In this study, we recruited 39 men (aged 27–43 years, referred by a physician) from Project ELEFANT (26, 31–35). At recruitment, all participants excluded autoimmune diseases, cardiovascular diseases, severe liver diseases, thyroid diseases, and blood diseases, and completed a physical examination under the guidance of nurses, signed an informed consent form, and filled in basic information (including age and BMI) listed in Table 1. This project complies with the Human Medical Ethic Review Law of the Ministry of Health and the Declaration of Helsinki. All procedures and study protocols were authorized by the ethics committee of Tianjin Medical University.

In this study, we monitored 9 PAEs and 10 mPAEs, including di-methyl phthalate (DMP), di-ethyl phthalate (DEP), di-iso-butyl phthalate (DiBP), di-butyl phthalate (DBP), 1,2-benzenedicarboxylicacid (BMPP), di-pentyl phthalate (DPP), diethylhexyl phthalate (DEHP), di-n-octyl phthalate (DNOP), di-phenyl phthalate (DPHP), mono-methyl phthalate (MMP), mono-ethyl phthalate (MEP), mono-isobutyl phthalate (MiBP), mono-n-butyl phthalate (MBP), mono-ethyl phthalate (MEHP), mono-benzyl phthalate (MBzP), mono-octyl phthalate (MOP), mono-(2-ethyl-5-hydroxy-hexyl) phthalate (MEHHP), mono-(2-ethyl-5-oxo-hexyl) phthalate (MEOHP), and mono-(2-ethyl-5-carboxy-pentyl) phthalate (MECPP). To avoid the transfer of PAEs from PVC into urine samples, all materials were kept away from plastic vessels. Urine samples were collected, stored, and analyzed using glass tubes. Urine samples were gathered and stored at −20°C until analysis. To determine PAEs, 10 μl of internal standard (D4-DEHP, D4-DOP, and D4-DBP mixed solution, 10 μg/ml, Sigma) and 0.5 ml n-hexane was added to 0.5 ml of urine sample, and the sample mix solution was vortexed for 10 min. The sample mixture solution was centrifuged at 9,000 r/min for 10 min. The supernatant was collected and filtered through a 0.22-μm glass fiber. Finally, each sample was measured using gas chromatography-mass spectrometry (GC-MS, Thermo Fisher Scientific, Waltham, MA, USA) and calibrated and evaluated using the internal standard method. To determine mPAEs, 1.0 ml of urine sample was mixed with 0.25 ml ammonium acetate buffer solution (pH = 6.5, Sigma) and 10 μl β-glucuronidase standard solution (85,000 U/ml, Sigma), and placed in a 37°C water bath for 2 h. Then, the sample mix solution was filtered by a 0.22-μm glass fiber filter for further analysis.

After a medical examination, fasting blood samples were collected from 39 individuals by venipuncture using vacuum anticoagulant tubes with 2 ml EDTA, followed by centrifugation at 3,000 rpm for 10 min to separate plasma. Then 39 samples were stored at −80°C until mtDNA methylation analysis for platelets.

First, 300 μl of plasma was removed from each sample, and platelet mitochondria were extracted using a DNA methylation kit (Zymo Research, Orange, CA, USA) and treated with bisulfite. A 24-μl system was prepared from the bisulfite-treated DNA, and the DNA was amplified by polymerase chain reaction (PCR). Then the DNA was amplified 45 times under the conditions of 5 min at 95°C, 15 s at 95°C, 30 s at 52°C, 15 s at 72°C, and 5 min at 72°C. The PCR amplifications were conducted in a PCR amplifier (BIO-RAD, USA). The treated samples were run on 0.8% agarose gel and visualized under UV light. Finally, the rate of mtDNA methylation was tested by the PyroMark Q48 autoprep pyrophosphate sequencer (QIAGEN, Hilden, Germany) (26).

Phthalates were determined by Agilent 8890-5977B GC-MS. The gas chromatography (GC) conditions were as follows: column, DB-5MS UI (30 m × 0.25 mm × 0.25 μm); carrier gas, helium. The injection volume was 1 mul splitless, with an injection port temperature of 250°C. The carrier gas was helium, with a flow rate of 1.0 ml/min (constant flow mode). The temperature program was as follows: the initial temperature was 80°C for 60 s, followed by 300°C (10°C/min) for 120 s, and then 340°C (5°C/min) for 60 s. MS conditions were as follows: scan mode: SIM, electron energy: 70 eV; interface temperature: 280°C; ion source temperature: 230°C.

