Deciphering mechanisms of male reproductive toxicity: How azole fungicides disrupt fetal development

Incidence rates of many male reproductive disorders are increasing. These disorders include genital malformations such as hypospadias and cryptorchidism, but also reduced fertility and reproductive cancers. Since male reproductive development and function depend on androgens synthesized by the fetal testes, these disorders are hypothesized to be symptoms of a common testicular dysgenesis syndrome (TDS) originating in fetal life, where aberrant development ofthe testes is suggested to give rise to reproductive disorders at birth or later in life. Exposure to endocrine disrupting chemicals (EDCs), particularly during sensitive developmental windows, has been suggested as a significant contributor to these adverse trends in disease incidents.
EDCs are ubiquitous in our everyday lives and are found in pharmaceuticals, pesticides, personal care products, food contact materials, and much more. To safeguard human reproductive health against EDCs, international standardized test guidelines, for testing endocrine disrupting effects and reproductive toxicity of chemicals, have been established. However, modern toxicology faces a challenge. As the push towards using non-animal test data for assessment of chemical toxicity is increasing, so does the need for detailed mechanistic knowledge. This knowledge is essential for predictive toxicology, where we use alternative test methods in place of animal toxicity studies. While adverse reproductive effects independent of the underlying mechanisms would in most cases be measurable in the intact animal we need elaborate mechanistic understanding of chemical mode of action to assess chemical safety from non-animal test data. In many cases, however, this fundamental mechanistic knowledge is still lacking.
Azoles are used as antifungal agents in both agriculture and medicine. Unfortunately, many azole fungicides have endocrine disrupting potential. They display a diverse pattern of adverse effect outcomes in intact animals that, in many cases, do not mirror what is predicted by alternative test method data.
This thesis explores further the endocrine disrupting activities of azoles with particular focus on male reproductive development. Using rats and mice as model organisms, alongside in silico, in vitro, and ex vivo methodologies, I show that several azoles act as endocrine disruptors with consequences for reproductive development and, importantly, that they act through different types of mechanisms.
These findings help underscore the utility of the non-animal test methods we already have for detecting endocrine disrupting chemicals, but more importantly, they further highlight insufficiencies that must be addressed if we are to develop robust test strategies that will adequately help protect human reproductive health in the future. For instance, in this thesis we assess whether azole fungicides are capable of disrupting a non-classical mode of action for endocrine disruption, namely retinoid signaling in the fetal testis. The retinoid signaling pathway has not been widely considered in the origin of testicular toxicity, albeit developmental biologists for long have known of its role in testis development. We show that the retinoid signaling system in the fetal rodent testis is a target for azole fungicides, and likely other EDCs, and therefore should be included in future non-animal test strategies.
Another outcome of this thesis is a detailed examination of transcriptional footprints indifferent tissues and organs following fetal exposure to azole fungicides using high-throughput transcriptomics methods. This is important, since omics approaches are already being integrated as valuable additions in the assessment of chemicals, particularly in predictive toxicology. Although, there are still many challenges that must be overcome when using transcriptomics as a predictive measure in toxicology. One of these challenges is to look in the right tissue at the right time. This is evidenced by my studies of feminized male rat offspring following in utero azole exposure where few transcriptional changes are observed in the fetal testes, but where the same fetuses display marked transcriptional changes in androgen-sensitive target tissues, the external genitalia (phallus) and muscle tissues (perineum). Studies in this thesis clearly illustrate the great variability in transcriptional changes between tissues, but also between developmental stages. This knowledge is of utmost importance for the optimization of ‘omics’ data gathering and interpretation in predicting toxicological outcomes following chemical exposures.
Overall, this PhD thesis contributes new knowledge on how azole fungicides can act as endocrine disruptors and cause reproductive disease, and highlights important gaps in bothknowledge and test strategies pertaining to the assessment of endocrine disrupting potentials and reproductive toxicity of chemicals.