Di-(2-ethylhexyl) phthalate (DEHP) is the world's most widely used polyvinyl chloride (PVC) plasticizer and is used in virtually every category of flexible PVC. In fact, DEHP is extensively used in food cosmetics and medical packaging. It has become a serious problem in recent years. DEHP can be absorbed into the human body through the air, food, water, and skin. The current study involved intraperitoneal injection of DEHP dissolved in corn oil once daily for 21 consecutive days to investigate the effects of DEHP on the thyroid and the reproductive system in female rats. Results show that ovarian hormones (progesterone and estrogen) decreased significantly in the rats treated with DEHP compared to control. This result is supported by the alteration of folliculogenesis, the decrease of the follicles viability, and the apoptosis of the granulosa cells observed on histological sections of ovary and thyroid in female rats exposed to low doses of DEHP. Histopathological study revealed that DEHP could damage thyroid tissue and disrupt these functions. We also observed cellular damage, particularly in the liver cells, and a significant increase in biochemical parameters such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) compared to the control group.

  • Occurrence of contaminants di-(2-ethylhexyl) phthalate (DEHP) is an emerging concern.

  • DHEP can disrupt reproductive function, thyroid, and hepatic synthesis.

  • In vivo toxicity of DEHP.

  • This study investigated the effects of exposure to DEHP on the thyroid and the reproductive system in female rats.

  • Environmental pollution with phthalates.

Graphical Abstract

Graphical Abstract
Graphical Abstract

Increased industrial and agricultural developments have resulted in an increase in the release of pollutants into the environment, posing a major threat to human health and the ecosystem. Endocrine-disruptors (EDs) are chemical contaminants that are classed as possibly endocrine disruptive compounds (EDCs) (Beltifa et al. 2017). ED has many negative effects on reproduction such as the reduction in fertility, spontaneous abortions, biased sex ratio, abnormalities of the male and female genital tracts, precocious puberty, polycystic ovary syndromes, and behavioral disorders (McKinlay et al. 2008). Among these EDs, phthalates, commonly used as plasticizers, induce sperm lesions (Beltifa et al. 2018a, 2018b; Di Bella et al. 2018; Beltifa et al. 2019; Chebbi et al. 2022), early puberty in women (Wolff et al. 2010; Beltifa et al. 2019), reproductive system anomalies and false pregnancies (Whyatt et al. 2009; Desdoits-Lethimonier et al. 2012), neurological development disorders (Engel et al. 2010), allergies (Bornehag et al. 2004), and disturbed thyroid function (Lyn 2009). Phthalates are biodegradable organic pollutants that can remain longer in some environments, such as the aquatic environment, where they combine with sediments, delaying their breakdown in an aerobic environment and accumulating in adipose tissue (Gugliandolo et al. 2020; Jebara et al. 2021). Di-2-ethylhexyl phthalate (DEHP) is the most common member of the class of phthalates, which are used as plasticizers, cosmetics, medicines, dispersants, emulsifying agents, and kitchen plastic ware (Kimber & Dearman 2010; Beltifa et al. 2017, 2018a, 2018b, 2019, 2020). An aslipophilic compound, DEHP is not chemically combined with polyvinyl chloride (PVC) and it can be easily released from plastic (Herreros et al. 2010). Many studies showed the presence of the DEHP at high concentrations in different environmental and food matrices (Beltifa et al. 2018a, 2018b, 2021; Jebara et al. 2021). A recent study, conducted by Beltifa et al. (2019), indicates the presence of phthalates, especially DEHP, in urine samples collected from Tunisian men and women. Given the increasing evidence about the adverse health and environmental effects of phthalate exposure, we aimed to see how DEHP contamination affects health. In the present work, we conducted the in vivo toxicities using female rats, which were exposed to various doses of DEHP. The focus is to detect variations in biochemical biomarkers and histopathological changes in the ovary and thyroid of female rats exposed to low doses of DEHP intraperitoneally for 21 days.

Animal

Sixteen female rats (2 months old, 180–240 g) were purchased from Pasteur Institute, Tunisia. After 1 week of acclimatization, all rats were divided into four groups C, T1, T2, and T4 (n = 4/group) and hosted in a plastic cage under controlled temperature (22 ± 2 °C), 12 h light/dark cycle, and 70% relative humidity with free access to standard laboratory food (SNA, Sfax), water, and libitum.

  • Animals of the group (C) received the maize oil (control group).