High-performance liquid chromatography-mass spectrometry (HPLC-MS; Agilent 1290, USA) was used to detect mPAEs. HPLC Nexera LC-30A high-performance liquid chromatograph (Shimadzu). The parameters of HPLC were: chromatographic column: Kinetex XB-C18-100A LC column (100 × 3 mm, 2.6 μm; Phenomenex, USA); flow rate: 0.3 ml/min; injection volume: 10 μl; column temperature: 40°C. Mobile phase A contained an aqueous solution with 0.1% formic acid, and mobile phase B contained methanol (HPLC grade; Fisher Scientific, Houston, TX, USA). The mobile phase gradient program is listed in Supplementary Table 1.

mtDNA methylation was determined using a pyrosequencer (PyroMark Q48 Autoprep, QIAGEN, Hilden, Germany). The process was as follows: (1) 200 μl of enzyme-free water was added to the instrument's reagent clip for cleaning before sequencing each time, and the cleaning step was repeated once; (2) dNTP, denaturation buffer, enzyme mixture, mixed substrate, and annealing buffer were added to the instrument; (3) After the instrument was detected, 3 μl of magnetic beads (PyroMark Q48 Magnetic Beads) and 10 μl of the sample to be tested were added. The 48-well sample tray was added to the instrument, and then it was put on the instrument for mtDNA methylation sequencing; (4) The reagent clips were cleaned again after the test. mtDNA sequences used for targeted bisulfite sequencing are listed in Supplementary Table 2.

Blank analysis was performed on each batch of samples. The recoveries of all the target analytes in the matrix-spiked samples were 85–112% (PAEs) and 89–115% (mPAEs). The relative standard deviations were below 10%. No obvious ionization restraint or enhancement was discovered, and the matrix effects of the target analyte were on the scale of 86–110% (PAEs) and 85–106% (mPAEs). The limit of quantification (LOQ) and limit of detection (LOD) were defined as 10 and three times the signal-to-noise ratio (S/N), respectively. The LOQ and LOD were estimated at 0.02–1.27 ng/ml and 0.006–0.4 ng/ml for PAEs, and 0.01–6.54 ng/ml and 0.003–2.1 ng/ml for mPAEs. Concentrations below the LOQ were specified as zero values for statistical analysis.

Strict QA/QC procedures were also carried out during the analysis of mtDNA methylation. Every equipment was sterilized, non-pyrogenic, and DNAse/RNAse-free during the analysis of mtDNA methylation, and then two quality controls were included. In each 96-well plate, two blank samples were set as negative controls in PCR. Two samples with known global DNA methylation levels, namely, low (1%) and high (99%), were added as positive controls in pyrophosphate sequencing. Finally, two duplicate pyrosequencing analyses were performed for each DNA sequence (26).

This study used R 4.1.0 software from Origin 2018 and SPSS software for data analysis. In this study, the concentrations of analytes below LOD were set to zero. Considering that PAE exposure may affect mtDNA methylation levels and lipid levels, we analyzed the relationships between PAE exposure, mtDNA methylation, and lipids by adjusting a generalized linear model of several covariates (including age, BMI, and seniority). Different PAEs and mPAEs were included in the generalized linear model together with age, BMI, and seniority covariates as independent variables. Since the levels of triglycerides and some mtDNA did not correspond to the normal distribution, the natural logarithm was used for statistical analysis (Shapiro-test P < 0.05). In addition, generalized linear models were used to analyze the relationships between mtDNA methylation, in vivo pollutants, and lipid levels after adjusting for age, BMI, and seniority. The p-value of < 0.05 was considered to indicate statistical significance and the p-value of < 0.01 was considered to indicate high significance.

The EDI (μg/kg bw/day) of PAE exposure was evaluated by the mPAEs values in urinary samples using the following equation (1, 5, 36):

Where UC_m (ng/ml) is the concentration of urinary mPAE, UV (2.0 L/day) is the average adult volume of urine every day (37). Bw (kg) is the body weight of the participant. UC_p (ng/ml) is the urinary PAE concentration. The EDI of DEHP is the sum of the calculated DEHP concentrations based on DEHP, MEHP, MEHHP, MECPP, and MEOHP (11). MW_p is the molecular weight of PAE; MW_m (g/mol) is the molecular weight of mPAE; and F_ue is the molar fraction of mPAE. The values of MW_p, MW_m, and F_ue are listed in Supplementary Table 3.

The hazard quotient (HQ) for exposure to PAEs was computed using equation (38):

EDI (μg/kg bw/day) is the estimated total daily intake of PAE; TDI (μg/kg bw/day) is recommended by the European Food Safety Authority (EFSA); the TDI values of DiBP and DEHP were 10 and 50 μg/kg bw/day, respectively. RfD (μg/kg bw/day) is recommended by the Environmental Protection Agency (EPA), the RfD values for DEP, DiBP, and DEHP were 800, 20, and 100 μg/kg bw/day. An HQ < 1 is considered safe, whereas an HQ > 1 indicates that the chemical may have potential adverse health effects on humans.

HI was estimated by summing the different values of HQ as follows:

Out of 16 PAEs, nine were detected in urine samples in this study, including DMP, DEP, DiBP, DBP, BMPP, DPP, DEHP, DPHP, and DNOP. The detection rates ranged from 2.56 to 92.31%. The highest median concentration was DIBP (2.43 ng/ml), followed by DBP (2.40 ng/ml), DPP (0.55 ng/ml), DMP (0.51 ng/ml), and DPHP (0.06 ng/ml) as shown in Table 2.

