The p-Phthalates Terephthalic Acid and Dimethyl Terephthalate Used in the Manufacture of PET Induce In Vitro Adipocytes Dysfunction by Altering Adipogenesis and Thermogenesis Mechanisms

Public health concerns associated with the potential leaching of substances from Polyethylene terephthalate (PET) packaging have been raised due to the role of phthalates as endocrine-disrupting chemicals or obesogens. In particular, changes in the environment such as pH, temperature, and irradiation can improve contaminant migration from PET food packaging. In this study, the in vitro effects of p-phthalates terephthalic acid (TPA) and dimethyl terephthalate (DMT) on murine adipocytes (3T3-L1) were evaluated using concentrations that might be obtained in adult humans exposed to contaminated sources. TPA and, in particular, DMT exposure during 3T3-L1 differentiation increased the cellular lipid content and induced adipogenic markers PPAR-γ, C/EBPß, FABP4, and FASN, starting from low nanomolar concentrations. Interestingly, the adipogenic action of TPA- and DMT-induced PPAR-γ was reverted by ICI 182,780, a specific antagonist of the estrogen receptor. Furthermore, TPA and DMT affected adipocytes’ thermogenic program, reducing pAMPK and PGC-1α levels, and induced the NF-κB proinflammatory pathway. Given the observed effects of biologically relevant chronic concentrations of these p-phthalates and taking into account humans’ close and constant contact with plastics, it seems appropriate that ascertaining safe levels of TPA and DMT exposure is considered a high priority.


Introduction
Polyethylene terephthalate (PET) is a thermoplastic polymer broadly used for food and beverage packaging applications, due to gas barrier properties, good thermal and mechanical properties, light weight, transparency, strength, good processability, and good recyclability [1]. PET is produced by polymerizing ethylene glycol with terephthalic acid (TPA) or via transesterification with dimethyl terephthalate (DMT), using antimony trioxide as the catalyst [2]. A more recent development in PET manufacturing is the incorporation of an appropriate comonomer, such as isophthalic acid or DMT, to slow down the rate of crystallization, which allows the manufacture of thicker bottle walls, sheets, and films [3].
Despite the convenience plastic packaging offers to the consumer, it has been subjected to many debates regarding environmental and health issues. Generally, the migration is an undesirable process where polymerization residues or stabilizers can diffuse through the polymer matrix to the surface and then to food. Due to the increasing awareness of consumers in terms of health matters, the importance of the migration of substances from PET food packaging materials to foods attracted the interest of the scientific and legislative communities. According to European Regulation No. 10/2011 [4], the EU addresses the obtained in adult humans through the consumption of foods in PET packages or exposure to other contaminated sources [23,24]. Furthermore, we tested TPA and DMT at 10 nM, taking into consideration the environmentally relevant concentrations of phthalates as measured in the blood of human subjects [25,26]. Of course, the doses used in other studies may be relevant in the case of other routes of exposure to p-phthalates, such as inhalation or cutaneous absorption, or also if these compounds are hypothesized to be ingested through products other than food, for example, dust [27,28]. With this aim, 3T3-L1 adipocytes were selected as an in vitro model to clarify the potential obesogenic effects of these phthalate isomers, evaluating the adipogenesis process and tissue inflammation and the potential mechanisms of the observed effects, and thus providing an experimental basis for the correlation between p-phthalates and obesity.

p-Phthalates In Vitro Toxicity and In Silico ADME
The cytotoxic potential of DMT and TPA (10 nM and 10 µM) has been investigated by sulforhodamine B assay on NIH/3T3 mouse fibroblasts, after 48 h of exposure. The results obtained showed that the tested p-phthalates do not have any cytotoxic effect ( Figure 1).
In this paper, we intended to evaluate, for the first time, the effects of the p-phtha TPA and DMT on adipose tissue using concentrations mimicking those that might b tained in adult humans through the consumption of foods in PET packages or expo to other contaminated sources [23,24]. Furthermore, we tested TPA and DMT at 10 taking into consideration the environmentally relevant concentrations of phthalat measured in the blood of human subjects [25,26]. Of course, the doses used in other ies may be relevant in the case of other routes of exposure to p-phthalates, such as in tion or cutaneous absorption, or also if these compounds are hypothesized to be inge through products other than food, for example, dust [27,28]. With this aim, 3T3-L1 ad cytes were selected as an in vitro model to clarify the potential obesogenic effects of phthalate isomers, evaluating the adipogenesis process and tissue inflammation an potential mechanisms of the observed effects, and thus providing an experimental for the correlation between p-phthalates and obesity.

