Renal function and the exposure to melamine and phthalates in Shanghai adults

Journal Pre-proof Renal function and the exposure to melamine and phthalates in Shanghai adults JingSi Chen, XinLi Shi, XiaoFeng Zhou, RuiHua Dong, YaQun Yuan, Min Wu, WeiHua Chen, XiaoHong Liu, FuHuai Jia, ShuGuang Li, QiFan Yang, Bo Chen PII:

S0045-6535(20)30011-4

DOI:

https://doi.org/10.1016/j.chemosphere.2020.125820

Reference:

CHEM 125820

To appear in:

ECSN

Received Date: 20 August 2019 Revised Date:

19 December 2019

Accepted Date: 2 January 2020

Please cite this article as: Chen, J., Shi, X., Zhou, X., Dong, R., Yuan, Y., Wu, M., Chen, W., Liu, X., Jia, F., Li, S., Yang, Q., Chen, B., Renal function and the exposure to melamine and phthalates in Shanghai adults, Chemosphere (2020), doi: https://doi.org/10.1016/j.chemosphere.2020.125820. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Author’s Contribution Jingsi Chen

Data curation, Formal analysis, Investigation, Writing - original draft

Xinli Shi Data curation, Formal analysis, Investigation, Writing - original draft Xiaofeng Zhou

Data curation, Investigation

Ruihua Dong Data curation, Investigation Yaqun Yuan

Data curation, Investigation

Min Wu Data curation, Investigation WeiHua Chen Data curation, Investigation XiaoHong Liu Data curation, Investigation FuHuai Jia Writing - review & editing ShuGuang Li

Conceptualization, Writing - review & editing

QiFan Yang Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Supervision, Writing - review & editing

Bo Chen

Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration,

Supervision, Writing - review & editing

1

Renal Function and the Exposure to Melamine and Phthalates in Shanghai Adults.

2

JingSi Chena,1, XinLi Shia,1, XiaoFeng Zhoua, RuiHua Donga, YaQun Yuana, Min Wua,

3

WeiHua Chenb, XiaoHong Liub, FuHuai Jiac, ShuGuang Lia, QiFan Yangd,*, Bo Chena,*

4

5

a

6

Center of Social Risks Governance in Health, School of Public Health, Fudan University,

7

Shanghai, 200032, China

8

b

9

c

10

d

11

China

Key Laboratory of Public Health Safety of Ministry of Education, Collaborative Innovation

Community health service center of Nanjing (E) road, Shanghai, 200003, China

Ningbo Yu Fang Tang Biological Science and Technology Co., Ltd., Ningbo, 315012, China. Shanghai Jingan District Center for Disease Control and Prevention, Shanghai, 200072,

12

13

1

14

*

15

86-21-54237146, or to QiFan Yang, E-mail: [email protected], Tel/Fax: 86-21-56659090.

JingSi Chen and XinLi Shi had equal contribution. Correspondence should be addressed to Bo Chen, E-mail: [email protected], Tel/Fax:

1

16

Abstract

17

[Background] Melamine and phthalates have been reported to damage renal function in

18

children. This association is scarce in general adults.

19

[Method] A cross-sectional subsample population of 611 adults participating in the 2012

20

Shanghai Food Consumption Survey (SHFCS) was analyzed for urinary biomarkers of

21

melamine, metabolites of phthalates, and renal function parameters. The correlations between

22

renal function parameters and chemical exposure (either independently or interactively) were

23

explored by linear regression models. To simplify the analysis, phthalate metabolites were

24

dimensionally reduced using principal component analysis (PCA) method.

25

[Result] Urinary melamine was positively associated with renal function parameters of both

26

albumin-to-creatinine ratio (ACR) and β2-microglobulin (B2M) in multivariate linear

27

regression models (P < 0.05). A PCA pattern characterized by high-molecular-weight

28

phthalates (HMWP) was positively associated with all three parameters of renal function

29

(ACR, B2M, and N-acetyl-β-d-glucosaminidase (NAG)). The co-exposure to melamine and

30

HMWP presented an additive effect on increasing these parameters (ACR, B2M, and NAG).

31

[Conclusion] Impaired renal function in Shanghai adults was associated with exposure to

32

both melamine and HMWP.

