INTRODUCTION

Phthalates are one of the most important synthetic chemicals used in various materials such as children’s toys, drug coatings, cosmetics and solvents to give flexibility and solubility to the materials in which they are applied (Saeidnia and Abdollahi, 2013a; Wang et al., 2012a). More than 8.1 million tons of phthalates are released in the world annually (Crinnion, 2010). Phthalates-exposure may happen through oral, dermal and inhalation routes in human (Bahadar et al., 2014). It is also demonstrated that DEHP (Bis(2-ethylhexyl) phthalate) and MEHP (Mono-(2-ethylhexyl) phthalate) are found in the environment and cause adverse health effects in humans (Yang et al., 2012). Phthalates cause multi-organ damage through a number of mechanisms. Some phthalates can cause hepatic toxicity through transactivation of Peroxisome Proliferator-Activated Receptor (PPARs). This activation could cause uncontrolled cell proliferation leading to hepatic tumorogenesis consequences by the alteration of hepatic enzymes activities (Yavasoglu et al., 2014). A recent study showed that DEHP induce necrosis and inflammation in the liver.

Fig. 1:	Phthalates induce toxicity via affecting the antioxidant enzymes activity and gene expression

Phthalate-induced oxidative stress results in decreasing the antioxidant capacity, especially in GPX (glutathione peroxidase) and GST (glutathione S-transferase). Moreover, increase in lipid peroxidation, CAT (Catalase) and SOD (Cu/Zn superoxide dismutase) activity were observed. Reactive Oxygen Species (ROS) can affect Peroxisome Proliferator (PP) leading to parenchymal cell proliferation. It is mentioned that Kupffer cells are a potential oxidant in rodent liver (Erkekoglu et al., 2014). Actually, phthalates induce toxicity not only via affecting the antioxidant enzymes activity but also through gene expression (Fig. 1).

In another study, Li et al. (2013) showed the immunotoxicity of DBP (Dibutyl Phthalate). The cytotoxicity in Peritoneal Exudate Macrophages (PEM) was observed. The PEM are used to express CD36 and CD80. Furthermore, DBP reduce phagocytosis on apoptotic cells. It also decreases cytokine production, immunogenicity and antigen presentation (Li et al., 2013).

In another study, neurotoxicity of phthalate was assessed. Impairment in thyroid homeostasis and activation of PPAR were reported as the most important mechanisms. Phthalates are able to change transcriptional activity of the sodium/iodine symporter leading to disruption of iodine uptake into the thyroid gland. It is shown that maternal hypothyroxinemia especially in a low level of free T4 is associated with belated cognitive and neuromotor development as well as reduced IQ. On the other hands, DEHP may overexpress PPAR-γ, resulting in apoptosis of undifferentiated neurons (Miodovnik et al., 2014).

The cardiotoxicity of phthalates has been also reported. A significant decrease in conduction velocity was noted 24 h after DEHP exposure. Moreover, human embryonic stem cell viability and cardiac differentiation were reduced following MEHP exposure (Posnack, 2014). In the kidney, DEHP exposure could decrease the number of nephrons in offspring at the weaning. Likewise, DEHP induce glomerulosclerosis and interstitial fibrosis in adulthood. The renin and angiotensin ll expression were decreased at birthday and enhanced at weaning (Wei et al., 2012).

The preliminary studies revealed that the reproductive system is more susceptible to phthalates rather than other organs (Martino Andrade and Chahoud, 2010). The mechanisms of reproductive toxicity have not been exactly elucidated so far. It is reported that these effects are correlated with anti-androgenic effect (Noriega et al., 2009) and activation of PPARα which can alter testosterone biosynthesis through inducing variation in the gene expression of the related enzymes. This results in down-regulation of nuclear receptors which are important in testis development (Gazouli et al., 2002; Ward et al., 1998), altering the expression and activity of antioxidant enzymes which may result in DNA damage by producing ROS (Wang et al., 2012b) and impairment in the function of Leydig and Sertoli cell (Martino Andrade and Chahoud, 2010).

