J Bone Metab.  2019 Aug;26(3):169-177. 10.11005/jbm.2019.26.3.169.

Effects of Di(2-ethylhexyl)phthalate on Bone Metabolism in Ovariectomized Mice

• Choi JI
• Cho HH

• KMID: 2457436
• DOI: http://doi.org/10.11005/jbm.2019.26.3.169

Abstract

BACKGROUND
The molecular pathways of how endocrine disruptors affect bone mineral density (BMD) and bone remodeling are still unclear. The purpose of this experimental study is to determine the effects of di(2-ethylhexyl)phthalate (DEHP) on bone metabolism in ovariectomized mice.
METHODS
Twenty-six-month-old female CD-1 mice were divided into 4 groups: control, low-dose DEHP, high-dose DEHP, and estrogen groups (n=5, each group). All mice were subjected to ovariectomy for the induction of artificial menopause and then exposed to corn oil, DEHP, and estrogen for 2 months. Micro-computed tomography (Micro-CT) of the bone and analysis of blood samples for bone markers were performed to observe the changes in bone metabolism.
RESULTS
Osteocalcin level was decreased in the control, low-dose and high-dose DEHP group, the reduction width was greater in the high-dose DEHP group (−0.219 ng/mL) than control group (−0.077 ng/mL, P<0.05). C-terminal telopeptide of type I collagen level was increased in the control, low-dose and high-dose DEHP group, the increase range of low-dose DEHP group (0.329 ng/mL) showed greater than control group (0.093 ng/mL, P<0.05). Micro-CT analysis revealed that the BMD was significantly lower in the high-dose DEHP group (19.8×10⁻² g/cm³) than control group (27.2×10⁻² g/cm³, P<0.05). The structure model index was significantly higher in the high-dose DEHP group (2.737) than low-dose DEHP group (2.648) and estrogen group (2.63, P<0.05). It means the progression of osteoporosis in the high-dose DEHP group.
CONCLUSIONS
These results confirm the negative effects of DEHP on bone health in ovariectomized mice. Further continuous studies on genetic pathways and other endocrine disruptors will be necessary to validate these findings.

Keyword

Diethylhexyl phthalate; Osteoporosis; Ovariectomy; X-ray microtomography

MeSH Terms

Animals
Bone Density
Bone Remodeling
Collagen Type I
Corn Oil
Diethylhexyl Phthalate
Endocrine Disruptors
Estrogens
Female
Humans
Menopause
Metabolism*
Mice*
Osteocalcin
Osteoporosis
Ovariectomy
X-Ray Microtomography
Collagen Type I
Corn Oil
Diethylhexyl Phthalate
Endocrine Disruptors
Estrogens
Osteocalcin

Figure

• 

  Fig. 1 Comparison of serum bone formation marker (osteocalcin) concentrations at month 1 and 2 after di(2-ethylhexyl)phthalate (DEHP) treatment in ovariectomized mice. A gradual increase was shown in the estrogen treatment group (E), but a decrease was shown in the control group (C), low-dose DEHP group (L), and high-dose DEHP group (H). The calculated subtraction value is statistically greater in the H (*) than the C. *P<0.05 vs. C.

• 

  Fig. 2 Change in serum bone resorption marker (C-terminal telopeptide of type 1 collagen [CTX-1]) concentrations at month 1 and 2 after di(2-ethylhexyl)phthalate (DEHP) treatment. CTX-1 level showed a decrease in the estrogen treatment group (E), while an increase the other groups. The calculated subtraction value of CTX-1 is statistically greater in the low-dose DEHP group (L) (*) than the control group (C). H, high-dose di(2-ethylhexyl)phthalate treatment group. *P<0.05 vs. C.

• 

  Fig. 3 Change in serum calcium (Ca), phosphorus (P), alkaline phosphatase (ALK-P), and magnesium (Mg). There was no significant difference in the serum Ca levels and Mg levels among the three groups. Although, there was a significant decrease in the serum P level in the high-dose di(2-ethylhexyl)phthalate (DEHP) group, and the serum ALK-P levels were decreased to a greater extent in the low-dose DEHP groups (L) and high-dose DEHP groups (H) than in the control group (C). *P<0.05 vs. C.

• 

  Fig. 4 Comparison of bone marrow density (BMD) in the tibia of ovariectomized mice. The BMD was significantly lower in the high-dose di(2-ethylhexyl)phthalate (DEHP) group (H) (*) and significantly higher in the estrogen-treated group (E) than control group (C). L, low-dose d di(2-ethylhexyl)phthalate treatment group. *P<0.05 vs. C.

• 

  Fig. 5 Tibia head microstructure analysis using micro-computed tomography scan. Trabecular area (yellow) is more prominent in estrogen treatment group (E). In high-dose di(2-ethylhexyl)phthalate (DEHP) group (H), trabecular area is smaller than the others (E, control group [C], low-dose DEHP treatment group [L]).

• 

  Fig. 6 Structure model index (SMI) analysis in the tibia of ovariectomized mice. SMI was significantly higher in the high-dose di(2-ethylhexyl) phthalate (DEHP) group (H) (*) than in the estrogen treatment (E) and low-dose DEHP groups (L). No significant differences were observed in the H and control group (C). *P<0.05 vs. C.

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