33

Key

34

N-acetyl-β-d-glucosaminidase; adults.

word:

Melamine;

metabolites

of

35

2

phthalates;

albumin;

β2-microglobulin;

36

1. Introduction

37

In the past few decades, kidney disease diagnosed with objective measurements of structural

38

damage and dysfunction has been recognized as a significant public health problem around

39

the world [1]. The worldwide prevalence of chronic kidney disease (CKD) has exceeded 10%

40

from 2006 to 2016 [2]. An increasing amount of evidence indicates that kidney injury could

41

induce many systemic complications and increase all-cause mortality [3].

42

Kidney disease has been reported to be affected by several factors including age, sex, ethnic,

43

comorbidity (diabetes and hypertension), genetic susceptibility, et al. [1, 4]. In addition, the

44

exposure to environmental chemicals, such as melamine and phthalates, has also been

45

suggested to play an important role [5–10].

46

Melamine, an organic base synthesized from urea, is widely used as an industrial chemical to

47

produce melamine resin, melamine foam, countertops, tableware, and other commercial

48

products [11]. Due to the wide use of melamine-containing products, melamine can be

49

detected ubiquitously in human urine, even after the 2008 melamine baby formula scandal

50

from China [12]. Melamine has been mainly reported to have renal toxicity [11]. In the events

51

of both 2007 pet food recall and 2008 baby formula scandal, high exposure to adulterated

52

melamine was found to result in urolithiasis [13]. Furthermore, environmental exposure to

53

melamine was also found to be possible reason behind impaired renal function, although such

54

reports were scarce and limited to children, melamine tableware manufacturing workers, and

55

adult patients of calcium urolithiasis [6, 8, 10, 14, 15]. Currently, it is unknown that whether

56

or not low exposure to melamine from the living environment may cause impaired renal

57

function in general adult population.

58

Another type of nephrotoxic chemicals, phthalates, is widely used as plasticizers to provide

59

flexibility and durability in plastic products [16]. Phthalates are well known as environmental

60

endocrine disruptors, but our recent study also suggested that the exposure to

61

high-molecular-weight phthalates (HMWP) could increase the risk of impaired renal function

62

in adults [9]. In addition, reports from Taiwan and USA also found an impaired

63

albumin-to-creatinine ratio (ACR) in urine in association with the exposure to dis 3

64

(2-ethylhexyl) phthalate (DEHP) in children [5, 7].

65

It is interesting that the renal toxicity of melamine and phthalates may share a common

66

mechanism, such as oxidative stress—a common pathway leading to renal function

67

deterioration. Oral exposure to dis (2-ethylhexyl) phthalate (DEHP) and di-iso-nonyl

68

phthalate (DiNP) are reported to cause renal histopathologic alterations in mice by upsetting

69

the balanced status between oxidants and antioxidants [17–20]. Similarly, melamine is also

70

demonstrated to increase the production of reactive oxygen species (ROS) and activate the

71

p38 mitogen-activated protein kinase (MAPK) pathway, which results in apoptosis in rat

72

kidney epithelial cells [21]. The shared mechanisms between melamine and phthalates make

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the risk assessment of co-exposure to these chemicals being important, since people are

74

generally co-exposed to them. In this study, we investigated the renal function of Shanghai

75

adults in association with both melamine and phthalates. To the best of our knowledge, this is

76

the first epidemiological study reporting the nephrotoxic effect of exposure to melamine in

77

general adults.

78

2. Method

79

2.1 Study population

80

The participants in this study were enrolled in the Shanghai Food Consumption Survey

81

(SHFCS), which has been described extensively elsewhere [22–25]. Briefly, the SHFCS was

82

conducted four times within two years (from September 2012 to August 2014) to acquire

83

knowledge of the nutritional status of residents in Shanghai, and urine samples were collected

84

to measure the exposure to environmental chemicals related to diets. As urine samples were

85

collected only in the first survey (fall 2012), data collected in fall 2012 were ultimately

86

included in this study. Participants were eligible if they were aged ≥18 and had no history of

87

serious disease (cancer) at enrollment. Spot urine samples were obtained from 3082

88

participants, but 224 were aged ≤18 years, 89 were lacking weight or height information, 25

89

had unreasonable creatinine concentration (<20 µmol/L or >30,000 µmol/L), 326 had

90

insufficient samples for the detection of any biomarkers of this study, and only 2418 samples

91

of adults had been measured for the metabolites of phthalates. Among the 2418 samples, only

4

92

1663 samples were of sufficient volume for measuring renal function parameters, 908 had a

93

sufficient volume for measuring melamine and only 611 samples were measured for both

94

melamine and renal function parameters (Supplementary Figure 1). The Ethics Committee

95

of the School of Public Health at Fudan University approved this study. All participants gave

96

informed consent at enrollment.

