Characteristics of Participants
A total of 347 subjects were collected in this study, including 116 males (33.4%) and 231 females (66.6%).Age distribution ranged from 16 to 19 years old.69.7% of the subjects lived in urban and 30.3% lived in rural areas. Those whose household per capita income was less than 3,000 Yuan/month and those whose household per capita income was more than 3,000 Yuan/month accounted for 51.3% and 48.7%, respectively. The subjects' BMI was (21.54±3.67) kg/m2.As shown in table 1.
Table. 1 Demographic and socioeconomic characteristics of participants (N=347)
Variable
|
Categories
|
n (%)/±s
|
Gender
|
Males
|
116 (33.4)
|
|
Females
|
231 (66.6)
|
Age (years)
|
16~17
|
85 (24.5)
|
|
18~19
|
262 (75.5)
|
Household location
|
Urban
|
242 (69.7)
|
|
Rural
|
105 (30.3)
|
Income(Yuan/month)
|
<3000
|
178 (51.3)
|
|
≥3000
|
169 (48.7)
|
BMI(kg/m2)
|
---
|
21.54±3.67
|
The distributions and exposure levels of DEHP in Participants
The DEHP exposure level and distributions of the study subjects are shown in Table 2.In this study, the limits of the detection of urinary metabolites MEHP, MEOHP, MEHHP, MEECPP and MCMHP were 0.50, 0.50, 0.50, 0.50 and 0.10μg/L, respectively. Except for MEOHP (99.2%) and MEHHP (98.7%), the detection rates of all other DEHP metabolites were 100%.Among DEHP metabolites, the GM of MECPP was 9.43μg/g Crea, and the geometric mean of other metabolites was MCMHP (5.79μg/g CREA), MEOHP (4.55μg/g CREA), MEHHP (3.37μg/g CREA) and MEHP (2.43μg/g CREA) from high to low, and the geometric mean of ∑DEHP was 94.72nmol/g.
There were significant differences in the levels of DEHP metabolites among subjects of different ages and Household location (P<0.05).The MEOHP level in urine of 18-19 year olds was significantly higher than that of 16-17 year olds. The levels of MEOHP, MEHHP, MCMHP and ∑DEHP in urine of subjects living in urban areas were significantly lower than those from rural areas (P<0.05).There was no significant difference in urinary DEHP metabolites among subjects of different genders and families with per capita monthly income (P>0.05), but the levels of DEHP metabolites in females were slightly higher than those in males except for MCMHP. Correlation analysis results among DEHP metabolites were shown in Table S1, and DEHP metabolites showed significant positive correlation (P<0.05).
The distributions and levels of Thyroid hormone in Participants
The blood thyroid hormone levels and distributions of the subjects were shown in Table 3.The serum levels of TT4 and FT3 were (124.05±24.56) nmol/L and (4.57±1.69) pmol/L, respectively. The geometric mean of TT3 was 1.89 nmol/L, that of FT4 was 10.01 pmol/L, and that of TSH was 2.56μUI/mL.
There were significant differences in the blood TT4 levels among the subjects of different ages (P<0.05), and there was no significant difference in serum thyroid hormone levels among different genders, family location and family per capita monthly income (P<0.05).
