The organophosphorus flame retardants impaired intracellular lipid

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The organophosphorus flame retardants impaired intracellular lipid metabolic function in human hepatocellular cells Zhengliang Hao, Zhijie Zhang, Dezhao Lu, Bin Ding, Shu Lin, Quan Zhang, and Cui Wang Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00058 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

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Chemical Research in Toxicology

The organophosphorus flame retardants impaired intracellular lipid metabolic function in human hepatocellular cells Zhengliang Hao 1, Zhijie Zhang 1, Dezhao Lu 1, Bin Ding 1, Lin Shu 2, Quan Zhang 2*, Cui Wang 1* 1 College

of Life Science, Zhejiang Chinese Medical University, Hangzhou 310053,

Zhejiang, People’s Republic of China 2

Key Laboratory of Microbial Technology for Industrial Pollution Control of

Zhejiang University of Technology, Hangzhou 310032, Zhejiang, People’s Republic of China

*To whom correspondence should be addressed. Phone: +86 571 8887 1579; Fax: +86 571 8887 1579; E-mail: [email protected] (C Wang), [email protected] (Q Zhang).

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Graphic abstract

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Abstract Organophosphorus flame retardants (OPFRs), a replacement for brominated flame retardants, are gradually accepted as endocrine disrupt chemicals (EDCs). Recently, many evidences revealed that these EDCs would cause chronic health problems such as obesity, referred as metabolic disruptors. However, the disturbance to lipid metabolism caused by OPFRs remains poorly understood, especially at biological molecular levels. Herein, we used the human hepatocellular cells (HepG2) to study the lipid metabolism disruption caused by nine OPFRs (halogenated-, aryl-, and alkyl-containing). All the tested OPFRs, excluding the long carbon chain alkyl-OPFRs, could cause intracellular triglyceride (TG) and/or total cholesterol (TC) accumulation. In detail, aryl-OPFRs (TPhP and TCP) induced both TC and TG deposition. Halogenated-OPFRs (TCEP, TBPP, TDCPP, and TCPP) induced intracellular TG accumulation, and only TDCPP also induced TC accumulation. Furthermore, TPhP induced lipid accumulation through regulation genes encoding proteins involved in fatty acid β-oxidation, lipid and fatty acid synthesis. All the halogenated-OPFRs cause TG accumulation only, enacted through β-oxidation rather than lipid synthesis. TPhP and TDCPP induced TC accumulation through both PPARγ and srebp2 signalling. Mitochondrial dysfunction including decreased oxygen consumption rate and ATP content may also contribute to lipid metabolic disruption by the tested OPFRs. Our data indicated that halogenated- and aryl-OPFRs may not be safe candidates, and further information should be made available on potential for,

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as well as the mechanism of, metabolic disruption. And long carbon chain alkyl-OPFRs may be safer than the other two groups.

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1. Introduction As a suitable substitute for brominated flame retardants (BFRs), world production and use of organophosphorus flame retardants (OPFRs) has been a dramatic increase in recent years of 5% annually 1. Unfortunately, the recognized “less persistent” OPFRs, such as tris(2-butoxyethyl)phosphate (TBEP), tris(2-chloroisopropyl)phosphate (TCPP), triphenyl phosphate (TPhP), tris(2-chloroethyl)phosphate (TCEP), tris(2-chloro-1-(chloromethyl)ethyl) phosphate (TDCPP) and tributyl phosphate (TBP), were found in air, household dust, sediment, aquatic systems 1-4 and even in human urine, blood and breast milk beings 4-7. Given the characters of ubiquity, the potential health risks of OPFRs had aroused great concerns and sometimes they were called as “regrettable substitution”. To date, several researches have been conducted on the endocrine disruption caused by OPFRs 8-12. Our recent publications have also observed several types of OPFRs can influence the steroidogenesis in human adrenocortical carcinoma cell (H295R) 13. They are prone to disturbing the metabolism of sex hormones and endocrine axis to xenopus 14. Several OPFRs such as TPhP, TDCPP and TCP, etc. have potential affinity for nuclear receptors (NRs), suggesting the endocrine disruption effects 13-17. It is widely recognized that the endocrine system plays a critical role in regulating the metabolism of nutrients. Thus, chronic health problems caused by EDCs are of great concern, especially with these new anthropogenic chemicals 18,19. Worldwide prevalence of chronic health problems such as obesity, diabetes, and 5



