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Oral contraceptives modify DNA methylation and monocyte-derived macrophage function.

Campesi I, Sanna M, Zinellu A, Carru C, Rubattu L, Bulzomi P, Seghieri G, Tonolo G, Palermo M, Rosano G, Marino M, Franconi F - Biol Sex Differ (2012)

Bottom Line: As is already known, the use of OCs changed numerous parameters: the number of lymphocytes, iron levels, total iron-binding capacity of transferrin, triglycerides, high-density lipoprotein, total cholesterol, and C-reactive protein increased, while prothrombin time and alkaline phosphatase decreased.OC did not modify the expression of androgen receptor but increased estrogen receptor α expression, more considerably in FOCA+, and decreased estrogen receptor β, more considerably in FOCA-.Some of the above activities were linked to the androgenic properties of progestin.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Laboratory of Sex-Gender Medicine of the National Institute of Biostructures and Biosystems, Osilo, Italy. ilacampesi79@yahoo.it.

ABSTRACT

Background: Fertile women may be encouraged to use contraception during clinical trials to avoid potential drug effects on fetuses. However, hormonal contraception interferes with pharmacokinetics and pharmacodynamics and modifies internal milieus. Macrophages depend on the milieu to which they are exposed. Therefore, we assessed whether macrophage function would be affected by the use of combined oral contraceptives (OCs) and if this influence depended on the androgenic or non-androgenic properties of progestin.

Methods: Healthy adult women were enrolled and stratified into two groups: women who did not use OCs (Fs) and women treated with OCs (FOCs). FOCs were further stratified as a function of androgenic (FOCA+) and non-androgenic (FOCA-) properties of progestins. Routine hematological, biochemical, inflammatory and endothelial dysfunction parameters were measured. Monocyte-derived macrophages (MDMs) were evaluated for the expression and activity of estrogen receptors and androgen receptors, and release of tumor necrosis factor α (TNFα) was measured from unstimulated and lipopolysaccharide-stimulated cells.

Results: As is already known, the use of OCs changed numerous parameters: the number of lymphocytes, iron levels, total iron-binding capacity of transferrin, triglycerides, high-density lipoprotein, total cholesterol, and C-reactive protein increased, while prothrombin time and alkaline phosphatase decreased. Hormonal levels also varied: cortisol was higher in FOCs, while luteinizing hormone, follicle-stimulating hormone, and testosterone were lower in FOCs. Asymmetric dimethylarginine, an index of endothelial function, was lower in FOC than in Fs, as were cysteine and bilirubin. The androgenic properties of progestins affected the activity of OCs: in particular, white blood cell count, hemoglobin, high-density lipoprotein and calcium were higher in FOCA- than in FOCA+, whereas percentage oxygen saturation and γ-glutamyl transpeptidase were lower in FOCA- than in FOCA+. Importantly, FOCs had a lower global DNA methylation, indicating that OC may have epigenetic effects on gene expression. OC did not modify the expression of androgen receptor but increased estrogen receptor α expression, more considerably in FOCA+, and decreased estrogen receptor β, more considerably in FOCA-. Importantly, the activation state of estrogen receptor β in FOCs was decreased, while estrogen receptor α was not active in either Fs or FOCs. Unstimulated MDMs obtained from FOCs showed higher release of TNFα in comparison with Fs. After lipopolysaccharide stimulation, the release of TNFα was significantly higher in Fs than in FOCs.

Conclusions: OC use induced many changes in hematological and plasmatic markers, modifying hormonal levels, endothelial function, inflammation index and some redox state parameters, producing a perturbation of the internal milieu that impacted macrophagic function. In fact, different levels of estrogen receptor expression and release of TNFα were observed in macrophages derived from OC users. Some of the above activities were linked to the androgenic properties of progestin. Even though it is not known whether these effects are reversible, the results indicate that to avoid potential skewing of results only a single type of OC should be used during a single clinical trial.

No MeSH data available.


Related in: MedlinePlus

Activation status of estrogen receptors. Representative western blot for estrogen receptor α phosphorylation (A) and representative western blot with corresponding densitometric analysis of estrogen receptor β activity (measured as p38 phosphorylation) (B, C) in estrogen receptor α-expressing MCF-7 and estrogen receptor β-expressing DLD-1 cell lines stimulated with 10 nM estradiol (1 h) and in monocyte-derived macrophages (MDMs) from Fs (white bar), FOCA+ (circle bar) and FOCA- (striped bar). Data are expressed as the mean ± SD of at least four independent experiments. **P < 0.001 vs Fs. FOCA+/FOCA- = FOCs further stratified as a function of androgenic (FOCA+) and non-androgenic (FOCA-) properties of progestins; FOCs = women treated with OCs for at least 3 months; Fs = women who had not used OCs for at least 3 months to ensure a sufficient washout period; OC = oral contraceptive.
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Figure 3: Activation status of estrogen receptors. Representative western blot for estrogen receptor α phosphorylation (A) and representative western blot with corresponding densitometric analysis of estrogen receptor β activity (measured as p38 phosphorylation) (B, C) in estrogen receptor α-expressing MCF-7 and estrogen receptor β-expressing DLD-1 cell lines stimulated with 10 nM estradiol (1 h) and in monocyte-derived macrophages (MDMs) from Fs (white bar), FOCA+ (circle bar) and FOCA- (striped bar). Data are expressed as the mean ± SD of at least four independent experiments. **P < 0.001 vs Fs. FOCA+/FOCA- = FOCs further stratified as a function of androgenic (FOCA+) and non-androgenic (FOCA-) properties of progestins; FOCs = women treated with OCs for at least 3 months; Fs = women who had not used OCs for at least 3 months to ensure a sufficient washout period; OC = oral contraceptive.