p-Phthalates In Vitro Toxicity and In Silico ADME
The cytotoxic potential of DMT and TPA (10 nM and 10 µM) has been investigate sulforhodamine B assay on NIH/3T3 mouse fibroblasts, after 48 h of exposure. The re obtained showed that the tested p-phthalates do not have any cytotoxic effect ( Figure   Figure 1. Cell Viability. Cytotoxicity was evaluated by sulforhodamine B assay on NIH/3T3 treated with different concentrations of the tested compounds for 48 h. Control cells were tr with the vehicle (DMSO) alone. SDS was used as a positive control. a p < 0.05 vs. CTR.
As reported in Table 1, the tested compounds followed Lipinski's rules for drug ness with no violation. Regarding the in silico ADME analysis, the results may be i preted based on the marginal value compared with the resultant value as follows: Caco-2 permeability is predicted by a value > 0.90, and intestinal absorption less than is considered poorly absorbed; human VDss is low if below 0.71 L/kg and high if a 2.81 L/kg. In particular, both tested compounds appear to possess good oral bioavai ity. The percentage of intestinal absorption of all the compounds has been calculated values ranging from 75% (TPA) to 90% (DMT) and confirmed with Caco-2 permeabi As reported in Table 1, the tested compounds followed Lipinski's rules for druglikeness with no violation. Regarding the in silico ADME analysis, the results may be interpreted based on the marginal value compared with the resultant value as follows: High Caco-2 permeability is predicted by a value > 0.90, and intestinal absorption less than 30% is considered poorly absorbed; human VDss is low if below 0.71 L/kg and high if above 2.81 L/kg. In particular, both tested compounds appear to possess good oral bioavailability. The percentage of intestinal absorption of all the compounds has been calculated with values ranging from 75% (TPA) to 90% (DMT) and confirmed with Caco-2 permeability.

Effects of p-Phthalates on Adipogenesis Markers
Phthalates appear to play an essential role in the development of obesity, interfering at the level of the expression of key transcription factors in the adipogenesis process, such as SREBP-1c, PPAR-γ, C/EBPα, and LXRα, inducing important metabolic alterations [29]. Their activation determines, indeed, the upregulation of lipogenic genes resulting in the accumulation of intracellular lipids and adipocyte hypertrophy [30]. Therefore, in order to determine the effects of terephthalic acid and its dimethyl ester on adipocytes' differentiation, the 3T3-L1 preadipocytes were exposed to the tested compounds for 10 days, and the accumulation of intracellular lipids was evaluated by the histological technique of Oil Red O staining. The established adipogenesis activator rosiglitazone, a thiazolidinedione agonist of the PPAR-γ receptor, was used as the positive control. The results obtained showed that, under our experimental conditions, the treatment with the tested phthalates causes an increase in the size of lipid droplets compared to control cells in a dose-dependent manner. In particular, the extent of lipid accumulation induced by TPA was 1.27 ± 0.03 and 3.75 ± 0.05 vs. MDI (for 10 nM and 10 µM, respectively) and by DMT was 4.25 ± 0.03 and 5.04 ± 0.02 vs. MDI (for 10 nM and 10 µM, respectively) ( Figure 2). As

Effects of p-Phthalates on Adipogenesis Markers
Phthalates appear to play an essential role in the development of obesity, interfering at the level of the expression of key transcription factors in the adipogenesis process, such as SREBP-1c, PPAR-γ, C/EBPα, and LXRα, inducing important metabolic alterations [29]. Their activation determines, indeed, the upregulation of lipogenic genes resulting in the accumulation of intracellular lipids and adipocyte hypertrophy [30]. Therefore, in order to determine the effects of terephthalic acid and its dimethyl ester on adipocytes' differentiation, the 3T3-L1 preadipocytes were exposed to the tested compounds for 10 days, and the accumulation of intracellular lipids was evaluated by the histological technique of Oil Red O staining. The established adipogenesis activator rosiglitazone, a thiazolidinedione agonist of the PPAR-γ receptor, was used as the positive control. The results obtained showed that, under our experimental conditions, the treatment with the tested phthalates causes an increase in the size of lipid droplets compared to control cells in a dose-dependent manner. In particular, the extent of lipid accumulation induced by TPA was 1.27 ± 0.03 and 3.75 ± 0.05 vs. MDI (for 10 nM and 10 µM, respectively) and by DMT was 4.25 ± 0.03 and 5.04 ± 0.02 vs. MDI (for 10 nM and 10 µM, respectively) ( Figure 2