97

2.2 Dietary assessment

98

The SHFCS collected the data of the food frequency questionnaire (FFQ) survey. Since renal

99

function may be affected by protein or other nutrients, we used the data from the FFQ to

100

calculate the dietary intake of nutrients including protein, fat, carbohydrate, fiber, calcium,

101

phosphorus, potassium, and magnesium. The FFQ in SHFCS was self-designed and

102

semi-quantitative and has been described elsewhere [9].

103

2.3 Measurement of urinary melamine

104

Spot urine samples were collected in glass tubes capped with polypropylene lids and frozen at

105

-80 ℃ immediately after collection. The melamine in urine was measured by the

106

ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)

107

method coupled to off-line solid phrase extraction (SPE) according to Panuwet et al [26].

108

Briefly, 1 mL of each urine sample was thawed, transferred to a 50 mL plastic centrifuge tube,

109

and spiked with 125 µL isotopic (13C3) internal standard (400 µg/L), 150 µL hydrochloric

110

acid (1 mol/L), and 15 mL acetonitrile/water (70:30, v/v). Then, they were ultrasonically

111

extracted for 15 min, vortex-mixed and centrifuged at 12000 r/min for 15 min. The

112

supernatant was loaded into a PXC column (Dikma, China; 150mg/6mL) which was

113

previously activated with 6 mL methanol and 6 mL water. After sample loading, the column

114

was washed with 9 mL water and 6 mL methanol. Next, 8 mL ammonium methanol (5:95, v/v)

115

was added to elute melamine. The elute was concentrated under a stream of dry nitrogen at

116

50 °C. Finally, the residue was reconstituted with 1 mL acetonitrile/water (94:6, v/v), passed

117

through a 0.22 µm filter and analyzed (2µL) by a Waters ACQUITY UPLC coupled with a

118

Waters Xevo TQ-S micro triple quadrupole mass spectrometer (Waters, USA).

119

An internal standard method was used to quantify the target metabolite. The calibration range 5

120

was from 0.5 to 200 µg/L and the standard calibration curve had a regression coefficient (r) of

121

0.9999. For every 20 samples, two procedural blank and four pre-extraction matrix-spiked

122

samples by fortifying with known concentrations of standard (10 µg/L) were processed. The

123

average recoveries and relative standard deviations (RSD) of the target metabolite were

124

98.5% and 3.0% at 10 µg/L, and 98.2% and 1.0% at 50 µg/L, respectively. The limits of

125

detection (LOD) was calculated at a signal-to-noise (S/N) of 3, with a concentration of 0.1

126

µg/L (Supplementary Table 1).

127

The concentration of melamine was adjusted by creatinine to correct urine dilution.

128

Creatinine was measured using an automatic biochemical analyzer (ARCHITECT C8000,

129

Abbott Laboratories, Illinois, USA).

130

2.4 Measurement of urinary metabolites of phthalates

131

We used the method of liquid chromatography tandem mass spectrometry (LC-MS/MS) to

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determine 10 urinary metabolites of 6 parent phthalates, including metabolites of dimethyl

133

phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DnBP), di-iso-butyl phthalate

134

(DiBP), and benzyle butyl phthalate (BBP), which were monomethyl phthalate (MMP),

135

monoethylphthalate (MEP), mono-n-butylphthalate (MnBP), monoisobutylphthalate (MiBP),

136

and

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mono-2-ethylhexylphthalate

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mono-2-ethyl-5-hydroxyhexylphthalate (MEHHP), mono-2-ethyl-5-carboxypentylphthalate

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(MECPP), and mono-2-carboxymethyl-hexyl phthalate (MCMHP).