Table. 2 Distributions of the DEHP exposure levels among participants [GM (95%CI)]
Variable
|
MEHP
(μg/g Crea)
|
MEOHP
(μg/g Crea)
|
MEHHP
(μg/g Crea)
|
MECPP
(μg/g Crea)
|
MCMHP
(μg/g Crea)
|
∑DEHP
(nmol/L)
|
Total
|
2.43(2.23,2.65)
|
4.55(4.15,4.98)
|
3.37(3.06,3.69)
|
9.43(8.70,10.21)
|
5.79(5.30,6.36)
|
94.72(88.50,101.54)
|
Gender
|
|
Male
|
2.40(2.06,2.81)
|
4.29(3.70,5.01)
|
3.25(2.73,3.89)
|
9.02(7.88,10.32)
|
5.79(5.00,6.73)
|
90.46(79.92,103.03)
|
Female
|
2.43(2.20,2.72)
|
4.68(4.17,5.26)
|
3.43(3.03,3.88)
|
9.65(8.71,10.69)
|
5.79(5.17,6.50)
|
96.93(88.70,105.92)
|
Age(years)
|
|
16~17
|
2.14(1.75,2.67)
|
3.81(3.06,4.69)
|
2.96(2.34,3.76)
|
7.71(6.48,9.51)
|
5.37(4.47,6.55)
|
84.49(72.28,100.00)
|
18~19
|
2.52(2.29,2.80)
|
4.82(4.37,5.31)*
|
3.51(3.15,3.91)
|
10.07(9.21,11.04)*
|
5.94(5.38,6.56)
|
98.30(90.65,106.63)
|
Household location
|
|
Urban
|
2.40(2.16,2.68)
|
4.26(3.74,4.76)
|
3.12(2.72,3.53)
|
8.89(8.04,9.87)
|
5.54(4.97,6.21)
|
89.96(82.07,98.34)
|
Rural
|
2.46(2.10,2.92)
|
5.30(4.54,6.12)*
|
4.02(3.49,4.67)*
|
9.43(8.69,10.25)*
|
6.43(5.49,7.56)
|
106.67(93.95,121.35)*
|
Per capita income
(Yuan / month)
|
|
<3000
|
2.41(2.14,2.72)
|
4.83(4.27,5.50)
|
3.37(2.91,3.88)
|
9.86(8.85,10.98)
|
6.24(5.59,7.02)
|
98.28(88.89,108.34)
|
≥3000
|
2.44(2.13,2.78)
|
4.27(3.73,4.90)
|
3.37(2.88,3.94)
|
9.00(7.88,10.32)
|
5.36(4.71,6.18)
|
91.11(81.18,102.59)
|
*P<0.05
Table.3 Distributions of serum THs levels among participants [GM (95%CI)/]
Variable
|
TT3(nmol/L)
|
TT4(nmol/L)
|
TSH(μUI/mL)
|
FT3(pmol/L)
|
FT4(pmol/L)
|
Total
|
1.89(1.82,1.96)
|
124.05±24.56
|
2.56(2.41,2.73)
|
4.57±1.69
|
10.01(9.69,10.35)
|
Gender
|
|
|
Male
|
1.94(1.83,2.07)
|
123.73±24.40
|
2.42(2.19,2.67)
|
4.63±1.71
|
10.08(2.43,2.85)
|
Female
|
1.87(1.78,1.96)
|
124.21±24.69
|
2.63(2.43,2.85)
|
4.53±1.68
|
9.97(9.59,10.40)
|
Age(years)
|
|
|
16~17
|
1.84(1.71,1.98)
|
129.07±22.79
|
2.48(2.20,2.88)
|
4.40±1.60
|
10.12(9.48,10.74)
|
18~19
|
1.91(1.84,2.00)
|
122.43±24.93*
|
2.58(2.41,2.78)
|
4.62±1.72
|
9.97(9.56,10.34)
|
Household location
|
|
|
Urban
|
1.91(1.83,2.00)
|
125.07±24.76
|
2.60(2.43,2.83)
|
4.48±1.73
|
9.99(9.60,10.35)
|
Rural
|
1.85(1.76,1.96)
|
121.72±24.04
|
2.47(2.24,2.72)
|
4.76±1.59
|
10.04(9.32,10.80)
|
Per capita income
(Yuan / month)
|
|
|
<3000
|
1.89(1.80,1.98)
|
123.41±22.93
|
2.42(2.23,2.66)
|
4.56±1.62
|
9.98(9.50,10.51)
|
≥3000
|
1.90(1.79,2.01)
|
124.73±26.21
|
2.71(2.51,2.96)
|
4.57±1.77
|
10.03(9.59,10.48)
|
*P<0.05
Associations between Thyroid Hormones and Urinary DEHP Metabolites
Since metabolites of DEHP exhibit estrogen-like activity after entering the body, subsequent analyses of this study were further stratified according to gender
The results of multiple linear regression analysis of urinary DEHP metabolite levels and THs levels of all subjects after adjusting covariates are shown in Table 4. The levels of TT4 had a significant negative linear correlation with urine MEOHP [β =-3.599 (95% CI =-7.002, -0.625)]、MEHHP [β =-3.534 (95% CI =-6.774, -0.633)]、MECPP [β =-4.375 (95% CI =-7.697,-0.756)]、MCMHP [β =-4.397 (95% CI =-7.522,-1.174)] and ∑DEHP [β =-5.223 (95% CI =-9.880,-1.085)]=-9.880,-1.085)].