In the samples, 10 mPAEs, including MMP, MEP, MiBP, MBP, MBZP, and MOP, and four metabolites of DEHP (including MEHP, MEHHP, MECPP, and MEOHP) were detected. MiBP, MECPP, MEHHP, MEP, MEHP, MEOHP, MBP, and MOP were found in 100% of the samples, whereas MMP and MBZP were less frequently detected (30.77 and 82.05%, respectively). The highest median concentration was MBP (28.87 ng/ml), followed by MiBP (28.63 ng/ml), MECPP (7.23 ng/ml), MEP (4.74 ng/ml), MEHHP (4.00 ng/ml), MEOHP (2.11 ng/ml), MEHP (1.63 ng/ml), MOP (0.12 ng/ml), and MBZP (0.06 ng/ml). Figures 1A, B show that higher concentrations were found for MiBP and MBP, which correspond to the concentrations of their parent diesters (DIBP and DBP). The average contributions of nine PAEs (Σ9 PAEs) and 10 mPAEs (Σ10 mPAEs) were 12.3 and 87.7%, respectively. The results showed a significant positive correlation between the urinary metabolites of DEHP (MEHP, MEHHP, MEOHP, and MECPP) (p < 0.05), respectively (Figure 2).

Using a generalized linear model, we tested associations between PAEs and mPAEs with lipid levels (including TC, triglycerides, HDL-C, and LDL-C). PAEs and lipid levels did not have a significant relationship (Supplementary Table 4). Both MEP and MBZP showed a positive influence on triglycerides (β = 0.90, p < 0.05; β f= 0.85, p < 0.001, respectively) (Table 3). Because a high level of triglycerides is one of the causes of CVDs (18), MEP and MBZP may increase the risk of CVDs.

For methylation analysis, six genes, including MT-COX1, MT-COX2, MT-COX3, MT-ATP6, MT-ATP8, and MT-ND5, were selected. Genetic mutations in cyclooxygenase (COX) genes are significantly associated with fatal metabolic disorders (39). ATPase subunit 6 (ATP6) and ATPase subunit 8 (ATP8) firsthand affect ATP synthesis and energy metabolism (40). NADH dehydrogenase subunit 5 (ND5) is the vital gene encoding NADH dehydrogenase. Hence, these genes mainly affect organs with high energy demands (41), such as the heart.

In 39 plasma samples, the mean methylation level [interquartile range (IQR) 25th−75th percentile] in the COX1 gene was 14.02 (12.23–16.17)%, 14.73 (11.12–17.35)% for COX2, 2.34 (2.12–2.62)% for COX3, 15.59 (12.55–20.45)% for ATP6, 1.43 (1.25–1.59)% for ATP8, and 2.84 (2.21–2.89)% for ND5 (Supplementary Table 5; Figure 3). The average methylation rates of MT-COX2 and MT-ATP8 were positively correlated with triglycerides (p < 0.05, respectively), indicating that the mtDNA methylation of specific genes could induce CVDs, which needs to be further explored (Table 4).

Relationships of PAEs and mPAEs concentrations with mtDNA methylation levels were analyzed by generalized linear models. A 1-ng/ml increment in DEP concentration was associated with a 2.44 and 0.50% decrease in MT-COX1 and MT-COX3 methylation rates (p < 0.05, p = 0.001, respectively) among all participants (Table 5). Each 1 ng/ml increase in DBP and DiBP concentrations was associated with a 1.28 and 1.78% decrease in the MT-ATP6 methylation rates (p < 0.05, respectively). The MT-ND5 methylation rate was positively associated with BMPP (β = 0.41, p < 0.05), DPP (β = 0.66, p < 0.05), MiBP (β = 0.06, p < 0.05), and MBP (β = 0.06, p < 0.05). These results suggested that the mtDNA methylation level was susceptible to changes in response to PAE exposure.

The triglyceride levels were significantly associated with MEP concentration and MT-ATP8 methylation rate (p < 0.05, β = 0.90; p = 0.02, β = 0.25, respectively). To clarify the role of MEPs in the epigenetic regulation of the mitochondrial genome, we conducted a mediation analysis to examine the MT-ATP8 methylation rate with MEP and triglyceride levels (Table 6). However, we did not find the mediated effect of the above exposure levels on triglycerides through the mtDNA methylation pathway (p > 0.05).

The EDIs, HQs, and HIs were calculated based on the levels of PAEs (DEP, DiBP, and DEHP). The median EDIs of DEP, DiBP, and DEHP were 0.438, 3.676, and 4.374 μg/kg bw/day, with details listed in Table 7. HQ showed a risk for a single PAE, while HI indicated a cumulative risk for several PAEs (calculated by the sum of DEP, DiBP, and DEHP). HQ<1 and HI<1 are considered significant daily intake doses and safe adverse effects caused by PAEs. In this study, 10% of participants showed HI_RfD>1. Additionally, it should be noted that 12 of the 39 men in this study showed HI_TDI>1, indicating that ~31% of the population has potential health risks.