Effects of p-Phthalates on Adipogenesis Markers
Phthalates appear to play an essential role in the development of obesity, interfering at the level of the expression of key transcription factors in the adipogenesis process, such as SREBP-1c, PPAR-γ, C/EBPα, and LXRα, inducing important metabolic alterations [29]. Their activation determines, indeed, the upregulation of lipogenic genes resulting in the accumulation of intracellular lipids and adipocyte hypertrophy [30]. Therefore, in order to determine the effects of terephthalic acid and its dimethyl ester on adipocytes' differentiation, the 3T3-L1 preadipocytes were exposed to the tested compounds for 10 days, and the accumulation of intracellular lipids was evaluated by the histological technique of Oil Red O staining. The established adipogenesis activator rosiglitazone, a thiazolidinedione agonist of the PPAR-γ receptor, was used as the positive control. The results obtained showed that, under our experimental conditions, the treatment with the tested phthalates causes an increase in the size of lipid droplets compared to control cells in a dose-dependent manner. In particular, the extent of lipid accumulation induced by TPA was 1.27 ± 0.03 and 3.75 ± 0.05 vs. MDI (for 10 nM and 10 µM, respectively) and by DMT was 4.25 ± 0.03 and 5.04 ± 0.02 vs. MDI (for 10 nM and 10 µM, respectively) ( Figure 2). As expected, rosiglitazone-exposed cells showed strong enhancement (approximately 5.22-fold) in lipid accumulation compared to untreated cells. expected, rosiglitazone-exposed cells showed strong enhancement (approximately 5.22fold) in lipid accumulation compared to untreated cells. Although the exact mechanism of action remains unclear, several studies have shown how the PPARs may be involved in obesity following ortho-phthalate exposure [15,31,32]. These transcriptional factors regulate, indeed, a broad range of physiological processes including adipogenesis, fatty acid uptake, and cell proliferation, and are essential for the metabolism of lipids [33]. Therefore, in order to evaluate the mechanism through which p-phthalates determine the accumulation of lipid deposits, the expression of PPAR-γ was evaluated. The obtained results confirm that exposure to the tested phthalates induces a marked increase in PPAR-γ levels, with respect to unexposed cells, in a dose-dependent manner ( Figure 3A,B). In particular, low nanomolar levels were already able to activate this transcription factor, and the rank order of PPAR-γ induction was DMT ˃ TPA.
We also examined the level of expression of C/EBP-β, which is an important transcription factor expressed at the early stage of adipocyte differentiation, induced rapidly after the addition of adipogenic stimuli, and responsible for stimulating the expression of PPAR-γ [34]. The obtained results show a dose-dependent increase in C/EBP-β expression with respect to unexposed differentiated cells following treatment with the tested pphthalates. Additionally, in this case, DMT showed higher activity compared to TPA. These data demonstrate how the effects of these substances in the induction of the adipogenesis process occur from the early stage of adipocyte differentiation ( Figure 3A,C).
As further confirmation of the adipogenic effect induced by p-phthalates, we evaluated the gene expression of FABP4, a downstream gene of the PPAR-γ signaling pathway and a key regulator of fatty acid uptake and lipid accumulation during the adipogenic process [35]. FABP4-deficient adipocytes exhibit, in fact, reduced efficiency in adipogenesis and lipogenesis [36]. The data shown in Figure 4A also demonstrate how p-phthalates, already at nanomolar levels, are able to induce FABP4 gene expression in comparison to unexposed differentiated cells, confirming the activation of the PPAR-γ pathway. DMTexposed cells showed higher transcriptional activity compared to those exposed to TPA. Furthermore, the expression of the gene-encoding fatty acid synthase (FASN), a central enzyme involved in the lipogenesis process since it catalyzes the de novo biosynthesis of long-chain saturated fatty acids in adipose tissue, was evaluated. The tested p-phthalates Although the exact mechanism of action remains unclear, several studies have shown how the PPARs may be involved in obesity following ortho-phthalate exposure [15,31,32]. These transcriptional factors regulate, indeed, a broad range of physiological processes including adipogenesis, fatty acid uptake, and cell proliferation, and are essential for the metabolism of lipids [33]. Therefore, in order to evaluate the mechanism through which p-phthalates determine the accumulation of lipid deposits, the expression of PPAR-γ was evaluated. The obtained results confirm that exposure to the tested phthalates induces a marked increase in PPAR-γ levels, with respect to unexposed cells, in a dose-dependent manner ( Figure 3A,B). In particular, low nanomolar levels were already able to activate this transcription factor, and the rank order of PPAR-γ induction was DMT > TPA.
We also examined the level of expression of C/EBP-β, which is an important transcription factor expressed at the early stage of adipocyte differentiation, induced rapidly after the addition of adipogenic stimuli, and responsible for stimulating the expression of PPARγ [34]. The obtained results show a dose-dependent increase in C/EBP-β expression with respect to unexposed differentiated cells following treatment with the tested p-phthalates. Additionally, in this case, DMT showed higher activity compared to TPA. These data demonstrate how the effects of these substances in the induction of the adipogenesis process occur from the early stage of adipocyte differentiation ( Figure 3A,C).
As further confirmation of the adipogenic effect induced by p-phthalates, we evaluated the gene expression of FABP4, a downstream gene of the PPAR-γ signaling pathway and a key regulator of fatty acid uptake and lipid accumulation during the adipogenic process [35]. FABP4-deficient adipocytes exhibit, in fact, reduced efficiency in adipogenesis and lipogenesis [36]. The data shown in Figure 4A also demonstrate how p-phthalates, already at nanomolar levels, are able to induce FABP4 gene expression in comparison to unexposed differentiated cells, confirming the activation of the PPAR-γ pathway. DMTexposed cells showed higher transcriptional activity compared to those exposed to TPA. Furthermore, the expression of the gene-encoding fatty acid synthase (FASN), a central enzyme involved in the lipogenesis process since it catalyzes the de novo biosynthesis of long-chain saturated fatty acids in adipose tissue, was evaluated. The tested p-phthalates significantly induced a dose-dependent increase in FASN gene expression compared to unexposed differentiated cells ( Figure 4B), thus further suggesting the important role of these compounds in lipogenic pathway activation. significantly induced a dose-dependent increase in FASN gene expression compared to unexposed differentiated cells ( Figure 4B), thus further suggesting the important role of these compounds in lipogenic pathway activation.

