| 1. |
- Almamoun, Radwa, et al.
(författare)
-
Altered gut microbiota community structure and correlated immune system changes in dibutyl phthalate exposed mice
- 2023
-
Ingår i: Ecotoxicology and Environmental Safety. - 0147-6513 .- 1090-2414. ; 262
-
Tidskriftsartikel (refereegranskat)abstract
- Di-n-butyl phthalate (DBP) is a ubiquitous environmental contaminant linked with various adverse health effects, including immune system dysfunction. Gut microbial dysbiosis can contribute to a wide range of pathogenesis, particularly immune disease. Here, we investigated the impact of DBP on the gut microbiome and examined correlations with immune system changes after five weeks oral exposure (10 or 100 mg/kg/day) in adult male mice. The fecal microbiome composition was characterized using 16S rRNA sequencing. DBP-treated mice displayed a significantly distinct microbial community composition, indicated by Bray-Curtis distance. Numerous amplicon sequence variants (ASVs) at the genus level were altered. Compared to the vehicle control group, the 10 mg/kg/day DBP group had 63 more abundant and 65 less abundant ASVs, while 60 ASVs were increased and 76 ASVs were decreased in the 100 mg/kg/day DBP group. Both DBP treatment groups showed higher abundances of ASVs assigned to Desulfovibrio (Proteobacteria phylum) and Enterorhabdus genera, while ASVs belonging to Parabacteroides, Lachnospiraceae UCG-006 and Lachnoclostridium were less common compared to the control group. Interestingly, an ASV belonging to Rumniniclostridium 6, which was less abundant in DBP-treated mice, demonstrated a negative correlation with the increased number of non-classical monocytes observed in the blood of DBP-treated animals. In addition, an ASV from Lachnospiraceae UCG-001, which was more abundant in the DBP-treated animals, showed a positive correlation with the non-classical monocyte increase. This study shows that DBP exposure greatly modifies the gut bacterial microbiome and indicates a potential contribution of microbial dysbiosis to DBP-induced immune system impairment, illustrating the importance of investigating how interactions between exposome components can affect health.
|
|
| 2. |
|
|
| 3. |
- Almamoun, Radwa, et al.
(författare)
-
Mechanistic screening of reproductive toxicity in a novel 3D testicular co-culture model shows significant impairments following exposure to low-dibutyl phthalate concentrations
- 2024
-
Ingår i: Archives of Toxicology. - 0340-5761 .- 1432-0738.
-
Tidskriftsartikel (refereegranskat)abstract
- To improve the mechanistic screening of reproductive toxicants in chemical-risk assessment and drug development, we have developed a three-dimensional (3D) heterogenous testicular co-culture model from neonatal mice. Di-n-butyl phthalate (DBP), an environmental contaminant that can affect reproductive health negatively, was used as a model compound to illustrate the utility of the in vitro model. The cells were treated with DBP (1 nM to 100 µM) for 7 days. Automated high-content imaging confirmed the presence of cell-specific markers of Leydig cells (CYP11A1 +), Sertoli cells (SOX9 +), and germ cells (DAZL +). Steroidogenic activity of Leydig cells was demonstrated by analyzing testosterone levels in the culture medium. DBP induced a concentration-dependent reduction in testosterone levels and decreased the number of Leydig cells compared to vehicle control. The levels of steroidogenic regulator StAR and the steroidogenic enzyme CYP11A1 were decreased already at the lowest DBP concentration (1 nM), demonstrating upstream effects in the testosterone biosynthesis pathway. Furthermore, exposure to 10 nM DBP decreased the levels of the germ cell-specific RNA binding protein DAZL, central for the spermatogenesis. The 3D model also captured the development of the Sertoli cell junction proteins, N-cadherin and Zonula occludens protein 1 (ZO-1), critical for the blood–testis barrier. However, DBP exposure did not significantly alter the cadherin and ZO-1 levels. Altogether, this 3D in vitro system models testicular cellular signaling and function, making it a powerful tool for mechanistic screening of developmental testicular toxicity. This can open a new avenue for high throughput screening of chemically-induced reproductive toxicity during sensitive developmental phases.
|
|
| 4. |
|
|
| 5. |
- Almamoun, Radwa, 1989-
(författare)
-
Toxicological studies of di-n-butyl phthalate (DBP) : Impact on the reproductive system and gut microbiota
- 2024
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
|
|
| 6. |
- Cattani, Daiane, et al.
(författare)
-
Long-Term Effects of Perinatal Exposure to a Glyphosate-Based Herbicide on Melatonin Levels and Oxidative Brain Damage in Adult Male Rats
- 2023
-
Ingår i: Antioxidants. - : MDPI. - 2076-3921. ; 12:10
-
Tidskriftsartikel (refereegranskat)abstract
- Concerns have been raised regarding the potential adverse health effects of the ubiquitous herbicide glyphosate. Here, we investigated long-term effects of developmental exposure to a glyphosate-based herbicide (GBH) by analyzing serum melatonin levels and cellular changes in the striatum of adult male rats (90 days old). Pregnant and lactating rats were exposed to 3% GBH (0.36% glyphosate) through drinking water from gestational day 5 to postnatal day 15. The offspring showed reduced serum melatonin levels (43%) at the adult age compared with the control group. The perinatal exposure to GBH also induced long-term oxidative stress-related changes in the striatum demonstrated by increased lipid peroxidation (45%) and DNA/RNA oxidation (39%) together with increased protein levels of the antioxidant enzymes, superoxide dismutase (SOD1, 24%), glutamate-cysteine ligase (GCLC, 58%), and glutathione peroxidase 1 (GPx1, 31%). Moreover, perinatal GBH exposure significantly increased the total number of neurons (20%) and tyrosine hydroxylase (TH)-positive neurons (38%) in the adult striatum. Mechanistic in vitro studies with primary rat pinealocytes exposed to 50 mu M glyphosate demonstrated a decreased melatonin secretion partially through activation of metabotropic glutamate receptor 3 (mGluR3), while higher glyphosate levels (100 or 500 mu M) also reduced the pinealocyte viability. Since decreased levels of the important antioxidant and neuroprotector melatonin have been associated with an increased risk of developing neurodegenerative disorders, this demonstrates the need to consider the melatonin hormone system as a central endocrine-related target of glyphosate and other environmental contaminants.
|
|
| 7. |
- Källsten, Liselott, et al.
(författare)
-
Adult Exposure to Di-N-Butyl Phthalate (DBP) Induces Persistent Effects on Testicular Cell Markers and Testosterone Biosynthesis in Mice
- 2022
-
Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:15
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 8. |
- Källsten, Liselott, 1992-, et al.
(författare)
-
Di-n-Butyl Phthalate and Its Monoester Metabolite Impairs Steroid Hormone Biosynthesis in Human Cells : Mechanistic In Vitro Studies
- 2022
-
Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 11:19
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 9. |
|
|
| 10. |
- Källsten, Liselott, 1992-
(författare)
-
In vitro and in vivo studies on the toxicology of di-n-butyl phthalate (DBP) : Effects on reproductive, endocrine, and immune systems
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
|
|