In another study, Davis et al. (1994) showed that MEHP decreased the estradiol level in cultured rat granulosa cells via inhibition of aromatase activity. In the mentioned study, MEHP also decreased aromatase transcript and protein level. Literature revealed that MEHP can act through activation of PPAR-α and PPAR-γ to reduce the level of estradiol and related genes expression (Lovekamp-Swan and Davis, 2003). In the same way, activation of PPAR-γ disrupted the growth time and follicle differentiation (Lovekamp-Swan et al., 2003). Many of the toxic compounds can act through oxidative stress causing genotoxicity and various chronic diseases (Mostafalou and Abdollahi, 2012, 2013; Saeidnia and Abdollahi, 2013b).

One of the most important mechanisms for phthalates toxicity is defined regarding oxidative stress. The most common ROS are superoxide and H₂O₂ that can be converted into H₂O and O₂ by antioxidant enzymes such as SOD, GPX and CAT (Abdollahi et al., 2014). Phthalates are demonstrated to alter the expression and activity of these enzymes leading to disruption of the cell function (Wang et al., 2012a). Although there are several reports demonstrating the role of oxidative stress in this regard, the presence of a clear relationship between phthalate toxicity in some organs and oxidative stress as the main cause is still under question. Therefore, the main purpose of the present study is to systematically review the related reports and papers published in the valid and credible journals to confirm whether there is a correlation between phthalate toxicity and oxidative stress as a main mechanism of toxicity for these compounds.

APPROACH TO A SYSTEMATIC REVIEW

Electronic searches were performed on Science Finder, PubMed, Science Direct and Google Scholar. Key words included: Phthalate, oxidative stress, antioxidant enzymes, liver toxicity and reproductive toxicity. In vitro and in vivo animal and human studies have been evaluated and included in this review. Also, oxidative stress as one of the mechanisms for phthalate toxicity has been discussed. The results of those selected reports are categorized into four sections including histopathology, hormone analysis, oxidative stress parameters and gene expression.

HISTOPATHOLOGICAL ALTERATIONS

Reproductive toxicity: Recently, the reproductive toxicity of phthalate has been evaluated by Wang et al. (2012b). In that study the antral follicles isolated from CD-1 mice which were received DEHP and MEHP at doses: 1, 10 and 100 μg mL‾¹ during 96 h. The results showed that the antral follicle growth decreased using all doses of DEHP and MEHP. In another study, Erkekoglu et al. (2010) examined the influence of DEHP and MEHP on MA-10 Leydig cell in mice. Resulted data showed that about 60-80% of the cells survived at high dose of DEHP but there is no cell survival at the dose 10 μM of MEHP.

Furthermore, Zhou et al. (2010) investigated the effects of DBP (dibutyl phthalate) in rats. Animals were given DBP at the doses of 100, 250 and 500 mg/kg/day orally during two weeks. Sperm count and motility significantly decreased at 250 and 500 mg kg‾¹. Moreover, seminiferous tubules atrophy was observed in 500 mg kg‾¹. In addition, decreased testis weight was observed in several reports (Kasahara et al., 2002; Shono and Taguchi, 2014). Moreover, Botelho et al. (2009) investigated the DEHP toxicity on the reproductive system. The results revealed that the number of multinucleated gonocytes was increased.

Liver toxicity: Seo et al. (2004) assessed the effects of DEHP (Bis(2-ethylhexyl) phthalate), DBP (dibutyl phthalate) and BBP (benzyl butyl phthalate) at the doses of 50, 200 and 1000 mg kg‾¹ orally during 14 days in the rat. Hepatomegaly was observed by the DEHP administration at all doses while it happened at a dose of 1000 mg kg‾¹ for BBP and DBP. Moreover, Yavasoglu et al. (2014) investigated the effects of BCP (butyl cyclohexyl phthalate) toxicity in mouse liver. The animals received BCP orally at three doses of 100, 200 and 400 mg kg‾¹ for 20 days. The results showed congestions in vena centralis, an enlargement of the sinusoids, degeneration in hepatocytes, vacuole formations and presence of lipid droplets in hepatocytes in all groups (Yavasoglu et al., 2014). Additionally, Zhou et al. (2011) evaluated the effects of DBP at three doses of 100, 250 and 500 orally during 2 weeks. A decrease in epididymal weight, an atrophy of epididymal tubules and hyperemia of interstitial vascular were also noted.