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The method has been described in our previous study [24]: “Briefly, 1 mL of urine sample

141

was incubated with β-glucuronidase (Helix pomatia; Sigma, Louis, MO, USA; Type HP-2,

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aqueous solution, ≥ 100,000 units/mL) at 37 °C for 120 min. The sample was subsequently

143

acidified with 1 mL of aqueous 2% (v/v) acetic acid, mixed with 100 µL of internal standard

144

(100 µg/L), and loaded into a PLS column (Dikma, China; 60 mg/3 mL) previously activated

145

with 2 mL methanol and 2 mL of aqueous 0.5% (v/v) acetic acid. After sample loading, the

146

column was washed with 2 mL of aqueous 0.5% (v/v) acetic acid and eluted with 1 mL of

147

methanol. The eluate was passed through a 0.2-µm filter and analyzed (10 µL) by LC-MS/MS

mono-benzylphthalate

(MBzP) ； and (MEHP),

metabolites

of

DEHP,

mono-2-ethyl-5-oxohexylphthalate

6

which

are

(MEOHP),

148

(Shimadzu, USA; API 4000 LC/MS/MS system) coupled to an AQUASIL C18 column (150

149

× 4.6 mm; Thermo Fisher Scientific, Inc., USA). For each batch of 30 samples analyzed, two

150

procedural blanks and four matrix-spiked samples at two different spiking concentrations (10

151

and 25 ng/mL) were processed. The average recoveries and relative standard deviations (RSD)

152

of target metabolites in spiked samples respectively ranged from 71.5% to 109.1% and from

153

1.2% to 7.4% at 10 ng/mL, and ranged from 58.5% to 139.2% and from 0.8% to 8.1% at 25

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ng/mL. Trace concentrations of MEP, MnBP, MiBP, and MEHP were detected in procedural

155

blanks with average concentrations and RSDs ranging from 0.05 to 0.8 µg/L and from 3.7%

156

to 9.3%, respectively. Sample concentrations of these metabolites were determined after

157

subtraction of the blank values.” The results of quality assurance and quality control are

158

presented in Supplementary Table 1. The concentrations of 10 metabolites were adjusted by

159

creatinine and expressed as µg/g creatinine.

160

2.5 Measurement of biomarkers of renal function

161

Urinary

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N-acetyl-β-d-glucosaminidase (NAG), reported as good and early indicators for impaired

163

renal function, were measured according to previous methods [27–32]. Briefly, ALB and

164

B2M were measured using enzyme-linked immunosorbent assay (ELISA), while NAG was

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measured using the P-nitrophenol colorimetric method [27, 28]. The concentrations of ALB,

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B2M, and NAG were adjusted by urinary creatinine. The albumin-to-creatinine ratio (ACR)

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is one of the key markers for chronic kidney disease (CKD); thus, the creatinine-adjusted

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albumin was shown as ACR rather than ALB in this study [33].

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2.6 Statistical analyses

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All analyses were performed using SPSS version 23.0 (IBM SPSS, USA). The value of 1/2

171

LOD was assigned to chemical concentrations below the LOD. Because of the skewed

172

distribution, urinary melamine, phthalate metabolites, and renal function parameters were

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natural log–transformed. Two-sided p-values <0.05 were considered to be statistically

174

significant.

175

A 2-step procedure was used to explore the relationship between chemical exposure and

parameters

of

albumin

(ALB),

7

β2-microglobulin

(B2M),

and

176

impaired renal function:

177

(1) The first step was to assess the effects of melamine and phthalates independently using

178

linear regression models. We have previously reported phthalate exposure in association with

179

impaired renal function in a larger sample size of this population. In this study, we use

180

another strategy to explore the effects of phthalates on impaired renal function. Since

181

phthalate metabolites were strongly correlated with each other (Supplementary Table 2), we

182

used principal component analysis (PCA) to generate summary measures of co-exposure to

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phthalates before conducting the regression analyses. Briefly, the Kaiser–Meyer–Olkin

184

Measure of Sample Adequacy and the Bartlett Test of Sphericity were firstly used to assess

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the data adequacy of PCA; then, the PCA with varimax rotation was applied to the natural

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log–transformed concentration of metabolites, and the component scores for each participant

187

were calculated representing how closely the phthalate metabolites in a subject’s urine sample

188

conform to identify exposure patterns. Three patterns (HMWP pattern, DBP pattern, and the

189

DMP & DEP pattern) were ultimately selected in this study. We used multiple linear

190

regression to test whether or not these patterns were associated with renal function

191

biomarkers. Since the HMWP pattern and melamine exposure were both positively associated

192

with impaired renal function, they were selected for the second step analyses.

193

(2) The second step assessed the co-exposure to melamine and phthalates (HMWP pattern).

194

Both multiplicative and additive effects of such co-exposure were investigated in our data set.