  • Animals of group T1 received a dose of 0.10 mg/kg b.w. of DEHP dissolved in maize oil.

  • Animals of group T2 received a dose of 1.5 mg/kg b.w. of DEHP dissolved in maize oil.

  • Animals of group T3 received a dose of 27.25 mg/kg b.w. of DEHP dissolved in maize oil.

All rats were administered DEHP by intraperitoneal injection daily for 21 days.

Blood and organ collection

After exposure, the female rats were sacrificed by cervical decapitation under light ether anesthesia. The tube of blood collected may contain an anticoagulant in order to inhibit its coagulation. After centrifugation at 3,500 rpm for 15 min at 4 °C, the collected serum was stored at −20 °C until biochemical analysis.

Thyroid and ovarian organs were quickly excised, cleared and weighed, and immediately fixed in 10% formalin solution for histopathological examination.

Animals were treated according to its guidelines and to Medical Ethics Committee for the Care and Use of Laboratory Animals of the Pasteur Institute of Tunis, Tunisia (approval number: FST/LNFP/Pro 152012). We tried our best to minimize the animal's pain and the number of rats.

Determination of biochemical parameters

The concentration of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), alkaline phosphatase (ALP), and total bilirubin (TBIL) were determined in serum automatically using Cobas Kits for Roche/Hitachi Cobas c 501 systems autoanalyzer (Roche Diagnostics, Mannheim, Allemagne).

Determination of hormones concentrations

Estradiol and progesterone serum were measured by the Cobas® 6000 rock diagnoses 601 and Tosoh AIA-360 kits, respectively, following the manufacturer's instructions. The assay was carried out in the biochemistry laboratory CHU Fattouma Bourguibain Monastir.

Histopathological examination

Portions of tissue obtained from ovaries and thyroids were examined using a circulation machine and standard laboratory procedures. The ovaries and thyroid embedded in paraffin blocks were divided into 2-μm slices and stained with hematoxylin–eosin (H&E), the most basic of the combination colorings. The tissues reacted with hematoxylin for 10 min and eosin for 2–3 min (Hould 1984). Finally, the samples were analyzed by assessing the morphological changes under a light microscope.

Effect of DEHP treatment on sex hormone levels

We have observed a reduction in estrogen and progesterone levels with DEHP treatment (Figure 1). However, estrogen levels significantly reduced P < 0.05 (Table 1) in a dose–response dependent manner compared to controls if the mice were treated with 27, 25 mg/kg/day of DEHP (Figure 1(a)). Furthermore, Figure 1 shows a significant reduction of progesterone levels in rats treated with T1 = 0.10 mg/kg b.w. of DEHP (48.07 ± 66.05 μg/ml) compared to control. This concentration decreased with T2 and T3 (Figure 1(b), Table, P < 0.05). These parameters decrease in a dose-dependent manner of compounds tested.
Table 1

Result of analysis of variance (ANOVA) of hormonal dosages according to the injected dose using the Turkey's Post Hoc Test

dfSSMSFpTurkey's Post Hoc Test
Estrogen 
Treatment 304.88 101.625 4.135 0.031 C > T3 
Residuals 12 294.87 24.573    
Total 15 599.75     
Progesterone 
Treatment 210.91 70.303 5.729 0.011 C = T1 > T2 = T3 
Residuals 12 147.25 12.271    
Total 15 358.16     
TC  
Treatment 2.38 0.793 4.570 0.023 C = T1 = T2 > T3 
Residuals 12 2.08 0.173    
Total 15 4.47     
TG 
Treatment 2.32 0.773 1.290 0.322 n.s. 
Residuals 12 7.19 0.599    
Total 15 9.52     
TBIL  
Treatment 0.19 0.062 0.200 0.894 n.s. 
Residuals 12 3.75 0.312    
Total 15 3.94     
ALP  
Treatment 4652.19 1550.7 0.457 0.716 n.s. 
Residuals 12 40645.75 3387.1    
Total 15 45297.94     
ASAT 
Treatment 603807 201269 4.614 0.022 T3 > C = T1 = T2 
Residuals 12 523440 43620    
Total 15 1127247     
ALAT 
Treatment 449474 149824.5 3.108 0.036 T3 > C = T1 = T2 
Residuals 12 578470 48205.8    
Total 15 1027943     
dfSSMSFpTurkey's Post Hoc Test
Estrogen 
Treatment 304.88 101.625 4.135 0.031 C > T3 
Residuals 12 294.87 24.573    
Total 15 599.75     
Progesterone 
Treatment 210.91 70.303 5.729 0.011 C = T1 > T2 = T3 
Residuals 12 147.25 12.271    
Total 15 358.16     
TC  
Treatment 2.38 0.793 4.570 0.023 C = T1 = T2 > T3 
Residuals 12 2.08 0.173    
Total 15 4.47     
TG 
Treatment 2.32 0.773 1.290 0.322 n.s. 
Residuals 12 7.19 0.599    
Total 15 9.52     
TBIL  
Treatment 0.19 0.062 0.200 0.894 n.s. 
Residuals 12 3.75 0.312    
Total 15 3.94     
ALP  
Treatment 4652.19 1550.7 0.457 0.716 n.s. 
Residuals 12 40645.75 3387.1    
Total 15 45297.94     
ASAT 
Treatment 603807 201269 4.614 0.022 T3 > C = T1 = T2 
Residuals 12 523440 43620    
Total 15 1127247     
ALAT 
Treatment 449474 149824.5 3.108 0.036 T3 > C = T1 = T2 
Residuals 12 578470 48205.8    
Total 15 1027943     