Neurotoxicity: There are a few articles on neurotoxicity of phthalates through the oxidative stress. In a study, Tseng et al. (2013) evaluated the influence of DEHP (2 and 20 ppm), DBP (500 and 1000 ppm) and DIBP (100 and 1000 ppm) on nematode. The results showed that intracellular ROS was increased at all doses. In the recent study, Zuo et al. (2014) assessed the properties of DBP on antioxidant parameters. Mice were given DBP at doses of 0.45 and 45 mg kg‾¹ orally during 32 days. A significant increase of ROS, GSH and MDA (malondialdehyde) level was observed in the brain.

SEXUAL HORMONE ALTERATIONS

Botelho et al. (2009) investigated the effects of DEHP on sexual hormones at a dose of 500 mg kg‾¹ from gestational day (seventh) to lactation day (second) in rats. The results showed that DEHP had no effect on testosterone in offspring. In another study, Lee et al. (2007) assessed DBP exposure on rat. The animals received DBP in drinking water at a single dose of 750 mg/kg/day for 30 days. Statistical analysis identified a significant decrease in testosterone level but there was no effect on thyroid hormones.

CHANGING THE OXIDATIVE STRESS PARAMETERS

In a recent study, Wang et al. (2012a) investigated the effect of DEHP on the activity of antioxidant enzymes. At the beginning, SOD activity significantly increased during 24 h by DEHP (10 μg mL‾¹) but its level decreased at 72 and 96 h. GPX and CAT activities exhibited no alteration. Furthermore, Wang et al. (2012b) assessed MEHP properties on antioxidant enzymes activities at 0.1, 1, 10 and 100 μg mL‾¹. It was observed that GPX activity decreased at 1, 10 and 100 μg mL‾¹ and also at 0.1 μg mL‾¹ after 96 h. CAT activity increased at two doses of 0.1 and 1 μg mL‾¹. Moreover, SOD activity decreased at a single dose of 100 μg mL‾¹, whereas, SOD activity increased in a single dose of 10 μg mL‾¹. Moreover, Erkekoglu et al. (2010) examined the influence of DEHP and MEHP on MA-10 Leydig cell and measured the antioxidant enzymes activity. Reduction of the activity of GPX, TrxR and GST was observed with both DEHP and MEHP. Moreover, total GSH levels decreased. On the other side, Botelho et al. (2009) assessed the DEHP toxicity in rats. The animals were given DEHP in a dose of 500 mg kg‾¹ from gestational day (seventh day) to lactation day (second day) and the antioxidant enzymes activity was measured in offspring. Increase of CAT activity and a decrease in GST activity were noted. In another study, Zhou et al. (2010) evaluated the effect of DBP on antioxidant enzymes activity. The findings showed that SOD, GSH-Px and GSH activity decreased at two doses of 250 and 500 mg kg‾¹ and MDA (malondialdehyde) level was increased at the same doses in the testes. In a previous study, Lee et al. (2007) examined DBP properties at a dose of 750 mg kg‾¹ orally during 30 days and measured the antioxidant enzymes activity. A significant increase was noted in MDA and 8-OHdG (8-hydroxy-2-deoxyguanosine) levels. Additionally, SOD, CAT and GPx activity increased. Bibliography revealed that Kasahara et al. (2002) evaluated the influence of DEHP in rat. The animals were treated with DEHP at 1 and 2 g/kg/day in drinking water during seven days. Reduction of free thiol, GSH and ascorbic acid, were reported at all doses. In addition, CAT and GPX activity increased at all doses too.

