3.1 Exposure concentration
Measurement of the concentrations of di-(2-ethylhexyl) phthalate (DEHP) was displayed in Supplementary Information, Table. S3. The deviations between the nominal concentrations and experimental concentrations of DEHP was less than 20%. Thus, the nominal concentrations of DEHP can represent the experimental concentrations of DEHP used in the study.
3.2 Effects of DEHP on the hepatosomatic index of male Xenopus tropicalis
The hepatosomatic index was measured at the end of the exposure, to assess the chronic toxicity of DEHP on male X. tropicalis. Liver is an important organ involved in the metabolic process and detoxification process of vertebrates (Kabir et al. 2015). In the present study, HSI was significantly increased (Fig. 1, P < 0.05). The increase in HSI of the liver may be due to disturbed lipid metabolism and lipid accumulation (Meng et al. 2018; Mo et al. 2019). In a relevant study, Zhang et al. (2019) illustrated that DEHP could cause inhibition in HSI through restraining the metabolism of toxicants and amassing the toxic effect in the organisms. The results indicated that the liver may be one of targets for DEHP toxicity to male X. tropicalis. Moreover, the liver is an organ that is directly attacked by xenobiotics, making it a suitable organ for assessing the toxicity of external pollutants (Różanowska et al. 1999). Thus, further studies need to be conducted on the hepatotoxicity caused by DEHP in male frogs.
3.3 Effects of DEHP on the histopathology of livers
HE staining was applied to observe the cellular morphology, to determine whether DEHP caused structural damage to livers. On the other hand, Oil Red O stains the lipids, determining lipid content in livers. The toxic effects mediated by DEHP to livers was further explored through the histopathological alterations.
The liver samples were subjected to HE staining (Fig. 2). Normal hepatocytes, distributed in a concentric circle, with obvious and neat morphology, were observed in the control group (Fig. 2a). Exposed to DEHP, the hepatocytes exhibited vacuolization of cytoplasm and loose cytoplasm (b-f). In the histopathological result of 5.4 mg/L DEHP, some hepatocytes were filled with nuclear shrinkage, swelled cell and darkening of colour (f). These results indicated that long-term exposure to DEHP can damage the structure of the liver. Destruction of hepatocytes caused by DEHP have been reported in other animals. In a previous study on quail, severe vacuolization, disorderly permutation of hepatocytes, and squeezed nucleus were found following exposure to a dose of 1000 mg/kg DEHP (Zhang et al. 2019). Our findings were in line with the outcome of a study by Ito et al. (2007), which showed that DEHP can cause fragmentation and necrosis of hepatocytes in Rattus norvegicus.
The liver samples were subjected to Oil Red O (Fig. 3) staining. DEHP caused pathological changes in the form of the larger melanin and larger lipids (Fig. 3a-e). Very small lipid drops were observed in the control group (a). Conspicuous and larger melanin was observed in the 0.2 and 0.6 mg/L DEHP treatments, because the important role of melanin is an important scavenger of xenobiotics and a powerful antioxidant for prevention of oxidative stress (Fig. 3b-c; Barni et al. 2002; Fenoglio et al. 2005). The melanin in livers was probably present in macrophage aggregates (Cicero et al. 1982; Bani 2009). On the contrary, the amount of melanin decreased in the 1.8 and 5.4 mg/L DEHP treatments, possibly due to the occurrence of oxidative stress and severe damage to the antioxidant system (Fenoglio et al. 2005; Fig. 3d-e). Figure 3f shows that the area of lipid droplets in different DEHP-treated (0.2, 0.6, 1.8, and 5.4 mg/L) livers samples markedly increased in a dose-dependent manner by 0.61%, 0.69%, 0.83%, and 1.04% (P < 0.01), respectively, compared with the controls. The results reflected the accumulation of lipids following DEHP exposure. Our study was agreed with a previous study that indicated DEHP could increase the lipid content in livers of Zebrafish (Danio rerio) (Forner-Piquer et al. 2017). The histopathological alterations of liver caused by DEHP, such as abnormalities of hepatocyte and accumulation of lipids, may be attributed to the disturbed metabolism of toxicants in the liver (Zhang et al., 2019). Furthermore, the alterations of the structure could possibly cause liver dysfunction and lead to necrosis of cells (Li et al., 2018), which emphasized the importance of the ultrastructural analysis of livers.
