Analgesics and Endocrine Disruption in the Adult Testis
Study question: Do mild analgesics affect the endocrine system of the human adult testis?
Summary answer: Mild analgesics induce multiple endocrine disturbances in the human adult testis in vitro.
What is known already: Mild analgesics have recently been incriminated as potential endocrine disruptors. Studies of the effects of these widely used molecules on the androgenic status of men are limited and somewhat contradictory. This prompted us to investigate whether these compounds could alter the adult human testicular function. We therefore assessed in parallel the effects of paracetamol, aspirin and indomethacin on organo-cultured adult human testis and on the NCI-H295R steroid-producing human cell line.
Study design, size, duration: Adult human testis explants or NCI-H295R adrenocortical human cells were cultured with 10 or 10 M paracetamol, aspirin or indomethacin for 24–48 h. The effect of 10 M ketoconazole, used as an anti-androgenic reference molecule, was also assessed.
Participants/materials, setting, methods: Testes were obtained from prostate cancer patients, who had not received any hormone therapy. The protocol was approved by the local ethics committee of Rennes, France and informed consent was given by the donors. Only testes displaying spermatogenesis, as assessed by transillumination, were used in this study. Hormone levels in the culture media were determined by radioimmunoassay (testosterone, insulin-like factor 3), Enzyme-Linked Immunosorbent Assay (inhibin B) or Enzyme Immunosorbent Assay [prostaglandin (PG) D2, and PGE2]. Tissues were observed and cells counted using classical immunohistochemical methods.
Main results and the role of chance: The three mild analgesics caused multiple endocrine disturbances in the adult human testis. This was particularly apparent in the interstitial compartment. Effective doses were in the same range as those measured in blood plasma following standard analgesic treatment. The production of testosterone and insulin-like factor 3 by Leydig cells was altered by exposure to all these drugs. Inhibin B production by Sertoli cells was marginally affected by aspirin only. Our experiments also revealed that mild analgesics display direct anti-PG activity, which varied depending on the drug used, the dose and the duration of exposure. Nevertheless, associations between the alteration of the PG and testosterone profiles were not systematically observed, suggesting that a combination of mechanisms of endocrine disruption is at play.
Limitations, reasons for caution: Our studies were performed in vitro.
Wider implications of the findings: We provide the first evidence that direct exposure to mild analgesics can result in multiple endocrine disturbances in the human adult testis. Caution, concerning the consumption of mild analgesics by men, should be strengthened, particularly in high-risk population subgroups such as elite athletes.
Study funding/competing interest(s): This work was funded by Inserm (Institut national de la santé et de la recherche médicale), EHESP (Ecole des Hautes Etudes en Santé Publique), Ministère de l'Enseignement Supérieur et de la Recherche, Région Bretagne and Agence Nationale de sécurité du médicament et des produits de santé (IMPACTESTIS, Project no. AAP-2012-037). The authors declare they have no competing interests, be it financial, personal or professional.
Paracetamol and over-the-counter non-steroidal anti-inflammatory drugs (NSAIDs), herein referred to collectively as mild analgesics, are among the most sold pharmaceutical drugs worldwide. These molecules have excellent reputations within the general population and are widely used for self-medication and prophylaxis, and are also present in the environment (Verlicchi et al., 2012). However, they are often mis- or over-consumed, including in population subgroups, such as elite athletes, to prevent pain and treat injuries (Gorsline and Kaeding, 2005; Ziltener et al., 2010). The risks associated with the misuse of mild analgesics, such as hepatotoxicity (Bessone, 2010), cardiovascular side effects (Chan et al., 2006) or inducing asthma (Jenkins et al., 2004) are well known. Recent findings indicate that paracetamol and NSAIDs have endocrine disruptive potential during fetal life. Indeed, several independent epidemiological studies indicate the existence of a significant association between the intake of paracetamol alone or combined with NSAIDs during pregnancy, and an increased risk of cryptorchidism in newborn boys (Berkowitz and Lapinski, 1996; Jensen et al., 2010; Kristensen et al., 2011b; Snijder et al., 2012). Mild analgesics were also found to display anti-androgenic effects in the rat fetal testis both in utero and in vitro (Kristensen et al., 2011a,b).
During adulthood, a number of deleterious effects of aspirin and NSAIDs exposure on testicular function have also been described. Indeed, abnormal spermatogenesis was observed in adult rats following aspirin exposure (Scott and Persaud, 1977; Biswas et al., 1978). Prolonged (>15 days) administration of 1–2.5 mg/day indomethacin (Saksena et al., 1975) or of 5 mg/100 g body weight aspirin (Didolkar et al., 1980) to adult rats also decreased their plasma testosterone levels and, respectively, decreased or increased LH secretion. In mice, administration of 600 mg/kg body weight paracetamol caused degeneration of spermatids as early as 6-h post-treatment (Placke et al., 1987).
In a placebo-controlled single-blinded study with normal male volunteers, aspirin treatment inhibited the human androgenic response to human chorionic gonadotrophin (hCG; Conte et al., 1999). Originally designed to explore the involvement of cyclooxygenase (COX) metabolites in the regulation of human testis steroidogenesis, this study implicates prostaglandins (PGs) and COX inhibitors in the observed endocrine disruption, and echoes recent findings showing that many putative endocrine disruptors inhibit PG synthesis (Kristensen et al., 2011a).
Here, the direct effects of paracetamol, aspirin and indomethacin exposure on the adult human testis were investigated. To this end, an experimental approach recently validated for the study of endocrine disruption, based on the analysis of both whole human testis in organotypic culture and the NCI-H295R human steroidogenic cell line (Desdoits-Lethimonier et al., 2012), was used.
