecting the body's endocrine homeostasis (Rahman et al., 2017). Antibiotics are emerging contaminants in the aquatic environment and might have potential adverse effects on human health. Wang et al. recently reported that oxytetracycline, chlortetracycline, and tetracycline antibiotics were detected at concentrations of 2626.7, 361.2, and 55.8 ng/mL, respectively, in the urine of Chinese school
Conclusions
Doxycycline induced steroidogenesis disturbances via mitochondrial dysfunction in Leydig cells, decreasing testosterone levels, and ultimately affecting sperm quality. Early-life exposure to low-doxycycline showed negative outcomes on testis health in later-life and associated with male mice obesity risk. To our knowledge, this is the first study to identify doxycycline as one antibiotic which could be an EDC. To further confirm the contribution of environmental pollution to health risk,
Conflicts of interest
The authors declare that they have no conflict of interest.
Acknowledgements
This study was supported by National agricultural product quality and safety risk assessment (GJFP2019013) and the National Natural Science Foundation of China (No21307044 and No31302009).
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These authors contributed equally to this manuscript.