p-Phthalates-Induced PPAR-γ Expression Is Dependent on Estrogen Receptor (ER)
To further demonstrate whether p-phthalates-induced effects on preadipocytes' differentiation are mediated through the ER, adipocytes were cotreated with the tested com-Molecules 2022, 27, 7645 7 of 17 pounds (10 nM or 10 µM) and the ER antagonist ICI (10 nM) during differentiation. ER antagonists provide a good tool to study the effect of a lack of estrogen action on the target tissue. In our experimental conditions, treatment with ICI alone did not affect the basal levels of PPAR-γ (data not shown). On the contrary, the ER antagonist prevented TPA-and DMT-induced protein expression, restoring PPAR-γ levels to that of the control ( Figure 5). Interestingly, PPAR-γ expression was inhibited with both 10 nM and 10 µM concentrations of TPA and DMT. These data suggest that the mechanism of p-phthalates-induced differentiation of 3T3-L1 preadipocytes may involve an ER-mediated mechanism. undifferentiated cells; b p < 0.05 vs. MDI; c p < 0.05 vs. DMT 10 nM; d p < 0.05 vs. DMT 10 µM; e p < 0.05 vs. TPA 10 nM.

p-Phthalates-Induced PPAR-γ Expression Is Dependent on Estrogen Receptor (ER)
To further demonstrate whether p-phthalates-induced effects on preadipocytes' differentiation are mediated through the ER, adipocytes were cotreated with the tested compounds (10 nM or 10 µM) and the ER antagonist ICI (10 nM) during differentiation. ER antagonists provide a good tool to study the effect of a lack of estrogen action on the target tissue. In our experimental conditions, treatment with ICI alone did not affect the basal levels of PPAR-γ (data not shown). On the contrary, the ER antagonist prevented TPAand DMT-induced protein expression, restoring PPAR-γ levels to that of the control (Figure 5). Interestingly, PPAR-γ expression was inhibited with both 10 nM and 10 µM concentrations of TPA and DMT. These data suggest that the mechanism of p-phthalatesinduced differentiation of 3T3-L1 preadipocytes may involve an ER-mediated mechanism. Figure 5. Effect of the ER antagonist ICI on the obesogenic effect of p-phthalates: PPAR-γ expression. 3T3-L1 preadipocytes were cultured in differentiation medium (MDI) containing p-phthalates (10 nM and 10 µM) and ICI 10 nM for 10 days. Cells treated with MDI alone were used as controls. Undifferentiated cells were cultured in standard medium for 10 days. The densitometry results are reported as fold change compared to MDI. The intensity values of the PPAR-γ were normalized to the corresponding value of β-actin. All data are expressed as mean ± SD of three independent experiments. a p < 0.05 vs. undifferentiated cells; b p < 0.05 vs. MDI; c p < 0.05 vs. DMT 10 nM; d p < 0.05 vs. DMT 10 µM; g p < 0.05 vs. TPA 10 nM; h p < 0.05 vs. TPA 10 µM.

p-Phthalates Reduce the Thermogenic Pathway
Recent insights into adipocyte function recognized the potential to contribute to energy turnover through thermogenic brown adipose tissue (BAT), which is specialized in dissipated energy compared to white adipose tissue (WAT), which accumulates it as triglycerides. However, WAT is also able to express a "brown-like" phenotype promoting mitochondrial biogenesis modulated by the AMP-activated protein kinase (AMPK) pathway, a crucial energy sensor that regulates energy metabolism by activating PGC-1α [37]. Some evidence showed that obesogens are associated with the disruption of thermogenic Figure 5. Effect of the ER antagonist ICI on the obesogenic effect of p-phthalates: PPAR-γ expression. 3T3-L1 preadipocytes were cultured in differentiation medium (MDI) containing p-phthalates (10 nM and 10 µM) and ICI 10 nM for 10 days. Cells treated with MDI alone were used as controls. Undifferentiated cells were cultured in standard medium for 10 days. The densitometry results are reported as fold change compared to MDI. The intensity values of the PPAR-γ were normalized to the corresponding value of β-actin. All data are expressed as mean ± SD of three independent experiments. a p < 0.05 vs. undifferentiated cells; b p < 0.05 vs. MDI; c p < 0.05 vs. DMT 10 nM; d p < 0.05 vs. DMT 10 µM; g p < 0.05 vs. TPA 10 nM; h p < 0.05 vs. TPA 10 µM.