195

The additive effect was assessed by analyzing a combination score of co-exposure. Firstly,

196

both melamine and HWMP pattern were categorized as 0, 1, 2 and 3 according to their

197

step-by-step quartile concentrations, respectively; secondly, a combination score was created

198

by summarizing the category score of both melamine and HWMP pattern; finally, linear

199

regression analyses between the combination score and renal function parameters were used

200

to explore the additive effect of co-exposure.

201

All regression analyses in this study were adjusted for age, sex, education, occupation,

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ethnicity, physical activity, marital status, smoking status, drinking status, body mass index

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(BMI), diabetes, systolic blood pressure, diastolic blood pressure, and nutrients (protein, fat,

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carbohydrate, fiber, calcium, phosphorus, potassium, and magnesium). Among these 8

205

covariates, the age, sex, disease status of diabetes and high blood pressure, and nutrition

206

intake have been well investigated to be associated with renal function [1, 4, 34-38].

207

As nutrients were correlated, we also used PCA to generate summary nutrient consumption

208

patterns. Sensitivity analysis was done using nutrient patterns adjusted in linear regression

209

models to investigate the association between renal function and environmental chemicals

210

(Supplementary Table 3)

211

3. Result

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3.1 Basic characteristics

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The general characteristics of the study participants are shown in Table 1. Of the 611

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participants, 286 (46.8%) were male. The prevalence of diabetes was 7.2%. The median

215

urinary melamine was 3.2 µg/g Cr (interquartile range: 0.11–20.51 µg/g Cr).

216

Supplementary Table 4 compares the basic characteristics of included and excluded adult

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participants. Excluded participants tended to be younger, were more often Han Chinese, and

218

had a lower education level. In addition, both groups were also significantly different in the

219

characteristics of occupation and physical activity.

9

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Table 1. Demographic characteristics, clinical parameters, and nutrient intake of study

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population (n=611). Characteristic Age, n (%) ≤ 45 45–60 > 60 Sex, n (%) Male Female Ethnicity, n (%) Han Other Education, n (%) ≤ Middle school ≥ High school Occupation, n (%) Student Official or intellectual Worker Retiree Other Physical activity, n (%) Light Moderate Vigorous Marital status, n (%) Married Others Smoking status, n (%) Never smoked Current/past smoker Drinking status, n (%) Never drank Current/past drinker Diabetes, n (%) No Yes Systolic blood pressure, median (IQR), mm Hg Diastolic blood pressure, median (IQR), mm Hg BMI, median (IQR), kg/m2 10

Total 158 (25.9) 234 (38.3) 219 (35.8) 286 (46.8) 325 (53.2) 599 (98.0) 12 (2.0) 253 (41.7) 358 (58.3) 18 (1.3) 116 (19.0) 23 (3.8) 295 (48.3) 159 (27.6) 272 (44.5) 138 (22.6) 201 (32.9) 531 (86.9) 80 (13.1) 458 (75.0) 153 (25.0) 395 (64.6) 216 (35.4) 567 (92.8) 44 (7.2) 120 (120, 130) 80 (78, 80) 23.2 (21.1, 25.4)

Protein, median (IQR), g Fat, median (IQR), g Carbohydrate, median (IQR), g Fiber, median (IQR), g Calcium, median (IQR), mg Phosphorus, median (IQR), mg Potassium, median (IQR), mg Magnesium, median (IQR), mg Melamine, median (IQR), µg/g Cr ACR, median (IQR), mg/mmol Cr B2M, median (IQR), µg/mmol Cr NAG, median (IQR), U/mmol Cr

71.42 (55.61, 88.70) 31.29 (21.82, 47.61) 234.09 (178.62, 288.34) 10.70 (7.56, 14.40) 475.13 (340.46, 649.41) 906.92 (701.85, 1142.65) 1693.18 (1259.37, 2107.98) 220.38 (159.90, 278.65) 3.25 (0.11, 20.51) 1.64 (0.77, 3.19) 9.31 (2.17, 28.81) 0.65 (0.32, 1.27)

222

BMI, body mass index; ACR, albumin–creatinine ratio; B2M, β2-microglobulin; NAG,

223

N-acetyl-β-d-glucosaminidase; IQR, interquartile range.

224

3.2 Relationship between urinary biomarkers of melamine exposure and renal function

225

Linear regression analyses revealed that melamine exposure was significantly associated with

226

ACR and B2M in the three different models adjusted for specific covariates and with NAG

227

only in model 1 (Figure 1).