non-alcoholic fatty liver disease were reported to correspond with a subclass of EDCs, referred as metabolic disruptors (MDs) 20. Over the last few decades, a group of MDs including BPA, PBDEs, and organochlorine pesticides were reportedly associated with liver lipid accumulation, glucose resistance and even liver steatosis 21-23. Although experimental results are still emerging, an association between several OPFRs and energy imbalance seems to be suggested. For instance, prenatal and postnatal exposure to a commercial product FM550 (containing 10-20% of TPhP) was linked to susceptibility to obesity, adipogenesis, hypertriglyceridemia and disruption of energy balance both in in vitro and in vivo 24-26. In addition to this aryl-phosphorus compound, acute exposure to alkyl-phosphorus flame retardant TBP also induced disorders of the TCA cycle and energy metabolism in male Sprague-Dawley rats 27. Given the widespread exposure and endocrine disrupting potential for humans, the disturbance to lipid metabolism caused by OPFRs is a critical issue to be addressed, especially at biological molecular levels. Hepatic cells metabolic disorder is related to liver lipid metabolism. We chose human hepatocellular cells (HepG2) cell lines as a research model. The HepG2 cell line is currently the most used cell line as their similarities to human hepatocyte especially in lipid and glucose metabolism 28. We aimed to evaluate the potential of lipid accumulation including triglyceride (TG) and cholesterol (TC) in cells after exposure to nine different OPFRs. The genetic transcription levels of lipid synthesis, fatty acid synthesis and β-oxidation were measured. Moreover, we also focused on the PPARs and the mitochondrial functions, which tightly participated in cellular lipid 6


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homeostasis, after treatment with OPFRs.

2. Materials and Methods 2.1 Materials The organophosphorus flame retardants (OPFRs) including tris(2-butoxyethyl)phosphate (TBEP, 95.8%),tris(2-chloroisopropyl)phosphate (TCPP, 96%), triphenyl phosphate (TPhP, 99.5%) , tris(2-chloroethyl)phosphate (TCEP, 98.5%), tris(2-chloro-1-(chloromethyl)ethyl) phosphate (TDCPP, 95.5%) and tributyl phosphate (TBP, 99.5%), tris(2-ethylhexyl)phosphate (TEHP), tricresyl phosphate (TCP, 99.4%), tris(2-chloroethyl)phosphate (TCEP, 98.5%), were purchased from Bestown (Beijing, China). The stock solution was prepared in DMSO. Human multi-clonal antibodies to PPARα, PPARγ, and actin were obtained from Proteintech (Wuhan, China). The secondary antibody was from CWbio (Beijing, China). 2.2 Cell culture and treatment HepG2 obtained from American Type Culture Collection were cultured in RPMI 1640 containing 10% foetal bovine serum and 25 mM HEPES (Gibco, New York, USA) in a 37 °C incubator. The culture medium was changed to 10% of charcoal-dextran treated foetal calf serum (CDFBS, HyClone, Logan, UT, USA) during the chemical treatment. Cells (density: 2105/mL) were seeded in 96-well microplates for cell viability and oxygen consumption rate with six and five replicates, 6-wells for lipid, ATP content, and target protein expression measurement 7