p-Phthalates Reduce the Thermogenic Pathway
Recent insights into adipocyte function recognized the potential to contribute to energy turnover through thermogenic brown adipose tissue (BAT), which is specialized in dissipated energy compared to white adipose tissue (WAT), which accumulates it as triglycerides. However, WAT is also able to express a "brown-like" phenotype promoting mitochondrial biogenesis modulated by the AMP-activated protein kinase (AMPK) pathway, a crucial energy sensor that regulates energy metabolism by activating PGC-1α [37]. Some evidence showed that obesogens are associated with the disruption of thermogenic fat and adipose tissue, and these effects are related to the altered AMPK pathway [20,37]. Reduced thermogenesis is associated with lower energy expenditure, higher adiposity, and increased risk of insulin resistance [38].
To confirm and further extend these findings, we examined the effects of p-phthalates exposure on thermogenesis during adipogenesis differentiation. The results showed that, in our experimental conditions, DMT and TPA reduced AMPK levels in a dose-dependent way compared to unexposed differentiated control cells ( Figure 6A). Interestingly, these effects were also evident at nanomolar concentrations, with DMT being more effective than TPA in AMPK inhibition.
terestingly, these effects were also evident at nanomolar concentrations, with DMT being more effective than TPA in AMPK inhibition.
Thermogenesis activated by AMPK relies on PGC-1α, a cAMP-dependent protein that promotes mitochondrial biogenesis and oxidative metabolism [39]. PGC-1α coactivates different nuclear receptors involved in the transcriptional induction of markers of thermogenesis such as UCP-1 [40]. Data confirmed lower PGC-1α mRNA levels in DMTand TPA-treated cells with a dose-dependent effect, corroborating the impaired thermogenesis pathway ( Figure 6B).

p-Phthalates Induce the NF-κB Proinflammatory Pathway
Excessive storage of fatty acids in adipose tissue produces hypertrophic and dysfunctional adipocytes, thereby secreting several proinflammatory adipokines such as IL-6, IL-8, IFN-γ, and TNF-α and inducing a state of chronic low-grade inflammation [41]. In particular, nuclear factor (NF)-κB, the main transcriptional factor involved in inflammatory processes [42], plays a central role in inflammation-mediated metabolic disorders. Therefore, to evaluate the effects of p-phthalates on adipocytes' inflammation, the NF-κB pathway was studied. Data showed that p-phthalates induce NF-κB p65 nuclear translocation in a dose-dependent way ( Figure 7A). Interestingly, low nanomolar concentrations were already able to activate the NF-κB pathway, and this effect was more evident for DMT. Thermogenesis activated by AMPK relies on PGC-1α, a cAMP-dependent protein that promotes mitochondrial biogenesis and oxidative metabolism [39]. PGC-1α coactivates different nuclear receptors involved in the transcriptional induction of markers of thermogenesis such as UCP-1 [40]. Data confirmed lower PGC-1α mRNA levels in DMT-and TPA-treated cells with a dose-dependent effect, corroborating the impaired thermogenesis pathway ( Figure 6B).

p-Phthalates Induce the NF-κB Proinflammatory Pathway
Excessive storage of fatty acids in adipose tissue produces hypertrophic and dysfunctional adipocytes, thereby secreting several proinflammatory adipokines such as IL-6, IL-8, IFN-γ, and TNF-α and inducing a state of chronic low-grade inflammation [41]. In particular, nuclear factor (NF)-κB, the main transcriptional factor involved in inflammatory processes [42], plays a central role in inflammation-mediated metabolic disorders. Therefore, to evaluate the effects of p-phthalates on adipocytes' inflammation, the NF-κB pathway was studied. Data showed that p-phthalates induce NF-κB p65 nuclear translocation in a dose-dependent way ( Figure 7A). Interestingly, low nanomolar concentrations were already able to activate the NF-κB pathway, and this effect was more evident for DMT.
The canonical pathway of NF-κB is triggered through the enzymatic activity of an IκB kinase (IKK), which induces the disengagement of the IκB inhibitor from NF-κB, allowing its translocation to the cell nucleus. p-Phthalates dose-dependently induced pIKK levels, confirming that these compounds modulate the NF-κB pathway, improving the phosphorylation of the IκB inhibitor by IKK. Additionally, in this case, the effect was more prominent for DMT compared to TPA ( Figure 7B). Additionally, the transcriptional activity of the NF-κB pathway was studied evaluating IL-6 gene expression, an NF-κBregulated cytokine connected to the activation of chronic inflammatory pathways in obese people [43]. Results showed that DMT and TPA induce IL-6 gene expression at low nanomolar concentrations, and this effect was dose-dependent ( Figure 7C). Moreover, in this case, DMT 10 µM was able to induce NF-κB transcriptional activity better than TPA (10 µM).
The canonical pathway of NF-κB is triggered through the enzymatic activity of an IκB kinase (IKK), which induces the disengagement of the IκB inhibitor from NF-κB, allowing its translocation to the cell nucleus. p-Phthalates dose-dependently induced pIKK levels, confirming that these compounds modulate the NF-κB pathway, improving the phosphorylation of the IκB inhibitor by IKK. Additionally, in this case, the effect was more prominent for DMT compared to TPA ( Figure 7B). Additionally, the transcriptional activity of the NF-κB pathway was studied evaluating IL-6 gene expression, an NF-κB-regulated cytokine connected to the activation of chronic inflammatory pathways in obese people [43]. Results showed that DMT and TPA induce IL-6 gene expression at low nanomolar concentrations, and this effect was dose-dependent ( Figure 7C). Moreover, in this case, DMT 10 µM was able to induce NF-κB transcriptional activity better than TPA (10 µM).