11

228 229

Figure 1. Linear regression analyses of urinary melamine in association with renal function

230

parameters. Model 1: unadjusted; Model 2: adjusted for age, sex, ethnicity, education,

231

occupation, physical activity, marital status, smoking status, drinking, body mass index,

232

diabetes, systolic blood pressure, diastolic blood pressure; Model 3: adjusted for age, sex,

233

ethnicity, education, occupation, physical activity, marital status, smoking status, drinking,

234

body mass index, diabetes, systolic blood pressure, diastolic blood pressure, and nutrients

235

(protein, fat, carbohydrate, fiber, calcium, phosphorus, potassium, and magnesium).

12

236

3.3 Impaired renal function in association with co-exposure to multiple phthalates

237

For phthalate metabolites, PCA identified three patterns with eigenvalues over 1 (Figure 2),

238

named the “HWMP pattern”, “DBP pattern” and “DMP & DEP pattern”, which accounted for

239

40%, 17% and 12% of the total variance, respectively. These three patterns were mainly

240

loaded with the metabolites of HMWP, DBP, and two low-molecular-weight phthalates (DMP

241

and DEP), respectively.

242

The component scores of the HMWP pattern were positively associated with renal function

243

parameters (ACR, B2M, and NAG), while DBP pattern were negatively associated with these

244

parameters (Supplementary Table 3).

245 A.

B.

246 247

Figure 2. Principal and varimax-rotated component loading weights for metabolites of

248

phthalates in urine. (A) Principal component loading; (B) varimax-rotated component loading.

249

HMWP: high-molecular weight phthalates.

250 251

3.4 Impaired renal function in association with co-exposure to melamine and phthalates

252

Since impaired renal function was found to be independently associated with the exposure to

253

both melamine and phthalates of the HWMP pattern, we further assessed the interaction 13

254

effects of co-exposure. Table 2 presents the results of multiplicative analyses, showing no

255

significant effect on both ACR and NAG, but a negative effect on B2M (Table 2). Figure 3

256

presented the results of additive analyses, showing significant associations between

257

co-exposure and all three parameters of impaired renal function (ACR, B2M, and NAG).

258 259

Table 2. The interactive analyses of co-exposure to urinary metabolites of melamine and

260

high-molecular-weight phthalates (HMWP) on renal function parameters by linear regression

261

models. ACR

B2M

NAG

β

p-value

β

p-value

β

p-value

Melamine

0.018

0.033

0.029

0.019

0.012

0.100

HMWP Pattern

0.083

0.001

0.125

0.001

0.092

<0.001

Melamine*HMWP Pattern

-0.012

0.143

-0.025

0.038

0.008

0.255

262

Data were adjusted for age, sex, ethnicity, education, occupation, physical activity, marital

263

status, smoking status, drinking, body mass index, diabetes, systolic blood pressure, diastolic

264

blood pressure, and nutrients (protein, fat, carbohydrate, fiber, calcium, phosphorus,

265

potassium, and magnesium).

14

A.

266 B.

267 C.

268 269

Figure 3. Linear regression analyses of a combination score of co-exposure in association

270

with renal function parameters. (A) ACR; (B) B2M; (C) NAG. Results were presented as

271

median scores with 95% confidence interval. N: number of cases; S: a combination score for

272

ranking the co-exposure to melamine and phthalates of the HMWP pattern, where a higher

273

number represents a higher level of co-exposure. Data were adjusted for age, sex, ethnicity,

274

education, occupation, physical activity, marital status, smoking status, drinking, body mass

275

index, diabetes, systolic blood pressure, diastolic blood pressure, and nutrients (protein, fat,

276

carbohydrate, fiber, calcium, phosphorus, potassium, and magnesium). 15

277

4. Discussion

278

In this cross-sectional study, we found the risk of impaired renal function was associated with

279

independent exposure to melamine and HMW phthalates, respectively. Their co-exposure

280

increased this risk by mainly showing an additive effect.

281

4.1 Melamine exposure and impaired renal function

282

In this study, melamine had a median concentration of 0.38 µg/mmol Cr (unadjusted median

283

concentration: 2.60 µg/L, geometric mean: 1.14 µg/L). Previous studies reported a geometric

284

mean of 2.37 µg/L in 477 samples from the U.S. general population, and a median

285

concentration of 4.7 µg/L in 109 U.S. children, as well as a median concentration ranging

286

from 0.78 to 1.7 µg/mmol Cr in children from Taiwan and Hongkong [15, 26, 39, 40]. These

287

data indicated that the melamine exposure was at a similar level in our study to that in

288

previous reports.