3.4 Effects of DEHP on the ultrastructure of livers
The liver slices were further scanned using transmission electron microscopy (TEM), to observe the hepatocyte microstructure of the hepatic cell membrane and organelles. The control treatment showed an integral structure of hepatocyte, including well-defined nucleus, a large number of oval-shaped mitochondria and endoplasmic reticulum, and tiny lipid droplets. Moreover, the nuclear membrane and chromatin of the nucleus were evident and closely arranged, with no diffusion of chromatin (Fig. 4a). A reduction of mitochondria and the larger lipid droplets within the same field of view were observed in the 0.2 and 0.6 mg/L DEHP treatments (Fig. 4b-c). With exposure to 1.8 and 5.4 mg/L DEHP treatments, swollen and deformed mitochondria were observed, and the chromatin margination dispersing the nuclear membrane was slightly occurred. (Fig. 4d-e). As a result, it can be seen that DEHP could cause mitochondrial abnormalities and accumulation of lipid. Lipid accumulation caused by DEHP corroborated the histopathological findings about production of lipid droplets. Mitochondria are the target of exogenous toxic substances (Li et al. 2014), which clarifies that liver is the target of DEHP on male X.tropicalis. Previous studies have demonstrated that the mechanism of lipid accumulation was related to damage to hepatocyte mitochondria, and mitochondria contained proteins involved in lipid metabolism and regulated energy generation by decomposing lipids. (Jiang et al. 2014; Regnault et al. 2016; Chai et al. 2017). In addition, mitochondrial damage is related to changes in the antioxidant system (Li et al. 2014). Antioxidants, existing in mitochondrial inner membrane, efficiently inhibited lipid peroxidation in cells and prevented oxidative damage (James et al., 2004; Li et al. 2014). To some extent, damage to hepatocyte mitochondria induced by DEHP may be harmful to the antioxidant capacity of livers. Therefore, the results suggest that DEHP may induce hepatotoxicity by inhibiting mitochondrial function, leading to the accumulation of lipids and inhibition of antioxidant capacity.
3.5 Effects of DEHP on oxidative stress
Antioxidants include antioxidant enzymes and non-enzymatic scavengers, which adjust the production and removal of ROS to maintain dynamic homeostasis (Liang and Yan 2020). However, excessive ROS production is stimulated by external pollutants (Dogan et al. 2011), which may attack the biological macromolecules of the cell and produce lipid peroxidation products, such as MDA. Due to the inhibition of antioxidant functions, there is occurrence of oxidative stress and damage (Pandey et al. 2003). In order to examine whether DEHP caused oxidative stress in livers of male X. tropicalis, we determined the ROS content (Fig. 5), activities of antioxidants (Fig. 6a-e), and MDA content (Fig. 6f) in the livers subjected to different DEHP treatments.
As shown in Fig. 5a-e, the amount of ROS in the liver was determined by measuring the intensity of red fluorescence. The fluorescence intensity in 0.2, 0.6, 1.8, and 5.4 mg/L DEHP treatments were significantly increased in a dose-dependent manner by 6.4%, 56%, 71%, and 76%, respectively, relative to the controls (P < 0.05 and P < 0.01) (Fig. 5f). Huang et al. (2019) noted that the ROS in Mus musculus livers increased following exposure to DEHP at doses of 125, 250, and 375 mg/kg/day. In another relevant study, zebrafish juveniles, which were aquatic vertebrates along with X.tropicalis, showed 1.13-fold increase in ROS levels after DEHP exposure at 9.75 mg/L, compared to the control group (Lu et al. 2021). Compared to zebrafish juveniles, adult frogs showed a significant increase in ROS levels at lower DEHP concentrations (0.2 mg/L). It suggests that adult X.tropicalis may be more sensitive to DEHP than zebrafish larvae and more suitable for studies of DEHP pollution in the water environment, especially in case of lower concentrations of DEHP. Moreover, this result indicated that excessive ROS was produced after DEHP exposure and it is possible that lack of antioxidants was used to eliminate the ROS (Amado and Monserrat 2010; Gavrilovic et al. 2021).