Erkekoglu et al. (2011) examined the oral administration of DEHP at a single dose of 1000 mg kg‾¹ in rat testis during 10 days. The results showed a decrease in both Cu.Zn-SOD activity and GSH level. Also, an increase of TBARS level was noted. In a previous study, Ambruosi et al. (2011) investigated the influence of DEHP on Equine COCs (cumulus-oocyte complexes) at three doses of 0.12, 12 and 1200 μM during 1 h. An increase in CC apoptosis was observed at all doses. At the lowest dose, oocyte maturation was inhibited and ROS level was decreased, whereas, at other doses, there were no changes in oocyte maturation or ROS levels.

Moreover, Zhou et al. (2011) evaluated the effects of DBP in rat testis. The animals received DBP orally at doses of 100, 250 and 500 mg kg‾¹ during 2 weeks. The results showed a decrease of SOD activity and an increase of MDA level in all groups. A further decrease of GSH-Px was observed at a dose of 500 mg kg‾¹. In another study, Seo et al. (2004) investigated the influence of DEHP, DBP and BBP at three doses of 50, 200 and 1000 mg kg‾¹ in rat liver. The MDA level increased significantly with all compounds at doses of 200 and 1000 mg kg‾¹. It is also increased in a dose of 50 mg kg‾¹ with DEHP. Moreover, enhancement of 8-OHdG was observed at a dose of 1000 mg kg‾¹ with DEHP. Kang et al. (2010) examined the effects of DEP by three consecutive daily intraperitoneal (i.p.) injections at doses of 100, 300 and 900 mg kg‾¹ in fish liver. The results showed an increase in LPO levels in all groups and decreasing the activity of CAT, AST and A LT at a dose of 900 mg kg‾¹.

In a recent study, Yavasoglu et al. (2014) assessed the effects of BCP orally for 20 days at doses of 100, 200 and 400 mg kg‾¹ in mouse liver. A significant decrease was observed in all groups. In addition decrease of SOD activity and increase of TBARS level were noted at doses of 200 and 400 mg kg‾¹.

Recently, phthalate toxicity has been evaluated by Yang et al. (2012). In that study, the HepG₂ cells were received MEHP at doses of 6.25, 12.5, 25, 50 and 100 μM during 36 h. The results showed an increase of MDA level and a decrease of GPx activity at all doses. It was also noted an increase of 8-OHdG at doses of 25, 50 and 100 μM. In another study, Erkekoglu et al. (2010) investigated the effects of DEHP (0.01-10 mM) and MEHP (3-30 μM) on LNCaP cells during 72 h. Furthermore, decrease of GPx activity and increase of GR activity (glutathione reductase) as well as DNA damage were all observed.

ALTERATIONS IN GENE EXPRESSION OF THE ANTIOXIDANT ENZYMES

Literature revealed that Wang et al. (2012a) assessed the effect of DEHP on gene expression of antioxidant enzymes. A decrease in SOD₁ expression was noted. In another study, Wang et al. (2012b) evaluated the effects of MEHP on the gene expression. The results revealed that SOD1 and GPX expression decreased at 100 μg mL‾¹. Decrease of anti-apoptotic factor (Bcl-2) expression and increase of pro-apoptotic factor (Bax) expression were noted at three doses of 1, 10 and 100 μg mL‾¹. Moreover, expression of cell cycle genes decreased at the same doses.

Erkekoglu et al. (2010) evaluated the DEHP and MEHP toxicity on gene expression too. In that study, p53 expression increased by MEHP (IC₅₀ = 3 mM). In addition, DNA damage was observed by increased tail intensity and tail moment. Also, these authors (Erkekoglu et al., 2011) investigated the effects of MEHP at a dose of 3 μM on LNCaP cell line. The results showed an increase in expression of p53 and p21. Additionally, in a previous study by Lee et al. (2007), the influence of DBP was evaluated on gene expression in rat. A significant increase of PPAR-r protein expression and PXRr (Retinoid X Receptors) expression were noted. All the mentioned data are exhibited in Table 1-5. Moreover, the diagrammatic representation designed for collecting data from the studies is shown in Fig. 1.