289

People may be exposed to melamine through several pathways. During the milk scandal,

290

melamine was illegally added to milk products to mimic the high protein content [11]. If

291

people continue to use the Kjeldahl method of measuring nitrogen content to assess the

292

protein content, this adulteration might still persist. Melamine has been reported to be capable

293

of migrating from melamine–formaldehyde tableware when heating food inside it (especially

294

when microwaving sour food) [41]. Melamine was also reported to come from the use of

295

cyromazine pesticide, which was used for insectcontrol and able to be metabolized into

296

melamine via a dealkylation reaction in both plants and animals [42-44]. Melamine is not

297

easily degraded and is mainly excreted via the urine tract, and therefore may consistently

298

persist in the environment [11, 39, 45]. Former studies have reported melamine

299

contamination in different environmental media including air, indoor dust, soil, sewage

300

sludge, sediments, et al. [6, 46-49]. It is currently unknown which sources and pathways are

301

the main reasons for extensive exposure to melamine in humans.

302

Kidney disease is the most common health outcome related to melamine exposure. Most

303

previous studies have reported melamine-induced urinary tract calculi in infants for the

304

reason of consuming adulterated formula, while little is known about other nephrotoxic 16

305

effects of chronic exposure to a low level of melamine from the living environment,

306

especially in general adults [50]. Compared with urinary tract calculi, impaired renal function

307

may serve as an earlier indicator before the calculi are able to be found in the clinic (e.g., by

308

ultrasonography). Several epidemiology studies have reported that melamine exposure was

309

positively associated with impaired renal function. A study involving 44 workers (16

310

manufacturers, 8 grinders, 10 packers, and 10 administrators) at two melamine

311

tableware-manufacturing factories in Taiwan found that urinary B2M and NAG were

312

significantly raised in manufacturers when compared to non-exposed workers, but this

313

significant association was not found for urinary ACR [6]. Similar results were found in

314

another study which enrolled 309 patients in Taiwan, which reported that melamine exposure

315

was significantly associated with higher concentrations of urinary B2M and NAG, but not

316

ACR [8]. Unlike these two studies in Taiwan, our data showed positive results of not only

317

B2M and NAG, but also ACR. Both B2M and NAG have been recognized as useful

318

biomarkers for early renal tubular injury, while urinary ACR usually serves as an early

319

indicator of glomerular dysfunction [29-32]. These three biomarkers represent different target

320

tissues of nephrotoxicity. Thus, most previous studies suggested environmental melamine

321

mainly damaged the renal tubular area rather than the glomerular area. However, a recent

322

Taiwanese study reported a positive association between melamine exposure and urinary

323

ACR in children, which corresponded with our observation [10]. More studies are wanted for

324

exploring the effect of melamine exposure on glomerulus.

325

No mater tubular or glomerular injury, they may share common pathways. Two possible

326

mechanisms have been proposed for the impaired renal function: (1) melamine may serve as

327

a nidus to aggressive stone formation, while patients with calcium urolithiasis were suggested

328

to have significantly higher urinary NAG, which indirectly supported the mechanism that

329

melamine exposure affects renal function via the promotion of stone formation [51, 52]; (2)

330

chronic exposure to melamine could induce impaired renal function via oxidative stress.

331

Hsieh et al. stimulated human renal proximal tubular HK-2 cells with melamine and

332

demonstrated that melamine could active MAPK, nuclear factor kappa beta (NFκB), and

333

ROS, which results in the upregulation of inflammatory factors (interleukin-6 (IL-6),

17

334

monocyte chemoattractant protein-1, and vascular cell adhesion molecule-1) and tumor

335

growth factor beta-1 (TGF-β1) [53]. In macrophage-like cell line RAW 264.7 and human

336

embryonic kidney cell line HEK293, melamine was found to activate NADPH oxidase (NOX)

337

accompanied by an increase in ROS production [54]. In addition, Li et al. reported that

338

apocynin and catechin could prevent melamine-related urolithiasis by decreasing oxidative

339

stress in vitro (HK-2 cells) and in vivo (male Sprague–Dawley rats) [55, 56]. These studies

340

demonstrated the importance of oxidative stress in melamine-induced renal toxicity.