Antioxidants, including antioxidant enzymes (SOD, CAT, GST, and GPX) and non-enzymatic scavengers (GSH), are known biomarkers of oxidative stress (Kobayashi and Yamamoto 2006; Liu et al. 2019). Figure 6a shows that the activity of SOD treated with 0.2, 0.6, 1.8, and 5.4 mg/L DEHP increased by 30%, 29%, 14%, and 2%, respectively, as compared to that in the controls. Similarly, an increase was also found in CAT and GST activities. Significantly increased in CAT and SOD (P < 0.05 and P < 0.01) was observed at 0.2, 0.6, 1.8 mg/L DEHP, and a decrease was found at 5.4 mg/L DEHP (Fig. 6b-c). The GPX activity displayed an upward trend in all of the DEHP groups, compared to the control group (Fig. 6d). The decrease in antioxidant enzymes following DEHP exposure was in line with the findings in Mus musculus and human endometrial stromal cells (You et al. 2014; Cho et al. 2015). As an important scavenger of ROS, GSH is highly correlated with the antioxidant capacity of the liver (Liang and Yan 2020). In our study, the GSH content of 0.2, 0.6, 1.8, and 5.4 mg/L DEHP-treated groups markedly decreased by 27%, 56%, 63%, and 69%, respectively, relative to the controls (P < 0.01) (Fig. 6e). MDA content directly reflects the degree of oxidative damage, including lipid peroxidation (Zou et al. 2015). The MDA content increased at 5.4 mg/L DEHP (Fig. 6f, P < 0.05), however, decreased slightly after exposure to 0.6 mg/L DEHP, for the reason that enough antioxidant removed excessive ROS and maximised protection against oxidative stress (Zhao et al. 2014). Under the exposure at 1.8, and 5.4 mg/L DEHP, the antioxidants may be saturated which resulted in the increasing of MDA (Qiang et al., 2011).
Excessive ROS, imbalance in antioxidants (SOD, CAT, GPX, GST, GSH), and MDA content are used as major biomarkers of oxidative stress in organisms (Kobayashi et al. 2006; Marrocco et al. 2017). Our study suggested that DEHP induced hepatotoxicity through oxidative stress. In addition, oxidative stress is strongly related to the toxic effects of DEHP on the liver, especially in terms of the histopathological biomarkers and accumulation of lipid (Ben Ameur et al. 2012; Zhang et al. 2019; Yan et al., 2020). Therefore, oxidative stress was associated with hepatocyte structural and microstructural changes, which is the finding in HE staining, Oil Red O staining and mitochondrial abnormalities.
3.6 Effect of DEHP on genes related to oxidative stress and lipid metabolism
To further elucidate the molecular mechanism of oxidative stress and lipid accumulation, the transcription of related genes was investigated in the livers of male X. tropicalis (Fig. 7). Long-term exposure to DEHP (0.2, 0.6, 1.8, and 5.4 mg/L) significantly decreased the expression of nrf2 by 30%, 31%, 33%, and 39%, respectively, of that in the controls (P < 0.01) (Fig. 7a). Upon exposure to 0.2, 0.6, 1.8, and 5.4 mg/L DEHP, the mRNA expression of sod increased by 124%, 106%, 30%, and 28%, respectively, of that in the control treatment (Fig. 7b). The mRNA level of cat is significantly induced in 0.6 and 1.8 mg/L treatments (P < 0.01), while slightly inhibited in 5.4 mg/L DEHP-exposed group, as compared to that in the control group (Fig. 7c). Gene expression of gst was significantly increased in the 0.2 and 0.6 mg/L DEHP-exposed groups, while it decreased in the 1.8 and 5.4 mg/L DEHP-exposed groups, when compared to the control group (P < 0.01) (Fig. 7d). The mRNA level of gpx showed an obvious increase in all the DEHP treatments (P < 0.05 and P < 0.01) (Fig. 7e). From these results, it can be seen that the expression patterns of antioxidant genes correspond to the previous results of the antioxidants. Nuclear factor erythrocyte 2-related factor 2 (encoded by nrf2) is a nuclear transcription factor that plays a crucial role in activating the antioxidant defence system in response to oxidative stress and excessive ROS (Osburn and Kensler 2008; Shaw et al. 2019). Downstream genes of antioxidants (cat, sod, gpx, and gst), regulated by nrf2, also have an outstanding protective effect to oxidative stress (Cavin et al. 2008). Many studies have revealed that the activation of the nrf2 signalling pathway and downstream antioxidants genes prevents lipid peroxidation, structural damage, and disease in the livers (Gong and Cederbaum 2006; Gan et al. 2012). Therefore, the present study indicated that the livers of X. tropicalis males had a certain degree of resistance to the toxic effects of low concentrations of DEHP through activating genes related to oxidative stress and highly stimulating the antioxidants activities of the organism. However, the antioxidants activities were suppressed because of the decreased antioxidant genes at higher concentrations.