The levels of DEHP metabolites and THs of the subjects were divided into quartile groups, and the relationship between them was analyzed by ordered multi-classification logistic regression after the adjustment of covariables.The results showed that the levels of TT3 and TT4 in ∑DEHP Q4 group were at least one grade lower than those in Q1 group (P<0.05).The levels of TT4 in Q4 groups of MEOHP, MEHHP, MECPP and MCMHP were all decreased by at least one grade compared with those in Q1 group (P<0.05).The FT3 levels in MECPP Q3 group was significantly lower than that in Q1(P<0.05). The results were shown in the figure 1.
As shown in Table 5, there was a significant negative correlation between MEOHP and TT4 in male subjects (P < 0.05),and the levels of FT4 were significant positive correlated with MEHP[β =0.089 (95% CI =-0.021,0.157)]、MECPP[β =0.106 (95% CI =0.021,0.190)]、MCMHP[β =0.106 (95% CI =(0.029,0.183)] and ∑DEHP[β =0.121 (95% CI =0.032,0.209)].
The results of ordered multi-classification logistic regression analysis in male subjects showed in figure 2. The TT4 level of MEOHP Q4 group in male subjects was significantly lower than that of Q1 group by at least one grade. The levels of FT3 in MEHP Q3 group was significantly higher than that in Q1 (P < 0.05).Compared with Q1 group, the level of FT4 in MEHP, MECPP and ∑DEHP Q4 group was higher. The levels of FT4 in MCMHP Q3 and Q4 groups were increased by at least one grade compared with that in Q1 (P < 0.05).
Table 4. Associations of serum thyroid hormones with urinary DEHP metabolites in total participants [β (95%CI), N=347].
Total a
|
Ln TT3
|
TT4
|
Ln TSH
|
FT3
|
Ln FT4
|
Ln MEHP
|
-0.013(-0.061,0.043)
|
-1.279(-4.620,2.146)
|
-0.045(-0.120,0.023)
|
-0.042(-0.266,0.147)
|
0.017(-0.023,0.058)
|
Ln MEOHP
|
-0.007(-0.066,0.059)
|
-3.599(-7.002,-0.625) *
|
-0.039(-0.113,0.036)
|
0.051(-0.158,0.256)
|
0.006(-0.032,0.046)
|
Ln MEHHP
|
0.002(-0.045,0.050)
|
-3.534(-6.774,-0.633) *
|
-0.035(-0.109,0.038)
|
0.039(-0.157,0.227)
|
0.010(-0.026,0.046)
|
Ln MECPP
|
-0.007(-0.058,0.051)
|
-4.375(-7.697,-0.756) *
|
-0.020(-0.100,0.062)
|
-0.115(-0.349,0.116)
|
0.013(-0.031,0.057)
|
Ln MCMHP
|
-0.001(-0.045,0.048)
|
-4.397(-7.522,-1.174) **
|
-0.017(-0.084,0.051)
|
-0.109(-0.314,0.106)
|
0.028(-0.012,0.067)
|
Ln ∑DEHP
|
-0.015(-0.076,0.048)
|
-5.223(-9.880,-1.085) **
|
-0.054(-0.135,0.038)
|
-0.074(-0.337,0.185)
|
0.025(-0.025,0.074)
|
a Adjusted for age, gender, domicile, per capita income, and BMI,*P<0.05.**P<0.01.
Table 5. Associations of serum thyroid hormones with urinary DEHP metabolites in male participants [β (95%CI), N=116].