341

4.2 Phthalates exposure and impaired renal function

342

We previous reported that exposure to some HMW phthalates (DEHP and MBzP) is

343

positively associated with impaired renal function [9]. Here, we used the method of PCA to

344

assess patterns of exposure in association with impaired renal function. A positive association

345

between HMWP pattern score and renal function parameters (ACR, B2M, and NAG) was

346

found in this study, which was in accordance with the results of the single metabolite analyses

347

in our previous study. We have also discussed the finding of HMWP (DEHP and MBzP) in

348

association with both glomerular (ACR) and tubular (B2M and NAG) biomarkers of renal

349

function in the previous study [9]. In this study, a co-exposure index of multiple phthalates

350

(HMWP pattern) was generated using the PCA method for simplifying the analyses of the

351

impaired renal function of co-exposure to both melamine and phthalates.

352

4.3 Impaired renal function in association with the co-exposure to melamine and

353

phthalates

354

Our data showed that independent exposure to either melamine or HMW phthalates was

355

associated with impaired renal function. This association was also found when considering

356

the interactive effect of co-exposure to both types of chemicals. Such an interactive effect

357

was found to be additive but not multiplicative. The linear regression model of multiplicative

358

effects even found that co-exposure presented a negative association on B2M (Table 2). Such

359

an unexpected result was also found when the co-exposure group with the highest

360

combination score (category 6) had a lower concentration of urinary B2M than other groups

361

(category 4 and 5) (Figure 3). It might be possible that the small subsample of category 6 (N

18

362

= 47) had occasionally lower urinary B2M. It should be noted that an association across the

363

categories was statistically more reliable than the comparison between individual categories.

364

Therefore, the results of interactive effects in Table 2 and Figure 3 were more trustworthy to

365

be explained as additive but not multiplicative.

366

One study from Taiwan also reported the interactive effect between current melamine

367

concentrations and past DEHP exposure, but not current DEHP exposure on urinary ACR

368

[10]. Unlike Taiwanese reports, the correlations between renal function parameters and

369

chemical exposure (both melamine and phthalates) in our data existed in more parameters in

370

the analyses of either independent or additive models of chemical exposure. Since the

371

exposure levels in our data were similar in melamine but lower in phthalates than in

372

Taiwanese reports, the different findings in our data and Taiwanese reports may suggest that

373

the renal toxicity of these two types of chemicals was more sensitive in adults than in

374

children. However, this suggestion needs to be clarified in future studies.

375

4.4 Strengths and limitations

376

The main strength of this study is that this is the first study to our knowledge reporting

377

impaired renal function in healthy adult in association with low level of exposure to

378

melamine, while previous studies only investigated sensitive populations (children and

379

calcium urolithiasis patients) or workers at high-dose melamine exposure levels.

380

This study also has some limitations. Firstly, the study design is cross-sectional, thus a

381

reverse causality may exist. Secondly, biomarkers for melamine and phthalate exposure as

382

well as impaired renal function were assessed in single-spot urine samples. Since both

383

melamine and phthalates have short half-lives, a single-spot urine sample reflects the nature

384

of short-term exposure, unless the participants maintain their lifestyle (in this situation, a

385

single measurement may serve as the snapshot of long-term exposure) [11, 57, 58]. Thirdly,

386

blood samples were not collected in this survey, thus we were unable to assess other

387

important renal function parameters, e.g. glomerular filtration rate (GFR). Fourthly, 2436

388

participants were excluded from the analysis, while some demographic characteristics (age,

389

education, ethnicity, education, occupation, and physical activity) differed from those of

19

390

included participants. As no modified effect of these factors were found, results in the final

391

sample size of this study may still reflect the true effects.

392

5. Conclusion

393

In summary, this study presents associations of two environmental chemicals (melamine and

394

HWM phthalates) with impaired renal function. In addition, co-exposure to these toxins

395

increases the risk of impaired renal function.

396

6. Acknowledgments

397

We thank all participants for their participation and kind assistance. This work was supported

398

by the National Natural Science Foundation of China (grant number 31741105) and the

399

Major State Research Development Program of China (grant number 2016YFD0400602).

400 401

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25

Highlights


Melamine and phthalates nephrotoxicity in general adults was reported.




Urinary melamine was positively associated with not only β2-microglobulin (B2M) and N-acetyl-β-d-glucosaminidase (NAG), but also Albumin-to-creatinine ratio (ACR).




Co-exposure to melamine and high-molecular-weight (HMW) phthalates had additive effects on biomarkers of renal function.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: