Decrease, testosterone levels

Testosterone is an endogenous steroid hormone and a potent androgen. Androgens act by binding androgen receptors in androgen-responsive tissues (Murashima et al., 2015). Testosterone and other androgens such as dihydrotestosterone (DHT) are important for reproductive development and masculinization of the fetus. Androgens are also important for bone, brain, muscle and skin health (Alemany, 2022). Just like other steroid hormones, testosterone is produced through a process known as steroidogenesis which is controlled by enzymes converting cholesterol into all of the downstream steroid hormones. In steroidogenesis, androstenedione or androstenediol is converted to testosterone by the enzymes 17β-hydroxysteroid dehydrogenase (HSD) or 3β-HSD, respectively. Testosterone can then be converted to the more potent androgen, DHT, by 5α-reductase, or aromatized by aromatase (CYP19A1) into estrogens. Testosterone secreted in blood circulation can be found free but more frequently is found bound to SHBG or albumin (Trost & Mulhall, 2016).

Testosterone is produced mainly by the ovaries (in females ), testes (in males), and to  a lesser degree in the adrenal glands. During fetal development testosterone plays a crucial role in the differentiation of male reproductive tissues and the overall male phenotype. In adulthood, testosterone synthesis is controlled by the Hypothalamus-Pituitary-Gonadal (HPG) axis. GnRH is released from the hypothalamus inducing LH pulses secreted by the anterior pituitary. This LH surge leads to increased testosterone production. If testosterone reaches low levels, this axis is once again stimulated to provoke more testosterone synthesis. This feedback loop is essential for maintenance of appropriate testosterone levels (Chandrashekar & Bartke, 1998; Ellis et al., 1983; Rey, 2021).

Disruption of any of the aforementioned processes may result in reduced testosterone levels, such as inhibition of steroidogenic enzyme activity thereby inhibiting production of testosterone.

General role in biology

Androgens, the main male sex steroids, are the critical factors responsible for the development of the male phenotype during embryogenesis and for the achievement of sexual maturation at puberty. In adulthood, androgens remain essential for the maintenance of male reproductive function and behaviour. Apart from their effects on reproduction, androgens affect a wide variety of non-reproductive tissues such as skin, bone, muscle, and brain (Heemers, Verhoeven, & Swinnen, 2006). Androgens, principally T and 5α-dihydrotestosterone (DHT), exert most of their effects by interacting with a specific receptor, the androgen receptor (AR), for review see (Murashima, Kishigami, Thomson, & Yamada, 2015). On the one hand, testosterone can be reduced by 5α-reductase to produce 5α dihydrotestosterone (DHT). On the other hand, testosterone can be aromatized to generate estrogens. Testosterone effects can also be classified by the age of usual occurrence, postnatal effects in both males and females are mostly dependent on the levels and duration of circulating free testosterone.

Quantification of testosterone levels can be performed by various means (e.g. serum levels in vivo, cell culture medium levels in vitro, tissue ex vivo or in vitro). Traditional immunoassay methods (ELISA or RIA), and advanced instrumental techniques (e.g. LC-MS/MS) or liquid scintillation spectrometry (after radiolabeling) can be used (Shiraishi et al., 2008).

The H295R Steroidogenesis assay (OECD TG 456) is used to measure mainly the production of estradiol and testosterone. This is a validated OECD test guideline using adrenal H295R cells and hormone levels are then measured in the cell medium (OECD 2011). H295R adrenocortical carcinoma cells produce all the main enzymes and hormones of the steroidogenic pathway. Therefore, exposure to different stressors allows for broad analysis of their impact on steroidogenesis by measuring hormones in culture medium by LC-MS/MS. H295 assay was designed measure disruption to testosterone or estradiol levels but can now also be used to measure additional steroid hormones such as progesterone or pregnenolone. The U.S. EPA’s ToxCast program developed a high throughput method for the H295R assay which can measure a total of 11 hormones from the steroidogenesis pathway (Haggard et al., 2018). The H295R can be considered an indirect measurement as it provides information on a disruption of overall steroidogenesis that would result in a change of testosterone levels but not the underlying mechanism.

Testosterone can be measured by immunoassays and by isotope-dilution gas chromatography-mass spectrometry in serum (Taieb et al., 2003), (Paduch et al., 2014). Testosterone levels are measured i.a. in: Fish Lifecycle Toxicity Test (FLCTT) (US EPA OPPTS 850.1500), Male pubertal assay (PP Male Assay) (US EPA OPPTS 890.1500), OECD TG 441: Hershberger Bioassay in Rats (H Assay).

This KE is applicable to mammals since the role of testosterone and its synthesis are conserved (Vitousek et al., 2018). Both sexes need, and produce, testosterone and its role is observed throughout different life stages, from development to adulthood (Luetjens & Weinbauer, 2012; Naamneh Elzenaty et al., 2022). Therefore, this KE is also applicable to both males and females as well as throughout these life stages. Also of note, key enzymes needed for testosterone production first appear in the common ancestor of amphioxus and vertebrates (Baker 2011). Consequently, it is acknowledged that this KE most likely has a much broader domain of applicability extending to non-mammalian vertebrates. AOP developers are encouraged to add additional relevant knowledge to expand on the applicability beyond mammals to other vertebrates.

Key enzymes needed for testosterone production first appear in the common ancestor of amphioxus and vertebrates (Baker 2011). Consequently, this key event is applicable to most vertebrates, including humans.

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