C, control; T1, low dose (0.10 mg/kg/day); T2, medium dose (1.5 mg/kg/day); T3, high dose (27.25 mg/kg/day); p, p-value; F, F-test; df, degrees of freedom; SS, sums of squares; MS, mean square.

Figure 1

Average concentration (mean ± sd) of sex hormone: (a) estrogen and (b) progesterone. C: control, T1: low dose (0.10 mg/kg/day), T2: medium dose (1.5 mg/kg/day), T3: high dose (27.25mg/kg/day).

Figure 1

Average concentration (mean ± sd) of sex hormone: (a) estrogen and (b) progesterone. C: control, T1: low dose (0.10 mg/kg/day), T2: medium dose (1.5 mg/kg/day), T3: high dose (27.25mg/kg/day).

Close modal

Effect of phthalate DEHP on lipid and hepatic balance

Figure 2(a) and 2(b) shows that AST ALT activities induced significantly increased (p < 0.01) compared to the negative control. However, the AST and ALT concentration increases significantly in a dose-dependent manner if the mice are treated with a higher dose of 27.25mg/kg/day of DEHP compared to the control group (p < 0.01).
Figure 2

Measurement of (a) AST and (b) ALT following an injection of increasing doses of DEHP. C: control, T1: low dose (0.10 mg/kg/day), T2: medium dose (1.5 mg/kg/day), T3: high dose (27.25mg/kg/day).

Figure 2

Measurement of (a) AST and (b) ALT following an injection of increasing doses of DEHP. C: control, T1: low dose (0.10 mg/kg/day), T2: medium dose (1.5 mg/kg/day), T3: high dose (27.25mg/kg/day).

Close modal
Furthermore, serum ALP levels, TBIL, and TG did not vary between the control group and the treated group (p > 0.05: Table 1) which reveals that the injection of DEHP did not influence this parameter. In fact, these biochemical parameters reach 142 ± 125.86IU/l (Figure 3(a)), 1.56 ± 0.12 μmol/l (Figure 3(b)), 2.57 ± 0.69mmol/l (Figure 3(c)), respectively. On the other hand, cholesterol levels were increased at a dose of 0.10mg/kg/day of DEHP (2.12 ± 0.2343 and 3.01 ± 0.43mmol/l) and decreased at a dose of 27.25mg/kg/day (Figure 3(d)), and varied significantly from one group to another (Table 1).
Figure 3

Lipid balance following the injection of increasing dose of DEHP: (a) ALP, (b) TBIL, (c) TG, and (d) TC. C: control, T1: low dose(0.10 mg/kg/day), T2: medium dose (1.5 mg/kg/day), T3: high dose (27.25mg/kg/day).

Figure 3

Lipid balance following the injection of increasing dose of DEHP: (a) ALP, (b) TBIL, (c) TG, and (d) TC. C: control, T1: low dose(0.10 mg/kg/day), T2: medium dose (1.5 mg/kg/day), T3: high dose (27.25mg/kg/day).