with three replicates, 24-wells for genetic transcription with four replicates. After adherence, cells were treated with differing concentrations of OPFRs (dissolved in DMSO) and a negative control (16 carbons). Moreover, OPFRs also changed different molecule signalling involved in lipid metabolism, as discussed below. Lipid accumulation resulted from increased synthesis and decreased fatty acid β-oxidation and exportation. Triglyceride and de novo fatty acid synthesis acted as a critical contributor to lipid accumulation 36. Sterol regulatory element-binding protein 1 is the principle transcriptional factor in de novo fatty acid synthesis and primarily regulates genes related to TG production 36, 37. Aryl-, alkyl-, and several halogenated-OPFRs upregulated srebp1c, indicated the potential for fatty acid synthesis. The synthesised fatty acid would be repackaged by DGATs enzymes to TG. Between the two DGATs enzymes, dgat2 was principally expressed in liver and its upregulation was found to be closely related to hepatic steatosis 38. Among the tested chemicals, only TBPP, TCP, TBP and TPhP increased the gene encoded for the final step of TG synthesis. Thus, these four compounds may induce the TG accumulation through upregulation the potential genes of lipid synthesis. However, the reduction of dgat2 in TDCPP, TCEP, and TCPP, which also induced TG accumulation, implied other signalling pathway. In addition to TG, TC is another important lipid. As a ubiquitously expressed transcription factor, srebp2 encodes enzymes for cholesterol homeostasis, such as the rate limiting enzyme hmgcr 39. Environmental chemicals that induce serum TC increase have been observed in mice with a high expression of srebp2 and its target genes 37. TDCPP, TBP, TCP and TPhP upregulated both srebp2 15



and hmgcr, which accompanied with the intracellular TC accumulation herein. Though less abundant in liver tissue, the level of PPARγ is essential for liver disease when disturbed 40. Stimulation of PPARγ in mice adipose tissue, zebrafish adipocyte cell, and BMS2 cell by TPhP clearly induced adipogenesis, stereogenesis and lipid accumulation 11, 41, 42. Among OPFRs, only TPhP, EHDPP and TBP were reported as PPARγ agonists 10, 25, which were confirmed by our molecular docking results. Additionally, we observed that TPhP, TDCPP, TCEP and TCPP suppressed the protein level of PPARγ. This phenomenon was also observed in BPA and troglitazone (PPARγ agonist) treated HepG2 cells 43. Moreover, activation of PPARγ in HepG2 cells was proven to inhibit cellular cholesterol synthesis through downregulation of srebp2 44. Consequently, our study suggested that downregulation of PPARγ and upregulation of srebp2 concomitantly induced intracellular cholesterol deposition by both TPhP and TDCPP. Inhibition of fatty acid β-oxidation by chemicals was highly related to liver steatosis or hyperlipoidemia 45. The fatty acid β-oxidation reaction occurs in mitochondria. Ideally, the long-chain fatty acid acyl-CoA should be converted to acylcarnitine by cpt1α at the outer mitochondrial membrane. These derivatives would then be converted back to fatty acid acyl-CoA in the presence of cpt2 on the inner mitochondrial membrane. The transferase enzyme cpt1α was regarded as the rate-limiting enzyme in β-oxidation. Previous studies revealed that chronic exposure to OCPs and BPA reduced the level of cpt1α in both the in vivo and in vitro models 43, which finally caused the lipid accumulation. PPARα, predominantly expressed in the 16


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liver, is involved in fatty acid oxidation through the control of cpt1α 40. Our data show that all of the tested OPFRs, except the alkyl-OPFRs (TEHP, TBEP), downregulated the protein level of PPARα and its downstream cpt1α. However, the regulation mode of these chemicals to PPARα would be quite different. TBP has a lower binding affinity to PPARα when compared to TPhP and TDCPP. The transcription level of cpt1α in the TBP treatment was increased, while decreased by TPhP and TDCPP, which indicated the high rate for fatty acid degradation. Thus, intracellular accumulation of TG was not obvious in TBP. Our results suggested that aryl- and halogenated-OPFRs would induce the intracellular lipid accumulation partially through PPARα-dependent pathway. Mitochondrial dysfunction including impairment on mitochondrial membrane potential (MMP), ATP content, and OCR, etc. was usually observed in chemicalinduced the metabolic disorder 46. The previous data showed that aryl- and alkyl-OPFRs caused mitochondrial dysfunction on CHO cells at relatively high doses (>100 µM) 47. However, mitochondria are sensitive target for many chemicals at non-cytotoxic doses. For example, exposure to OCPs reduced MMP and ATP content, as well as the OCR and TCA cycle in HepG2 cells at non-cytotoxic doses 21. Our data also confirmed that the altered OCR and decreased levels of ATP suggested that the chosen chemicals would cause mitochondrial dysfunction at non-cytotoxic concentrations, which may also contribute to the intracellular lipid metabolic imbalance. In summary, the majority of the OPFRs tested in this study caused intracellular 17