Mentions: MDMs expressed androgen receptors, estrogen receptor α, and estrogen receptor β (Figure 2), with the β isoform being the most highly expressed, as indicated comparing the estrogen receptor β band with the band obtained by loading 5 ng of recombinant proteins. Consequently, MDMs presented a low estrogen receptor α/estrogen receptor β ratio. The use of OCs had a considerable impact on estrogen receptor levels: they increased estrogen receptor α approximately fivefold and decreased estrogen receptor β approximately 0.5-fold in comparison with Fs. This effect led to a significant increase in the estrogen receptor α/estrogen receptor β ratio in MDMs derived from FOCs compared to Fs (Figure 2). When the FOCs were stratified according to the androgenic and non-androgenic properties of progestin, we observed that estrogen receptor α and estrogen receptor β levels were at least twice as high in FOCA+ than in FOCA-. Consequently, the α/β ratio was significantly higher in FOCA- than in FOCA+ (Figure 2). The result obtained in FOCs prompted us to investigate whether the altered levels of estrogen receptor isoforms were paralleled by differences in their activation statuses by measuring the phosphorylation of estrogen receptor α Ser118 and the activation of p38 as a measure of estrogen receptor β activity [28,29]. The phosphorylated form of estrogen receptor α was undetectable in our samples, indicating that this receptor was inactive, whereas estrogen receptor α phosphorylation was evident in MCF-7 cells (used as a positive control). The phosphorylation of p38, and thus estrogen receptor β activation, was decreased in MDMs obtained from FOCs compared to Fs, indicating that OCs not only decreased the level but also the activity of estrogen receptor β (Figure 3). Finally, the levels of androgen receptors were not changed by the use of OCs, indicating that OCs have a specific impact on estrogen receptor levels and activities (Figure 2).


Oral contraceptives modify DNA methylation and monocyte-derived macrophage function.

Campesi I, Sanna M, Zinellu A, Carru C, Rubattu L, Bulzomi P, Seghieri G, Tonolo G, Palermo M, Rosano G, Marino M, Franconi F - Biol Sex Differ (2012)

Activation status of estrogen receptors. Representative western blot for estrogen receptor α phosphorylation (A) and representative western blot with corresponding densitometric analysis of estrogen receptor β activity (measured as p38 phosphorylation) (B, C) in estrogen receptor α-expressing MCF-7 and estrogen receptor β-expressing DLD-1 cell lines stimulated with 10 nM estradiol (1 h) and in monocyte-derived macrophages (MDMs) from Fs (white bar), FOCA+ (circle bar) and FOCA- (striped bar). Data are expressed as the mean ± SD of at least four independent experiments. **P < 0.001 vs Fs. FOCA+/FOCA- = FOCs further stratified as a function of androgenic (FOCA+) and non-androgenic (FOCA-) properties of progestins; FOCs = women treated with OCs for at least 3 months; Fs = women who had not used OCs for at least 3 months to ensure a sufficient washout period; OC = oral contraceptive.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298494&req=5