Close modal

Histopathological changes in the ovaries following exposure to DEHP

Histological sections of the control group ovaries are shown in Figure 4. Ovary sections from the control group of rats revealed normal follicular structure: primary, secondary, tertiary follicles, mature follicles, and corpus luteum follicles (Figure 4(a)–4(e)).
Figure 4

Normal histological structure stained with hematoxylin–eosin of the ovary (G 400×) in control female rats injected with corn oil. a: primary follicles; b: secondary follicles; c: tertiary follicles; d: mature follicle; e: yellow body.

Figure 4

Normal histological structure stained with hematoxylin–eosin of the ovary (G 400×) in control female rats injected with corn oil. a: primary follicles; b: secondary follicles; c: tertiary follicles; d: mature follicle; e: yellow body.

Close modal
The treatment with DEHP resulted in significant lesions such as multiple subcapsular cysts, atretic follicles, vascular proliferation, cystic follicles, para ovarian cyst, cell degeneration, thin granulosa layer, and missing cystic follicle syndrome when compared to the control group (Figure 5). The absence of the corpus luteum indicates anovulation. Furthermore, cellular infiltration is accompanied by several atretic follicles with an anus filled with fluid.
Figure 5

Histopathological changes in the ovaries following exposure to an increasing dose of DEHP (T1 = 0.1 mg/kg/day; T2 = 1.5 mg/kg/day and T3 = 27.25 mg/kg/day). T1-a: subcapsular cysts; T1-b: Atretic follicles; T1-c: vascular proliferation; T1-d: cystic follicles; T1-e: subcapsular cysts; T1-f: cellular degeneration and angiectasia; T2-g: atretic follicles; T2-h: vascular proliferation; T2-i: follicular cyst; T2-j: Subcapsular cysts; T3-k: cellular degeneration; T3-l: subcapsular cysts; T3-m: paraovarian cyst; T3-n: cellular infiltration; T3-o: anovulation.

Figure 5

Histopathological changes in the ovaries following exposure to an increasing dose of DEHP (T1 = 0.1 mg/kg/day; T2 = 1.5 mg/kg/day and T3 = 27.25 mg/kg/day). T1-a: subcapsular cysts; T1-b: Atretic follicles; T1-c: vascular proliferation; T1-d: cystic follicles; T1-e: subcapsular cysts; T1-f: cellular degeneration and angiectasia; T2-g: atretic follicles; T2-h: vascular proliferation; T2-i: follicular cyst; T2-j: Subcapsular cysts; T3-k: cellular degeneration; T3-l: subcapsular cysts; T3-m: paraovarian cyst; T3-n: cellular infiltration; T3-o: anovulation.

Close modal

Histopathological changes in the thyroid organs following exposure to DEHP

The photomicrographs of the thyroid from the control group showed that normal follicles with epithelium of unistratified follicular cells organized around a central lumen containing the colloid (Figure 6). However, the treatment with DHEP induces many modifications and perturbations as shown by vacuolation of the epithelium of the follicle mucosa, hyperemia between distended follicles, and some involute follicles with tiny colloid and cystic follicular development (Figure 6).
Figure 6

Histological structure of the thyroid of control and treated rats with DEHP at 100× magnification. T: control; T1-A1: distended follicle; T1-A2: involuted follicle; T1-B: vacuolation; T1-C: cystic follicle.

Figure 6

Histological structure of the thyroid of control and treated rats with DEHP at 100× magnification. T: control; T1-A1: distended follicle; T1-A2: involuted follicle; T1-B: vacuolation; T1-C: cystic follicle.

Close modal

In the present study, administration of DEHP induced a significant increase in the ALT and AST levels in treated rats with the higher dose. This increase is an indicator of liver toxicity induced by DEHP. Both Ghorpade et al. (2002) and Chatterjee (2017) have shown that DEHP toxicity leads to enhanced AST and ALT activity, which is indicative of high protein turnover and amino acid metabolism and may stimulate tissue repair. The same results were found by Giboney (2005) showing that the level of ALT in the blood is the most precise indicator for liver disease. DEHP can cause serious damage to the liver structure of rats and mice (cell infiltration, hepatocellular bleeding, and necrosis), as well as disrupting lipid metabolism and DNA damage as demonstrated in the previous work by Beltifa et al. (2018b).

The present work showed that the level of cholesterol in serum was lower in T3-exposed rats (27.25 mg/kg/day). Some studies such as Poon et al. (1997) and Ma et al. (2006) showed that DEHP has multiple effects on lipid metabolism in rats. A significant reduction in serum of cholesterol and a decrease in body weight in rats exposed to DEHP were reported in some studies on the toxicity of DEHP. These results are confirmed by other studies (Dalgaard et al. 2000; Ma et al. 2006). The mechanism involved in cholesterol metabolism was disturbed by DEHP, probably due to the inhibition of hepatic bile acid synthesis as hypothesized by Yu et al. (2021).