lipid accumulation at non-cytotoxic concentrations through differing ways. The tested halogenated-OPFRs caused TG accumulation primarily through fatty acid synthesis and β-oxidation inhibition rather than TG synthesis. TPhP induced intracellular lipid imbalance through inhibition fatty acid oxidation and increasing de novo fatty acid and TG synthesis. Both TDCPP and TPhP caused TC accumulation through the PPARγ and srebp2 pathway. The tested five chemicals which caused lipid metabolic disruption also caused mitochondrial dysfunction. Long-chain alkyl-containing OPFRs may safer than halogenated- and aryl-OPFRs. However, more information should be available on the effect and mechanism of OPFRs-induced lipid metabolic disruption.

Supporting information Materials listed in the supporting information include: Information of cell viability, genes related to lipid metabolism. Acknowledgements: This study was supported by Zhejiang Province Nature Science Foundation of China (LY18B070007) and the National Natural Science Foundation of China (21777147).

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Table 1 The structure of selected nine organophosphorus flame retardants Categories

chemicals

Substitute

tributyl phosphate (TBP)

Alkyl-OPFRs

tris(2-butoxyethyl) phosphate (TBEP) tris(2-ethylhexyl) phosphate (TEHP)

Aryl-OPFRs

triphenyl phosphate (TPhP) tricresyl phosphate (TCP) tris(2-chloroisopropyl) phosphate (TCPP)

Halogenated-OPFRs

tris(2-chloroethyl) phosphate (TCEP) tris(2-chloro-1-(chloromethyl)ethyl) phosphate (TDCPP) tris(2,3-dibromopropyl) phosphate (TBPP)

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logKow 4.0 3.75 9.49

4.59

5.11 2.59

1.44 3.65

4.29


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Table 2 Intracellular lipid accumulation caused by nine organophosphorus flame retardants Endpoints

Relative Total triglyceride (to negative control)

Relative Total cholesterol (to negative control)

0

1.00±0.099

1.00±0.19

0.1 µM

0.98±0.11

1.37±0.071*

1 µM

1.19±0.12

1.36±0.095*

10 µM

1.06±0.33

1.32±0.007*

0

1.00±0.34

1.00±0.29

0.1 µM

1.22±0.31

1.21±0.42

1 µM

1.21±0.33

1.19±0.056

10 µM

0.85±0.074

1.21±0.19

0

1.00±0.19

1.00±0.081

0.1 µM

0.81±0.19

0.85±0.096

1 µM

0.86±0.14

0.96±0.045

10 µM

0.76±0.19

0.89±0.047

0

1.00±0.083

1.00±0.21

0.1 µM

1.36±0.46

0.84±0.17

1 µM

1.82±0.39*

1.39±0.38

10 µM

2.69±0.14**

1.82±0.47*

0

1.00±0.17

1.00±0.19

0.1 µM

1.12±0.44

1.01±0.14

1 µM

1.55±0.42

1.00±0.13

10 µM

1.75±0.25*

2.07±0.34*

0

1.00±0.40

1.00±0.66

0.1 µM

1.97±0.46

1.16±0.02

1 µM

2.49±0.079*

0.90±0.14

10 µM

1.33±0.01

1.04±0.27

0

1.00±0.024

1.00±0.069

0.1 µM

1.01±0.11

0.84±0.019

1 µM

1.13±0.13*

0.93±0.084

10 µM

1. 05±0.21

0.94±0.021

0

1.00±0.056

1.00±0.12

0.1 µM

1.45±0.30

1.04±0.05

1 µM

1.05±0.14

0.97±0.19

10 µM

1.46±0.099**

1.15±0.12

0

1.00±0.091

1.00±0.19

0.1 µM

1.29±0.17

1.30±0.15

1 µM

1.07±0.097

2.11±0.06*

10 µM

1.50±0.16*

2.69±0.40*

Chemicals TBP

TEHP

TBEP

TPhP

TCP

TBPP

TCEP

TCPP

TDCPP

*, p

The organophosphorus flame retardants impaired intracellular lipid

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