On the Use and Interpretation of Areola/Nipple Retention as a Biomarker for Anti-androgenic Effects in Rat Toxicity Studies

Abstract

Areola/nipple retention (NR) is an established biomarker for an anti-androgenic mode of action in rat toxicity studies. It is a mandatory measurement under several OECD test guidelines and is typically assessed in combination with anogenital distance (AGD). Both NR and AGD are considered retrospective biomarkers of insufficient androgen signaling during the masculinization programming window in male fetuses. However, there are still aspects concerning NR as a biomarker for endocrine disruption that remains to be clarified. For instance, can NR be regarded a permanent adverse effect? Is it a redundant measurement if AGD is assessed in the same study? Is NR equally sensitive and specific to anti-androgenic chemical substances as a shortening of male AGD? In this review we discuss these and other aspects concerning the use of NR as a biomarker in toxicity studies. We have collected available literature from rat toxicity studies that have reported on NR and synthesized the data in order to draw a clearer picture about the sensitivity and specificity of NR as an effect biomarker for an anti-androgenic mode of action, including comparisons to AGD measurements. We carefully conclude that NR and AGD in rats for the most part display similar sensitivity and specificity, but that there are clear exceptions which support the continued assessment of both endpoints in relevant reproductive toxicity studies. Available literature also support the view that NR in infant male rats signifies a high risk for permanent nipples in adulthood. Finally, the literature suggests that the mechanisms of action leading from a chemical stressor event to either NR or short AGD in male offspring are overlapping with respect to canonical androgen signaling, yet differ with respect to other mechanisms of action.

Using alternative test methods to predict endocrine disruption and reproductive adverse outcomes: do we have enough knowledge?

“…doses on same adverse effect endpoints, as for instance for the androgensensitive endpoints AGD and NR as previously reviewed (Schwartz et al, 2021;Schwartz et al, 2019).…”

“…We also have a good understanding of how many chemicals can interfere with biomolecules and regulatory pathways. We even have an extensive database on how a sizeable number of chemicals cause various male reproductive diseases in experimental animals (Brehm & Flaws, 2019;Gray et al, 2001;Schwartz et al, 2021;Schwartz et al, 2019). In humans the evidence is less clear and still debated, yet many epidemiological studies associate chemical exposures with comparative disease to those observed in animal models (Bonde et al, 2016;Kristensen et al, 2016;Rodprasert et al, 2021;Skakkebaek et al, 2022;Stukenborg et al, 2021).…”

Azole Fungicides and Their Endocrine Disrupting Properties: Perspectives on Sex Hormone-Dependent Reproductive Development

“…Several azoles induce anti-androgenic effects such as short male AGD following developmental exposure ( Vinggaard et al, 2005 ; Laier et al, 2006 ; Taxvig et al, 2007 ; Taxvig et al, 2008 ; Hass et al, 2012 ; Johansson et al, 2021a ), but surprisingly also longer AGDs in both male and female offspring with certain azole fungicides ( Noriega et al, 2005 ; Laier et al, 2006 ; Goetz et al, 2007 ; Taxvig et al, 2007 ; Christiansen et al, 2009 ; Hass et al, 2012 ; Melching-Kollmuss et al, 2017 ). Developmental exposure to azoles have also been shown to cause nipple retention in male rat offspring ( Noriega et al, 2005 ; Vinggaard et al, 2005 ; Laier et al, 2006 ; Taxvig et al, 2007 ; Hass et al, 2012 ), which is a biomarker for compromised androgen action ( Schwartz et al, 2021 ). Azoles can also cause increased incidents of malformed external genitalia and hypospadias ( Noriega et al, 2005 ; Laier et al, 2006 ), as well as delayed pubertal onset ( Melching-Kollmuss et al, 2017 ; Johansson et al, 2021a ), and fewer motile sperm ( Taxvig et al, 2007 ; Melching-Kollmuss et al, 2017 ).…”

Building an adverse outcome pathway network for estrogen-, androgen- and steroidogenesis-mediated reproductive toxicity

“…However, there is a need for further refinement of existing AOPs to improve overall confidence, as well as to develop additional AOPs describing relevant MoAs that are still missing. This includes reproductive toxicity where effects of EDs on both male and female reproductive systems have been well documented ( Schwartz et al, 2019 ; 2021 ; Duursen van et al, 2020 ; Sharma et al, 2020 ; Rodprasert et al, 2021 ; You and Song, 2021 ; Delbes et al, 2022 ; Marlatt et al, 2022 ), yet where we still lack fundamental knowledge allowing us to predict with high confidence in vivo outcomes based on data from alternative test methods ( Svingen et al, 2022 ).…”