Discussion
PET is the most common plastic for thermoforming packaging because of its low cost, physical and chemical tolerance, and easy recycling procedure. PET is produced starting with the esterification of TPA or transesterification of DMT with ethylene glycol to produce monomers, followed by polycondensation in the presence of a catalyst. However, public health concerns associated with the potential leaching of substances from PET packaging have been raised due to their role as endocrine-disrupting obesogens. Phthalates can be released from plastic products into the environment during their manufacture, storage, use, or disposal, inducing exposure and accumulation in humans. In particular, the main source of human exposure is the consumption of food that has been in contact with containers and products containing phthalates or through other sources contaminated by PET wastes [23]. Phthalates have been demonstrated to induce negative effects on human health and are suspected carcinogens, as well as toxic to the liver, kidneys, and reproductive organs [44]. Additionally, phthalates have been considered potential endocrine-disrupting substances since they are able to interfere with hormone receptors, disrupting the homeostasis system in the body and interfering with regulatory processes in metabolism and in the control of adipocyte function [45].
Ortho-phthalates, generally known as phthalates, are one of the three isomeric forms of benzenedicarboxylic acid and are considered the most biologically active phthalates. There is a general lack of information in the scientific literature on p-phthalates (or terephthalates), with the exception of DEHT, generally supporting a different and lower mammalian toxicity profile compared to other isomers [46,47]. In vitro nanomolar TPA exposure induced cellular perturbations evident as the induction of DNA damage, cell cycle arrest, and subsequent escape from programmed cell death in non-malignant human high-risk donor breast epithelial cells (HRBECs) isolated from several donors [48]. Additionally, DMT inhibited in vitro WiL2-NS human B cell proliferation, likely by enhancing ROS production [49].
In our study, due to the similar structure between phthalate isomers, we hypothesized that p-phthalates can affect fat metabolism and adipogenesis at environmentally relevant low concentrations. With this aim, we evaluated adipogenic activity in differentiation experiments with murine preadipocyte 3T3-L1 cells.
In our experimental model, cells were exposed to TPA and DMT at levels mimicking those that might be obtained in adult humans with the consumption of foods contained in PET packages, similar to other findings reported in the literature [50,51] or environmentally relevant concentrations found in the blood of human subjects [25,26]. One has to take into consideration that intestinal absorption of 100% of the phthalates after oral ingestion may be hypothesized; as said before, the in silico ADME analysis showed intestinal absorption of p-phthalates ranging between 75% (TPA) and 90% (DMT) ( Table 1).
Our data showed that DMT and TPA exposure during 3T3-L1 differentiation dosedependently increased the lipid content compared to unexposed differentiated cells. Although we observed a significative effect starting from 10 nM, DMT 10 µM induced the maximal lipid droplet formation, similar to that of the positive control rosiglitazone. Furthermore, among the p-phthalates tested, DMT was more effective than TPA. Since DMT shows better lipophilic features than those of TPA, this could explain, at least partially, why the effects of DMT were more marked than that induced by TPA. In fact, one hypothesis is that DMT could easily be transported across lipidic membranes and reach intracellular targets. The molecular mechanisms controlling adipogenesis in the 3T3-L1 cells involve the expression of key adipogenic transcription factors. In particular, the expression of the transcription factor PPAR-γ is required for adipocyte formation and maturation, upregulating the expression of proteins that play a role in lipid metabolism, such as FABP4 and FASN. The cytoplasmic protein FABP4 binds long-chain fatty acids and other hydrophobic ligands and is involved in fatty acid uptake, transport, and metabolism. FASN is critical in the regulation of body weight since it is required for de novo synthesis of long-chain saturated fatty acids from acetyl coenzyme A (CoA), malonyl-CoA, and NADPH [52]. At the molecular level, DMT and TPA induced PPAR-γ in a dose-dependent way, confirming the effects of p-phthalates in the adipogenic process. To the same extent, p-phthalates activated PPAR-γ transcriptional machinery inducing FABP4 and FASN gene expression. However, p-phthalates increased C/EBPβ expression, a transcription factor acting during the induction of 3T3-L1 differentiation. Since C/EBPβ is required for binding to DNA adipogenic hotspots of other adipogenic transcription factors and is an inducer of PPAR-γ transcription, we can speculate that the effects of p-phthalates are not due to direct PPAR-γ activation but rather to the modulation of early events in preadipocytes' differentiation. This mechanism was also proposed for the ortho-phthalate DEHP, since relative mRNA and protein levels of CEBP/β in adipose tissue of rats exposed to DEHP were significantly higher than in the control group [53]. Interestingly, the adipogenic action of TPA-and DMT-induced PPAR-γ was reverted by ICI, a specific antagonist of the ER, suggesting that p-phthalates likely induce adipocyte differentiation through an ER-mediated mechanism. Phthalates, in fact, have been demonstrated to induce estrogenic effects [54,55] modulating ERα or ERß activities [56], even if few data are reported for these compounds. Additionally, adipogenesis induced by bisphenol A (BPA), a well-known endocrine disruptor, was reverted by ICI in 3T3-L1 adipocytes, although the involvement of other non-classical ER pathways was suggested [57]. However, our data, although preliminary, need further confirmation using specific in vitro and cell-based assays able to study direct or indirect ER targeting [58].
As part of the obesity phenotype, dysfunctional adipocytes show increased stores of energy such as triglycerides in unilocular lipid droplets, instead of dissipating it as heat through the activation of the thermogenic program. In particular, AMPK plays an important role in WATm inhibiting fatty acid synthesis and promoting fatty acid oxidation via thermogenic processes inducing a "brown-like" phenotype [37]. Our data demonstrated the dose-dependent inhibitory effects of DMT and TPA on AMPK phosphorylation at all the concentrations used. Interestingly, lower nanomolar concentrations were already able to significantly reduce basal pAMPK levels. The effects on the negative imbalance of AMPK exerted by p-phthalates were confirmed by the inhibition of PGC-1α, a cAMP-dependent protein able to induce mitochondrial biogenesis and dissipate lipids to generate heat, and these results support the hypothesis of a reduced "brown-like" phenotype in 3T3-L1. Our data find support from the literature since dibutyl phthalate inhibits the phosphorylation of AMPK in the liver, significantly promotes liver enlargement, and increases triglyceride and cholesterol levels in Sprague-Dawley rats [59]. Furthermore, DEHP reduced the AMPK downstream phosphorylation of ACC2 in human SGBS-adipocytes, the key enzyme of lipogenesis, improving fatty acid beta-oxidation [60]. Additionally, obesogen BPA reduced pAMPK and its downstream enzyme pACC levels in 3T3-L1 cells [61].
Obesity is characterized by a state of chronic inflammation in adipose tissue mediated by the secretion of a range of inflammatory cytokines. There is increasing evidence that inflammation affects the thermogenic activity of adipose tissue by impairing its capacity for energy expenditure, contributing to adipocytes' dysfunction in obesity [62]. Herein, we observed that DMT and TPA dose-dependently induced NF-κB pathway activation at low nanomolar levels with slight differences between the two compounds at 10 µM. The promotion of an inflammatory state in adipocytes has also been demonstrated for other phthalates and obesogens at nanomolar concentrations [63]. DEHP exposure was also shown to induce rat adipose tissue infiltration with macrophages with the subsequent secretion of TNF-α and IL-1b, which promoted adipose tissue dysfunction and altered lipid metabolism [53]. In agreement, in utero DEHP exposure in Sprague-Dawley rats induced both adipose tissue and systemic inflammation in parallel with increasing preadipocyte differentiation [64]. It is also proposed that PPAR-γ may be at least partially responsible for the proinflammatory response in differentiated murine adipocytes in parallel with the adipogenic effects [63].
The drug-likeness properties were screened using the online tool Molinspiration (https://www.molinspiration.com/cgi-bin/properties accessed on 5 April 2022) based on the Lipinski Rules of five [66]. Molinspiration supports the calculation of important molecular properties such as Log P, polar surface area, and the number of hydrogen bond donors and acceptors. The calculation of Log P is based on a formula considering the lipophilicity, hydrophobicity, and polarity of the compound [67].
The Rule of Five (or Lipinski's rule), stated for compounds that are not substrates for active transporters, is able to predict the absorption or permeation of a potential drug candidate by combining specific parameters [68]. According to this, poor oral bioavailability is more likely when there are more than 5 H-bond donors, 10 H-bond acceptors, the molecular weight is greater than 500, and the calculated Log P is greater than 5. In general, an orally active drug has no more than one violation of these criteria.

