Chemical pollution is an increasing societal problem and has a major impact on human and environmental health. One important source of chemical pollution is plastic, which contains many different compounds with often largely unknown hazards. Phthalates are one group of chemicals in plastic that has been associated with several adverse effects in humans, particularly reproductive system impairments. Studies have also suggested a link between exposure to phthalates and negative effects on the immune system. One of the most widely used phthalates is di-n-butyl phthalate (DBP), which is frequently detected in humans and the environment. DBP has been associated with decreased male fertility and reduced levels of testosterone. However, the mechanisms behind these anti-androgenic effects are not entirely understood, and most studies have focused only on developmental exposure.

This thesis aims to, for the first time, investigate persistent effects on the reproductive and immune systems of adult male mice after exposure to DBP. Adult male mice were orally exposed to DBP (0, 10 or 100 mg/kg/day) for 5 weeks. A persistent and significant decrease in testicular testosterone levels was shown together with an increase in the levels of several steroidogenic enzymes 1 week after the conclusion of exposure. The decrease in testosterone may be related to the demonstrated increase in oxidative stress, which may affect enzyme activity. Additional mechanistic studies were conducted in the human adrenal cell line H295R. The testosterone levels decreased also in vitro; however, the levels of several steroidogenic enzymes in the cells decreased, which is in contrast with the in vivo study. Several additional steroid hormones were affected in vitro, but not in vivo. The animal study further revealed significantly increased levels of the key testicular proteins DAZL, vimentin, SOX9, and SULT1E1.

Moreover, a persistent immunosuppressive effect was demonstrated in the DBP-exposed mice, supporting previous data indicating that endocrine disruptors can affect the immune system. DBP-induced leukopenia, reduced numbers of T helper cells, and increased levels of immunosuppressive cells were observed. In addition, the distribution of two main DBP metabolites to three proposed target tissues (liver, testes, and adipose tissue) was examined, and the presence of the metabolites was confirmed 24 h after the final dose. The glucuronidation pattern in the mice was shown to be more similar to that previously observed in humans than in rats.

In conclusion, the results in this thesis support that the testes and immune system are key targets for DBP-induced toxicity. DBP decreased the testosterone levels both in vivo and in vitro, but certain differences in the effects on steroidogenesis were observed between the experimental models. Further studies are required to determine the No Observed Adverse Effect Level (NOAEL) for the effects identified in the animal model and to understand the underlying mechanisms completely.

The potential health impact of exposure to anthropogenic chemicals has raised major concerns worldwide. Phthalates are mainly used in the plastic industry and have been associated with adverse effects in humans. Di-n-butyl phthalate (DBP) is one of the dominant phthalates with a ubiquitous presence in the environment. While many studies have examined the endocrine disrupting properties of DBP, with a focus on developmental and reproductive dysfunctions, studies of its effects on the adult reproductive system and gut microbiota are limited. This thesis aimed to determine persistent effects of DBP on the adult male reproductive system, provide a high-throughput screening tool for identifying reproductive toxicants, and characterize the effects of DBP on the gut microbiota.

   Paper I investigated if adult DBP exposure can induce persistent effects on the mature reproductive system. Adult male mice were orally exposed to 10 or 100 mg/kg/day for five weeks and testes were collected one week after the last dose. The results demonstrated a significant decrease in testosterone levels in the DBP-exposed groups. Mechanistically, the levels of steroidogenic enzymes, cell-specific markers and oxidative stress were increased. In paper II, elements of the in vivo testicular microenvironment, including functional testosterone production, were modeled using a three-dimensional (3D) heterogenous testicular cell co-culture derived from neonatal mice. Automated high-content imaging of cell-specific markers confirmed the presence of germ cells (DAZL+), Leydig cells (CYP11A1+), and Sertoli cells (SOX9+). DBP exposure decreased testosterone production, as well as levels of the steroidogenic enzyme CYP11A1, and the steroidogenic regulator StAR. Overall, this in vitro 3D model recapitulates the testicular pathways involved in DBP toxicity, making it a relevant tool for assessing reprotoxic effects of chemicals.

   Paper III investigated the impact of oral DBP exposure on the gut microbiota and the potential interplay with immune and testicular toxicity using 16S rRNA sequencing. DBP-treated mice showed a distinct microbial composition and numerous differentially abundant amplicon sequence variants. Interestingly, the microbial alterations correlated with an increase in non-classical monocytes observed in DBP-exposed mice. In paper IV, a shotgun metagenomic analysis was conducted to achieve a more comprehensive characterization of the DBP-induced effects on gut microbiota composition and function. The DBP-exposed mice had a higher abundance of Adlercreutzia mucosicola, a bacterium linked with intestinal inflammation. In contrast, the beneficial Akkermansia muciniphila was less abundant in DBP-exposed mice. Functional analysis demonstrated that DBP exposure increased the abundance of genes involved in environmental sensing and antimicrobial resistance.

   In conclusion, this doctoral thesis demonstrates the antiandrogenic effects of DBP as well as potential underlying mechanisms of testicular dysfunction in adult mice. In addition, we established a powerful in vitro tool for screening reprotoxic effects. The gut microbiota was also impaired by DBP exposure, which may play a potential role in initiating or exacerbating the DBP-induced toxicity. Overall, this work highlights the potential health impact of the interplay between the two exposome components, chemical exposure and gut microbiota.