On the other hand, the administration of DEHP by intraperitonial injection in rats caused lower estrogen and progesterone levels than in control rats. These results were also found in many surveys (Gupta et al. 2010; Liu et al. 2014; Kalo et al. 2015; Tripathi et al. 2019). This decrease can be explained by the alteration of folliculogenesis and ovulation of the treated rats (Figure 5).

In the same manner, exposure to phlatate causes structural changes in follicles in the ovaries compared to a control group. Our results are consistent with previous studies showing that phthalate suppressed follicular development. This suppression was accompanied by low viability of cultured rat ovaries and may even have unleashed apoptosis of granulosa cells (Inada et al. 2012). Moreover, in this study, the degenerative changes observed in the ovaries of rats treated with DEHP are consistent with the results of Davis et al. (1994) who showed that DEHP treatment of female rats produced small preovulatory follicles accompanied by suppressed estradiol production.

We found follicular and luteinic cysts in the present study. Zhou et al. (2017) suggested that injection of phthalates cocktail's, including DEHP, increased the prevalence of cystic ovaries in mice.

Compared to control, administration of DEHP by intraperitoneal injection caused many modifications in the thyroid such as follicular degeneration and dilation, loss of follicular form, and loss of colloidal color. Previous studies by Kim et al. (2019), Zhang et al. (2022) found histological changes in the thyroid caused by phthalates.

DEHP disrupts the endocrine system by affecting the levels of thyroid hormones causing changes within the thyroid structure and autoimmune thyroid sicknesses and even thyroid tumors (Huang et al. 2007; Caldwell 2012; Liu et al. 2015; Kim et al. 2021).

This result confirms that the thyroid is vulnerable to phthalates, particularly by affecting thyroid hormone secretion, biosynthesis, and homeostasis (Boas et al. 2006; Gore et al. 2015). It seems that DEHP disrupts thyroid hormone biotransport and thereby changing follicular sensitivity to thyroid-stimulating hormone/thyroid-stimulating hormone receptor (TSH/TSHR) signaling (De Felice et al. 2004). Our results showed follicular cell hyperplasia which can be explained by the increase in TSH levels and the hyperactivity of the Hypothalamus–Pituitary–Thyroid (HPT) axis according to the hypothesis of Finch et al. (2006).

In recent years, accumulating evidence from humans and animals has indicated that the thyroid is vulnerable to the endocrine-disrupting effects of DEHP (Liu et al. 2015). Thus, we proved with this study that DEHP is toxic to the ovaries and the thyroid even at low doses. Kim et al. (2019) showed that DEHP can affect thyroid function in children, adults, and pregnant women. DEHP may also reduce thyroid hormones and thyrotopin-releasing hormone levels (Liu et al. 2015).

In conclusion, in this study, Wistar rats were treated wth DEHP for 21 days and their thyrotoxic and reproductive effects were examined. We have found that DHEP can disrupt reproductive function, thyroid, and hepatic synthesis. Thus, the presence of this compound (and other phthalates) in the marine environment and its bioconcentration in the food chain in Mahdia, Tunisia (Jebara et al. 2021) may constitute a risk to human health following the consumption of products. In addition, several fish farms use PVC to strengthen fish cages, which is a potential source of phthalates near fish at several stages of their development. We recommend limiting the use of these compounds in industry, prohibiting their release, and prohibiting their uses in the food industry and aquaculture cages.

A.J. was involved in investigation, histological analysis, histological methodology, formal analysis, writing of the original draft; A.B. was involved in histological analysis, histological methodology, formal analysis, and conceptualization; data curation, formal analysis, software, and investigation; L.M. was involved in investigation, review, and editing, G.D.B. was involved in data curation and formal analysis; H.B.M. was involved in methodology, and project administration conceptualization; writing of the original draft, project supervision.

Animals were treated according to its guidelines and to Medical Ethics Committee for the Care and Use of Laboratory Animals of the Pasteur Institute of Tunis, Tunisia (approval number: FST/LNFP/Pro 152012). Consent for publication: This manuscript hasn't contained any individual person's data in any form.

The authors have declared that no competing interests exist.

Not applicable.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.

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Author notes

The two authors contributed equally to this article.

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