Cell Culture and Treatments
The 3T3-L1 murine preadipocyte cells, obtained from the American Tissue Culture Collection (Manassas, VA, USA), were grown in Dulbecco's modified essential medium (DMEM) supplemented with 10% newborn calf serum, 4 mM L-glutamine, 100 U/mL penicillin/streptomycin solution, and 25-mM HEPES buffer. Cells were maintained at 37 • C in an incubator with a humidified atmosphere containing 95% air and 5% CO 2 . To prepare 3T3-L1 monolayers, cells were plated at 1.3 × 10 4 cells/cm 2 in multiwell plates and cultured for 10 days after confluence in a differentiation medium to obtain totally differentiated cells, morphologically similar to white adipocytes. In particular, the cells were incubated for 4 days with DMEM supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 100 U/mL penicillin/streptomycin solution, and 25-mM HEPES buffer, containing prodifferentiating agents (MDI: 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, and 1 µg/mL insulin). The cells were then maintained in DMEM supplemented with fetal bovine serum (see above) containing only 1 µg/mL insulin for the following 6 days, up to the total differentiation in mature adipocytes. In this phase, the medium was replicated every 3 days. The cells were always used within the 20th passage because differentiation efficiency declines rapidly with higher passage numbers.
For all the experiments, 3T3-L1 preadipocytes were treated, throughout all 10 days of the differentiation process described above, with DMT and TPA at two different concentrations (10 nM and 10 µM). The tested p-phthalates were always freshly dissolved in DMSO and immediately used. Rosiglitazone (10 nM) was used as the positive control. The final concentration of DMSO in the culture medium during the different treatments was ≤0.1% (v/v), while the cells treated with the vehicle (DMSO 0.1% v/v) and then exposed only to the differentiation inducers were used as controls. At the end of the exposure time, cells were immediately processed and/or preserved at −80 • C until the analysis required for each test.
For the ER antagonist studies, 10 nM ICI-182,780 (ICI) was added during all differentiation periods in the presence or not of DMT and TPA (10 nM and 10 µM).
For the toxicity test, the murine fibroblast cell line NIH/3T3 (ATCC, Rockville, MD, USA) was employed. Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 4 mM L-glutamine, and a 100 U/mL penicillin/streptomycin solution, and maintained in an incubator with a humidified atmosphere containing 5% CO 2 at 37 • C. In the experiments, cells were plated in 24-well cell plates at an initial density of 9 × 10 4 cells/well, and after 24 h, semi-confluent monolayers were treated for 48 h with DMT and TPA (10 nM and 10 µM) added to the cell culture medium. Sodium dodecyl sulfate (SDS) was used as a positive control at the historical mean concentration established by the laboratory (0.085 mg/mL). The cells treated with the vehicle alone (DMSO 0.1% v/v) were used as controls.