Abstract and Introduction
Abstract
Study question: Do mild analgesics affect the endocrine system of the human adult testis?
Summary answer: Mild analgesics induce multiple endocrine disturbances in the human adult testis in vitro.
What is known already: Mild analgesics have recently been incriminated as potential endocrine disruptors. Studies of the effects of these widely used molecules on the androgenic status of men are limited and somewhat contradictory. This prompted us to investigate whether these compounds could alter the adult human testicular function. We therefore assessed in parallel the effects of paracetamol, aspirin and indomethacin on organo-cultured adult human testis and on the NCI-H295R steroid-producing human cell line.
Study design, size, duration: Adult human testis explants or NCI-H295R adrenocortical human cells were cultured with 10 or 10 M paracetamol, aspirin or indomethacin for 24–48 h. The effect of 10 M ketoconazole, used as an anti-androgenic reference molecule, was also assessed.
Participants/materials, setting, methods: Testes were obtained from prostate cancer patients, who had not received any hormone therapy. The protocol was approved by the local ethics committee of Rennes, France and informed consent was given by the donors. Only testes displaying spermatogenesis, as assessed by transillumination, were used in this study. Hormone levels in the culture media were determined by radioimmunoassay (testosterone, insulin-like factor 3), Enzyme-Linked Immunosorbent Assay (inhibin B) or Enzyme Immunosorbent Assay [prostaglandin (PG) D2, and PGE2]. Tissues were observed and cells counted using classical immunohistochemical methods.
Main results and the role of chance: The three mild analgesics caused multiple endocrine disturbances in the adult human testis. This was particularly apparent in the interstitial compartment. Effective doses were in the same range as those measured in blood plasma following standard analgesic treatment. The production of testosterone and insulin-like factor 3 by Leydig cells was altered by exposure to all these drugs. Inhibin B production by Sertoli cells was marginally affected by aspirin only. Our experiments also revealed that mild analgesics display direct anti-PG activity, which varied depending on the drug used, the dose and the duration of exposure. Nevertheless, associations between the alteration of the PG and testosterone profiles were not systematically observed, suggesting that a combination of mechanisms of endocrine disruption is at play.
Limitations, reasons for caution: Our studies were performed in vitro.
Wider implications of the findings: We provide the first evidence that direct exposure to mild analgesics can result in multiple endocrine disturbances in the human adult testis. Caution, concerning the consumption of mild analgesics by men, should be strengthened, particularly in high-risk population subgroups such as elite athletes.
Study funding/competing interest(s): This work was funded by Inserm (Institut national de la santé et de la recherche médicale), EHESP (Ecole des Hautes Etudes en Santé Publique), Ministère de l'Enseignement Supérieur et de la Recherche, Région Bretagne and Agence Nationale de sécurité du médicament et des produits de santé (IMPACTESTIS, Project no. AAP-2012-037). The authors declare they have no competing interests, be it financial, personal or professional.
Introduction
Paracetamol and over-the-counter non-steroidal anti-inflammatory drugs (NSAIDs), herein referred to collectively as mild analgesics, are among the most sold pharmaceutical drugs worldwide. These molecules have excellent reputations within the general population and are widely used for self-medication and prophylaxis, and are also present in the environment (Verlicchi et al., 2012). However, they are often mis- or over-consumed, including in population subgroups, such as elite athletes, to prevent pain and treat injuries (Gorsline and Kaeding, 2005; Ziltener et al., 2010). The risks associated with the misuse of mild analgesics, such as hepatotoxicity (Bessone, 2010), cardiovascular side effects (Chan et al., 2006) or inducing asthma (Jenkins et al., 2004) are well known. Recent findings indicate that paracetamol and NSAIDs have endocrine disruptive potential during fetal life. Indeed, several independent epidemiological studies indicate the existence of a significant association between the intake of paracetamol alone or combined with NSAIDs during pregnancy, and an increased risk of cryptorchidism in newborn boys (Berkowitz and Lapinski, 1996; Jensen et al., 2010; Kristensen et al., 2011b; Snijder et al., 2012). Mild analgesics were also found to display anti-androgenic effects in the rat fetal testis both in utero and in vitro (Kristensen et al., 2011a,b).
During adulthood, a number of deleterious effects of aspirin and NSAIDs exposure on testicular function have also been described. Indeed, abnormal spermatogenesis was observed in adult rats following aspirin exposure (Scott and Persaud, 1977; Biswas et al., 1978). Prolonged (>15 days) administration of 1–2.5 mg/day indomethacin (Saksena et al., 1975) or of 5 mg/100 g body weight aspirin (Didolkar et al., 1980) to adult rats also decreased their plasma testosterone levels and, respectively, decreased or increased LH secretion. In mice, administration of 600 mg/kg body weight paracetamol caused degeneration of spermatids as early as 6-h post-treatment (Placke et al., 1987).
In a placebo-controlled single-blinded study with normal male volunteers, aspirin treatment inhibited the human androgenic response to human chorionic gonadotrophin (hCG; Conte et al., 1999). Originally designed to explore the involvement of cyclooxygenase (COX) metabolites in the regulation of human testis steroidogenesis, this study implicates prostaglandins (PGs) and COX inhibitors in the observed endocrine disruption, and echoes recent findings showing that many putative endocrine disruptors inhibit PG synthesis (Kristensen et al., 2011a).
Here, the direct effects of paracetamol, aspirin and indomethacin exposure on the adult human testis were investigated. To this end, an experimental approach recently validated for the study of endocrine disruption, based on the analysis of both whole human testis in organotypic culture and the NCI-H295R human steroidogenic cell line (Desdoits-Lethimonier et al., 2012), was used.
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