Male
|
Ln TT3
|
TT4
|
Ln TSH
|
FT3
|
Ln FT4
|
Ln MEHP
|
-0.018(-0.553,0582)
|
-1.228(-7.859,5.932)
|
0.009(-0.113,0.130)
|
-0.017(-0.374,0.340)
|
0.089(0.021,0.157) *
|
Ln MEOHP
|
-0.050(-0.113,0.005)
|
-5.994(-10.484,-1.855) *
|
-0.019(-0.183,0.164)
|
0.114(-0.258,0.486)
|
0.057(-0.015,0.129)
|
Ln MEHHP
|
-0.010(-0.311,0.756)
|
-3.671(-7.929,1.051)
|
0.054(-0.090,0.194)
|
0.188(-0.148,0.525)
|
0.048(-0.018,0.114)
|
Ln MECPP
|
-0.046(-0.118,0.033)
|
-4.622(-11.052,1.738)
|
0.100(-0.040,0.266)
|
-0.027(-0.470,0.416)
|
0.106(0.021,0.190) *
|
Ln MCMHP
|
-0.027(-0.092,0.045)
|
-1.561(-7.519,3.920)
|
0.034(-0.090,0.160)
|
-0.075(-0.480,0.330)
|
0.106(0.029,0.183) **
|
Ln ∑DEHP
|
-0.039(-0.123,0.043)
|
-4.815(-11.840,2.183)
|
0.076(-0.054,0.243)
|
0.043(-0.423,0.510)
|
0.121(0.032,0.209) *
|
a Adjusted for age, domicile, per capita income, and BMI,*P<0.05,**P<0.01.
The results of multiple linear regression analysis of urinary DEHP metabolite levels and THs levels of female subjects and the ordered multi-classification logistic regression after the adjustment of covariables were showed in Table 6 and figure3.In female subjects, the levels of TT4 was significantly negatively correlated with MECPP [β =-4.158 (95% CI =-8.722,0.211)]、MCMHP [β =-5.284 (95% CI =(-9.133,-1.613)] and ∑DEHP [β =-5.235 (95% CI =-11.270,-0.034)] .The levels of TSH was significantly negatively correlated with ∑DEHP (P<0.05).
The TT3 level of female subjects in ∑DEHP Q4 group was at least one grade lower than that in Q1 group (P<0.05), and the TT3 level of female subjects in MEHHP Q3 group was also significantly lower than that in Q1 group (P<0.05). The levels of TT4 in MEOHP, MEHHP, McMHP and ∑DEHP Q4 groups of female subjects were significantly lower than those in Q1 group (P<0.05), and TT4 levels in MEHHP and ∑DEHP Q3 groups were also significantly lower than those in Q1 group (P<0.05).The TSH level of female subjects in MECPP and ∑DEHP Q4 group was significantly lower than that in Q1 group (P<0.05), and the level of TSH in MEHP Q3 group was significantly lower than that in Q1 group by at least one grade (P<0.05)
Table 6. Associations of serum thyroid hormones with urinary DEHP metabolites in female participants [β (95%CI), N=231].
Female
|
Ln TT3
|
TT4
|
Ln TSH
|
FT3
|
Ln FT4
|
Ln MEHP
|
-0.014(-0.074,0.048)
|
-1.232(-5.546,2.540)
|
-0.084(-0.174,0.008)
|
-0.085(-0.356,0.187)
|
-0.022(-0.071,0.027)
|
Ln MEOHP
|
0.014(-0.070,0.094)
|
-2.583(-7.444,1.337)
|
-0.044 (-0.131,0.038)
|
0.022(-0.230,0.274)
|
-0.017(-0.063,0.029)
|
Ln MEHHP
|
0.004(-0.062,0.079)
|
-3.345(-7.661,0.492)
|
-0.080(-0.159,-0.005)
|
-0.059(-0.295,0.178)
|
-0.004(-0.048,0.039)
|
Ln MECPP
|
-0.002(-0.071,0.064)
|
-4.158(-8.722,0.211) *
|
-0.086(-0.179,0.011)
|
-0.199(-0.478,0.080)
|
-0.019(-0.070,0.032)
|
Ln MCMHP
|
0.002(-0.052,0.063)
|
-5.284(-9.133,-1.613) **
|
-0.045(-0.122,0.035)
|
-0.149(-0.399,0.100)
|
0.006(-0.040,0.051)
|
Ln ∑DEHP
|
-0.011(-0.095,0.074)
|
-5.235(-11.270,-0.034) *
|
-0.123(-0.231,-0.018) *
|
-0.166(-0.485,0.154)
|
-0.014(-0.072,0.044)
|
a Adjusted for age, domicile, per capita income, and BMI.*P<0.05.**P<0.01
Correlation between thyroid hormone levels and mRNA expression of key genes in thyroid hormone metabolism.