Figure 3: Activation status of estrogen receptors. Representative western blot for estrogen receptor α phosphorylation (A) and representative western blot with corresponding densitometric analysis of estrogen receptor β activity (measured as p38 phosphorylation) (B, C) in estrogen receptor α-expressing MCF-7 and estrogen receptor β-expressing DLD-1 cell lines stimulated with 10 nM estradiol (1 h) and in monocyte-derived macrophages (MDMs) from Fs (white bar), FOCA+ (circle bar) and FOCA- (striped bar). Data are expressed as the mean ± SD of at least four independent experiments. **P < 0.001 vs Fs. FOCA+/FOCA- = FOCs further stratified as a function of androgenic (FOCA+) and non-androgenic (FOCA-) properties of progestins; FOCs = women treated with OCs for at least 3 months; Fs = women who had not used OCs for at least 3 months to ensure a sufficient washout period; OC = oral contraceptive.
Mentions: MDMs expressed androgen receptors, estrogen receptor α, and estrogen receptor β (Figure 2), with the β isoform being the most highly expressed, as indicated comparing the estrogen receptor β band with the band obtained by loading 5 ng of recombinant proteins. Consequently, MDMs presented a low estrogen receptor α/estrogen receptor β ratio. The use of OCs had a considerable impact on estrogen receptor levels: they increased estrogen receptor α approximately fivefold and decreased estrogen receptor β approximately 0.5-fold in comparison with Fs. This effect led to a significant increase in the estrogen receptor α/estrogen receptor β ratio in MDMs derived from FOCs compared to Fs (Figure 2). When the FOCs were stratified according to the androgenic and non-androgenic properties of progestin, we observed that estrogen receptor α and estrogen receptor β levels were at least twice as high in FOCA+ than in FOCA-. Consequently, the α/β ratio was significantly higher in FOCA- than in FOCA+ (Figure 2). The result obtained in FOCs prompted us to investigate whether the altered levels of estrogen receptor isoforms were paralleled by differences in their activation statuses by measuring the phosphorylation of estrogen receptor α Ser118 and the activation of p38 as a measure of estrogen receptor β activity [28,29]. The phosphorylated form of estrogen receptor α was undetectable in our samples, indicating that this receptor was inactive, whereas estrogen receptor α phosphorylation was evident in MCF-7 cells (used as a positive control). The phosphorylation of p38, and thus estrogen receptor β activation, was decreased in MDMs obtained from FOCs compared to Fs, indicating that OCs not only decreased the level but also the activity of estrogen receptor β (Figure 3). Finally, the levels of androgen receptors were not changed by the use of OCs, indicating that OCs have a specific impact on estrogen receptor levels and activities (Figure 2).

Bottom Line: As is already known, the use of OCs changed numerous parameters: the number of lymphocytes, iron levels, total iron-binding capacity of transferrin, triglycerides, high-density lipoprotein, total cholesterol, and C-reactive protein increased, while prothrombin time and alkaline phosphatase decreased.OC did not modify the expression of androgen receptor but increased estrogen receptor α expression, more considerably in FOCA+, and decreased estrogen receptor β, more considerably in FOCA-.Some of the above activities were linked to the androgenic properties of progestin.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Laboratory of Sex-Gender Medicine of the National Institute of Biostructures and Biosystems, Osilo, Italy. ilacampesi79@yahoo.it.

ABSTRACT

Background: Fertile women may be encouraged to use contraception during clinical trials to avoid potential drug effects on fetuses. However, hormonal contraception interferes with pharmacokinetics and pharmacodynamics and modifies internal milieus. Macrophages depend on the milieu to which they are exposed. Therefore, we assessed whether macrophage function would be affected by the use of combined oral contraceptives (OCs) and if this influence depended on the androgenic or non-androgenic properties of progestin.

Methods: Healthy adult women were enrolled and stratified into two groups: women who did not use OCs (Fs) and women treated with OCs (FOCs). FOCs were further stratified as a function of androgenic (FOCA+) and non-androgenic (FOCA-) properties of progestins. Routine hematological, biochemical, inflammatory and endothelial dysfunction parameters were measured. Monocyte-derived macrophages (MDMs) were evaluated for the expression and activity of estrogen receptors and androgen receptors, and release of tumor necrosis factor α (TNFα) was measured from unstimulated and lipopolysaccharide-stimulated cells.

Results: As is already known, the use of OCs changed numerous parameters: the number of lymphocytes, iron levels, total iron-binding capacity of transferrin, triglycerides, high-density lipoprotein, total cholesterol, and C-reactive protein increased, while prothrombin time and alkaline phosphatase decreased. Hormonal levels also varied: cortisol was higher in FOCs, while luteinizing hormone, follicle-stimulating hormone, and testosterone were lower in FOCs. Asymmetric dimethylarginine, an index of endothelial function, was lower in FOC than in Fs, as were cysteine and bilirubin. The androgenic properties of progestins affected the activity of OCs: in particular, white blood cell count, hemoglobin, high-density lipoprotein and calcium were higher in FOCA- than in FOCA+, whereas percentage oxygen saturation and γ-glutamyl transpeptidase were lower in FOCA- than in FOCA+. Importantly, FOCs had a lower global DNA methylation, indicating that OC may have epigenetic effects on gene expression. OC did not modify the expression of androgen receptor but increased estrogen receptor α expression, more considerably in FOCA+, and decreased estrogen receptor β, more considerably in FOCA-. Importantly, the activation state of estrogen receptor β in FOCs was decreased, while estrogen receptor α was not active in either Fs or FOCs. Unstimulated MDMs obtained from FOCs showed higher release of TNFα in comparison with Fs. After lipopolysaccharide stimulation, the release of TNFα was significantly higher in Fs than in FOCs.

Conclusions: OC use induced many changes in hematological and plasmatic markers, modifying hormonal levels, endothelial function, inflammation index and some redox state parameters, producing a perturbation of the internal milieu that impacted macrophagic function. In fact, different levels of estrogen receptor expression and release of TNFα were observed in macrophages derived from OC users. Some of the above activities were linked to the androgenic properties of progestin. Even though it is not known whether these effects are reversible, the results indicate that to avoid potential skewing of results only a single type of OC should be used during a single clinical trial.

No MeSH data available.


Related in: MedlinePlus