Sulforhodamine B Assay
The cytotoxic effect of TPA and DMT on NIH/3T3 cells was evaluated using the sulforhodamine B assay, based on the measurement of cellular protein content according to Anwar et al. [69]. In detail, following treatment, the cells were fixed for 1 h at 4 • C using 10% trichloroacetic acid (w/v). After this step, cells were washed twice with distilled water and incubated for 30 min at room temperature with sulforhodamine B (0.4% w/v in 1% acetic acid). Then the excess dye was removed by washing repeatedly with 1% (v/v) acetic acid, and the dye trapped in the cells was solubilized in a 10 mM Tris base solution. Therefore, the absorbance was measured at 565 nm using a microplate reader.

Cell Lysates Preparation
Following the appropriate treatments, 3T3-L1 cells were washed with DPBS and harvested with a scraper. For total and nuclear lysates' preparation, the cells were resuspended in a lysis buffer containing protease inhibitors and dithiothreitol (DTT) as previously described [70].
After centrifugation, the protein fraction was stored at −20 • C until use. The protein concentration in lysates was assessed using the Bradford assay [71].
Membranes were then incubated overnight with specific primary antibodies:  [75]), and 18S rRNA (FW 5 -GTAACCCGTTGAACCCCATT-3 , RV 5 -CCATCCAATCG GTAGTAGCG-3 [73]) was used as reference gene. For IL6 and 18S amplification, the following parameters were applied: 40 cycles of 94 • C denaturation (15 s), 60 • C annealing, and extension (60 s). FABP4 and FASN were instead amplified using 40 cycles of 95 • C denaturation (45 s), 60 • C annealing, and extension (60 s). Data were elaborated by SDS 1.3.1 software (Applied Biosystems, Foster City, CA, USA) and expressed as the threshold cycle (Ct). The fold increase in mRNA expression compared with the control cells not pre-treated and not exposed to PA was determined using the 2 −∆∆Ct method [76].

Oil Red O Staining
The phthalates' effect on lipid accumulation was assessed by Oil Red O staining according to the method described by Molonia et al. [77]. After staining, the adipogenic cultures were observed by an optical microscope and photographed. The degree of lipid droplets was determined with red pixel areas indicating fat vacuoles being divided by the total scanned area. The staining of the multiwell plate sections was quantified by ImageJ software [78] and results were expressed as the fold change against control.

Statistical Analysis
All the experiments were carried out in triplicate and repeated three times. Results are expressed as mean ± SD from three experiments and statistically analyzed by a one-way ANOVA test, followed by Tukey's HSD, using the statistical software ezANOVA (https: //people.cas.sc.edu/rorden/ezanova/index.html accessed on 5 April 2022). Differences in groups and treatments were considered significant for p < 0.05.

Conclusions
Lacking data on the potential adipogenic effects of p-phthalates, especially at environmentally relevant very low concentrations, produces a high level of uncertainty regarding their suitability as safe compounds. Given the observed effects of nanomolar concentrations of the p-phthalates studied and taking into account humans' close and constant contact with plastics, it seems appropriate that ascertaining safe levels of TPA and DMT exposure is considered a high priority.