The correlation analysis results of the subjects' blood thyroid hormone levels and mRNA expression of key genes of thyroid hormone metabolism are shown in Table 7.There was a negative correlation between FT3 level and TG mRNA expression level in blood of all subjects (P<0.05).The TT4 level of male was significantly correlated with the mRNA relative expression levels of TG, NIS and PAX-8 (P<0.05).In female subjects, there was a significant negative correlation between FT3 level and TG mRNA level, and a significant negative correlation between FT4 level and NIS mRNA level (Table.7, P<0.05).
Table. 7 The correlation of THs related genes mRNA levels and THs levels
Variable
|
TPO
|
TG
|
NIS
|
TTF-1
|
PAX-8
|
Total
|
|
|
|
|
|
TT3
|
0.086
|
0.057
|
0.068
|
0.019
|
0.077
|
TT4
|
-0.002
|
0.024
|
-0.035
|
-0.039
|
-0.055
|
TSH
|
-0.003
|
0.016
|
0.010
|
0.023
|
0.035
|
FT3
|
-0.036
|
-0.119*
|
0.044
|
-0.101
|
-0.012
|
FT4
|
-0.030
|
-0.031
|
-0.093
|
-0.019
|
-0.036
|
Male
|
|
|
|
|
|
TT3
|
0.005
|
-0.050
|
-0.015
|
-0.042
|
0.025
|
TT4
|
-0.175
|
-0.185*
|
-0.211*
|
-0.129
|
-0.252*
|
TSH
|
0.059
|
0.113
|
0.035
|
0.078
|
0.016
|
FT3
|
0.057
|
-0.097
|
0.074
|
-0.163
|
0.014
|
FT4
|
-0.045
|
-0.019
|
-0.014
|
0.05
|
0.031
|
Female
|
|
|
|
|
|
TT3
|
0.120
|
0.107
|
0.111
|
0.045
|
0.107
|
TT4
|
0.075
|
0.106
|
0.048
|
0.009
|
0.040
|
TSH
|
-0.076
|
-0.025
|
-0.001
|
-0.007
|
0.041
|
FT3
|
-0.020
|
-0.132*
|
0.030
|
-0.073
|
-0.036
|
FT4
|
-0.019
|
-0.019
|
-0.146*
|
-0.036
|
-0.081
|
*P<0.05
Thyroid hormones related genes mRNA expression at different levels of DEHP metabolites.
In this study, the mRNA expression levels of thyroid hormone related genes TPO, TG, NIS, TTF-1 and PAX-8 in blood samples were analyzed by ordered multi-classification logistic regression. In total subjects, the relative mRNA expression level of TG in MECPP and MCMHP Q4 groups were higher than that in Q1group, and the mRNA level of TTF-1 in MECPP, MCMHP, MCMHP and ∑DEHP Q4 groups were higher than that in the Q1 group (P < 0.05).The mRNA level of PAX-8 in the MEHHP Q4 group was at least one grade higher than that in the Q1 group (Figure 4, P < 0.05).
In male subjects, the relative mRNA level of TG in ∑DEHP Q4 group was at least one grade higher than that in Q1 group (P < 0.05).The mRNA level of TTF-1 in ∑DEHP Q2 and Q4 groups was at least one grade higher than that in Q1 group (P < 0.05).The mRNA level of PAX-8 in MEHHP and ∑DEHP Q4 group was higher than that in Q1 group (P < 0.05).The results were showed in Figure 5.
As shown in figure 6, the mRNA level of NIS of female subjects in MECPP and MCMHP Q4 groups were significantly higher than that in Q1 group (P < 0.05).