Based on the histopathological and ultrastructural findings of the study, it can be speculated that lipid accumulation occurred following DEHP exposure. Lipid accumulation contributes to the variation in lipid metabolism (Gorria et al. 2006; Circu and Aw 2010; Ramesh et al. 2015; Meng et al. 2018; Li et al. 2020). In this study, the transcription levels of gene related to lipid metabolism was used to determine the molecular mechanism of variation in lipid metabolism. Downregulation of pparα expression was observed in all the DEHP-exposed groups. DEHP concentrations of 0.2, 0.6, 1.8, and 5.4 mg/L caused a marked decrease in the expression, by 16%, 21%, 27%, and 40%, respectively, as compared to the controls (P < 0.05 and P < 0.01) (Fig. 7f). Peroxisome proliferator activated receptor-α (encoded by pparα) is a type of peroxisome proliferator-activated receptor that plays an important role in enhancing fatty acid β-oxidation capacity in the liver (Yang et al. 2007; Feige et al. 2010; Maradonna et al. 2013). Promotion of fatty acid β-oxidation is associated with the release of free fatty acids and inhibition of lipogenesis (Wei et al. 2018). The study by Adeogun et al. (2018) supported our results, indicating that there was a decrease in the expression of pparα in Clarias gariepinus, after dealing with similar concentrations of DEHP (200 and 400 µg/L). In addition, published studies have indicated that the restrained activity of fatty acid β-oxidation could lead to excessive lipid accumulation and oxidative stress in the liver of Rattus norvegicus (Wierzbicki et al. 2009; Jia et al. 2015; Qin et al., 2016). According to the expression of pparα, it may be determined that the mechanism of disordered fatty acid β-oxidation finally induced lipid accumulation and oxidative stress in the livers exposed to DEHP.
3.7 Potential mechanisms of DEHP-induced hepatotoxicity in X.tropicalis
Based on previous results and analyses, Fig. 8 demonstrates the potential mechanisms of hepatotoxicity to X.tropicalis. Excessive ROS is the primary factor of toxicity, and the potential mechanism of hepatotoxicity by excessive ROS is divided into the following three parts. Firstly, the attack of DEHP on hepatocytes disrupted the balance of ROS and induced excessive ROS production (Fig. 5). Part of the ROS attacked the structure of hepatocytes, causing an increase in MDA content (Fig. 6f) with structural damage to hepatocytes (Fig. 2). Secondly, a portion of ROS interfered with antioxidant-related pathways (NRF2 pathway, Fig. 7), thereby inducing oxidative stress, such as inhibition of antioxidant activity (Fig. 6), lipid peroxidation (Fig. 6f) and mitochondrial abnormalities (Fig. 4), and the occurrence of oxidative stress may further exacerbate the imbalance in ROS levels. Finally, a portion of ROS inhibited the expression of lipid metabolism gene (Fig. 7f), which may cause a disturbance in fatty acid metabolism. Mitochondrial abnormalities also potentially affect the process of catabolism of fatty acids which ultimately induces lipid accumulation (Fig. 3 and Fig. 4).