Increased exposure to manmade chemicals may be linked to an increase in immune-related diseases in humans and immune system dysfunction in wildlife. Phthalates are a group of endocrine-disrupting chemicals (EDCs) suspected to influence the immune system. The aim of this study was to characterize the persistent effects on leukocytes in the blood and spleen, as well as plasma cytokine and growth factor levels, one week after the end of five weeks of oral treatment with dibutyl phthalate (DBP; 10 or 100 mg/kg/d) in adult male mice. Flow cytometry analysis of the blood revealed that DBP exposure decreased the total leukocyte count, classical monocyte and T helper (Th) popula-tions, whereas it increased the non-classical monocyte population compared to the vehicle control (corn oil). Immuno-fluorescence analysis of the spleen showed increased CD11b+Ly6G+ (marker of polymorphonuclear myeloid-derived suppressor cells; PMN-MDSCs), and CD43+staining (marker of non-classical monocytes), whereas CD3+ (marker of total T cells) and CD4+ (marker of Th cells) staining decreased. To investigate the mechanisms of action, levels of plasma cytokines and chemokines were measured using multiplexed immunoassays and other key factors were ana-lyzed using western blotting. The observed increase in M-CSF levels and the activation of STAT3 may promote PMN-MDSC expansion and activity. Increased ARG1, NOX2 (gp91phox), and protein nitrotyrosine levels, as well as GCN2 and phosphor-eIRF alpha, suggest that oxidative stress and lymphocyte arrest drive the lymphocyte suppression caused by PMN-MDSCs. The plasma levels of IL-21 (promotes the differentiation of Th cells) and MCP-1 (regulates mi-gration and infiltration of monocytes/macrophages) also decreased. These findings show that adult DBP exposure can cause persistent immunosuppressive effects, which may increase susceptibility to infections, cancers, and immune dis-eases, and decrease vaccine efficacy.

Studies indicate that phthalates are endocrine disruptors affecting reproductive health. One of the most commonly used phthalates, di-n-butyl phthalate (DBP), has been linked with adverse reproductive health outcomes in men, but the mechanisms behind these effects are still poorly understood. Here, adult male mice were orally exposed to DBP (10 or 100 mg/kg/day) for five weeks, and the testis and adrenal glands were collected one week after the last dose, to examine more persistent effects. Quantification of testosterone, androstenedione, progesterone and corticosterone concentrations by liquid chromatography-mass spectrometry showed that testicular testosterone was significantly decreased in both DBP treatment groups, whereas the other steroids were not significantly altered. Western blot analysis of testis revealed that DBP exposure increased the levels of the steroidogenic enzymes CYP11A1, HSD3 beta 2, and CYP17A1, the oxidative stress marker nitrotyrosine, and the luteinizing hormone receptor (LHR). The analysis further demonstrated increased levels of the germ cell marker DAZL, the Sertoli cell markers vimentin and SOX9, and the Leydig cell marker SULT1E1. Overall, the present work provides more mechanistic understanding of how adult DBP exposure can induce effects on the male reproductive system by affecting several key cells and proteins important for testosterone biosynthesis and spermatogenesis, and for the first time shows that these effects persist at least one week after the last dose. It also demonstrates impairment of testosterone biosynthesis at a lower dose than previously reported.

The widespread environmental contaminant di-n-butyl phthalate (DBP) has been linked with reduced testosterone levels and adverse reproductive health outcomes in men. However, the underlying mechanisms of these anti-androgenic effects and the potential effects on other classes of steroid hormones remain to be elucidated. Here, we conducted mechanistic studies in human adrenocortical H295R cells exposed to 1–500 µM of DBP or its metabolite, mono-n-butyl phthalate (MBP), for 48 h. Quantification of steroid hormones in the cell medium by liquid chromatography-mass spectrometry revealed that both phthalates significantly decreased testosterone, androstenedione, corticosterone, and progesterone levels, in particular after dibutyryl-cyclic-AMP stimulation of steroidogenesis. Western blot analysis of key steroidogenic proteins showed that DBP induced a dose-dependent decrease of CYP11A1 and HSD3β2 levels, while MBP only significantly decreased CYP17A1 levels, indicating that the compounds affect early steps of the steroidogenesis differently. Both DBP and MBP exposure also lead to a dose-related decrease in HSD17β3, the enzyme which catalyzes the final step in the testosterone biosynthesis pathway, although these effects were not statistically significant. Interestingly, DBP increased the cortisol concentration, which may be due to the non-significant CYP11B1 increase in DBP-exposed cells. In contrast, MBP decreased cortisol concentration. Moreover, the analysis of superoxide generation and quantification of the protein oxidation marker nitrotyrosine demonstrated that DBP induced oxidative stress in H295R cells while MBP reduced protein nitrotyrosine levels. These findings confirm the anti-androgenic effects of DBP and MBP and reveal several differences in their toxicological mechanisms, with possible implications for future research on phthalate toxicity.

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.