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 developmental indicators of male Xenopus tropicalis
In order to explore the influence of DEHP on the growth and development of male X. tropicalis, in the current study, we measured the total body weight (Fig. 1a), HSI (Fig. 1b), and the relative weight of organs (heart, stomach, and intestines) (Fig. S2) of male X. tropicalis. The total body weight of the frogs in the different groups (0.2, 0.6, 1.8, and 5.4 mg/L) were reduced by 0.16, 1.03, 0.37, and 0.96 g, respectively, on exposure day 49, as compared to that on day 0 (Fig. 1a). These results were similar to those of peer studies, which found that long-term DEHP exposure reduced the weight of Poecilia reticulata and Pseudobagrus fulvidraco (Jee et al. 2009; Zanotelli et al. 2010). It has also been revealed that the endocrine disrupter possesses estrogen-like features, which inhibit growth-related endogenous hormones, resulting in restricted growth (Nugegoda and Kibria 2017; Jia et al. 2016). This may be the reason why the total weight of the frogs post-DEHP treatment was lower than that before treatment. 0.6 mg/L group caused the most obvious reduction of total body weight, contrasted with other groups (Fig. 1a). This difference may be related to the fact that male frogs ate more bloodworm feed, so that they can acquire more energy and resist severe interference from DEHP (Yuan et al. 2017).
There was no difference in relative weights of the lungs, stomach, and intestines between the control and DEHP groups (Fig. S2). Liver is an important organ involved in the metabolic process and detoxification process of vertebrates (Kabir et al. 2015). An increase in HSI was shown in Fig. 1b (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). Above results indicated that the liver is one of the target organs 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, and thus, the Oil Red O-stained area reflects the lipid content in livers. Liver is the potential organ that were most affected by DEHP on male X. tropicalis. Therefore, the toxic effects mediated by DEHP to livers was further explored through the histopathological alterations.
The liver samples were subjected to HE (Fig. 2) and Oil Red O (Fig. 3) staining. Normal hepatocytes, distributed in a concentric circle, with obvious and neat morphology, were observed in the control group (Fig. 2a). Vacuolization of cytoplasm occurred in 0.2 mg/L DEHP-treated group (Fig. 2b). However, the livers of the 0.6 mg/L DEHP-treated group not only displayed damage in terms of vacuolization of cytoplasm, but also severely loose cell cords (Fig. 2c). Vacuolization and diffuse margin of hepatocytes appeared in the 1.8 and 5.4 mg/L DEHP-treated groups (Fig. 2d-e). Moreover, our study exclusively observed atrophy and solidification of hepatocytes upon treatment with 5.4 mg/L DEHP (Fig. 2e). These results indicated that long-term exposure to DEHP can damage the structure of the liver. In addition, the types and frequencies of liver damage increased with increasing DEHP dose. A similar result was seen in a previous study on quail, involving severe vacuolization, disorderly permutation of hepatocytes, and squeezed nucleus, upon 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.
Our study observed pathological changes upon Oil Red O staining after DEHP exposure, characterised by an increase in melanin and accumulation of lipids (Fig. 3a-e). Conspicuous and increasing melanin was observed in the 0.2 and 0.6 mg/L DEHP-treated groups, 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-treated groups, possibly due to the occurrence of oxidative stress and severe damage to the antioxidant system (Fenoglio et al. 2005; Fig. 3d-e). Fig. 3f shows that the Oil Red O-stained area in livers treated with different doses of DEHP (0.2, 0.6, 1.8, and 5.4 mg/L) markedly increased by 0.61-, 0.69-, 0.83-, and 1.04-fold (P < 0.01), respectively, when compared to that in the control treatment. The results also reflected the accumulation of lipids upon DEHP exposure, in a dose-dependent manner. A similar outcome was observed in a previous study on Danio rerio, that is, DEHP could increase the lipid content of the liver (Forner-Piquer et al. 2017).
In a similar study, Zhang et al. (2019) illustrated that DEHP could restrain the metabolism of toxicants, thus amassing the toxic effect and causing injury to the liver. Therefore, the liver injury caused by DEHP at environmentally relevant concentrations, such as damaged hepatocyte structure and accumulation of lipids, may be attributed to the disturbed metabolism of toxicants in the liver.
3.4 Effects of DEHP on the ultrastructure of livers
The liver slices of male X. tropicalis were further scanned using transmission electron microscopy (TEM), to observe the hepatocyte microstructure of the hepatic cell membrane and organelles. The control group showed an integral liver cell structure, including tiny lipid droplets, obvious cell nuclei, and numerous oval mitochondria (Fig. 4a). A decrease in the number of mitochondria was observed in the 0.2 and 0.6 mg/L DEHP-treated groups (Fig. 4b-c). Swollen and deformed mitochondria were observed in both the 1.8 and 5.4 mg/L DEHP-treated groups, with more severe effects in the latter (Fig. 4d-e). As a result, it can be seen that exposure to DEHP can cause mitochondrial damage. In addition, the liver sections in all the DEHP-treated groups were full of large lipid droplets (Fig. 4a-e), which indicated that lipid accumulation occurred upon DEHP exposure. Mitochondria are the target of exogenous toxic substances, and mitochondrial damage is related to changes in the antioxidant system (Li et al. 2014). Previous studies have concluded that mitochondria contain proteins involved in lipid metabolism and regulate energy generation by decomposing lipids (Jiang et al. 2014; Regnault et al. 2016; Chai et al. 2017). Therefore, the results suggest that DEHP at environmentally relevant concentrations may cause toxic effects by inhibiting mitochondrial function in the liver, 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 antioxidant substances (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-treated groups dose-dependently and significantly increased by 0.064-, 0.56-, 0.71-, and 0.76-fold, respectively, relative to the control group (P < 0.05 and P < 0.01) (Fig. 5f). Huang et al. (2019) noted that the ROS in Mus musculus livers increased when they were exposed to DEHP at doses of 125, 250, and 375 mg/kg/day. Compared to the rats, the male X. tropicalis may be more sensitive to DEHP, because the ROS produced in their livers were in response to environmentally relevant concentrations of DEHP. This suggests that the male X. tropicalis is suitable for studying DEHP pollution in the water environment, especially in case of lower environmental concentrations of DEHP. Moreover, this result indicated that excessive ROS was produced upon DEHP treatment, and it is possible that a lack of antioxidant substances 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). Fig. 6a shows that the activity of SOD enzymes in the livers treated with 0.2, 0.6, 1.8, and 5.4 mg/L DEHP increased by 0.30-, 0.29-, 0.14-, and 0.02-fold, respectively, as compared to that in the control group. There was an activity peak at 0.6 mg/L DEHP, and SOD enzymes presented significant differences upon DEHP treatment, except the group treated with 5.4 mg/L DEHP (P < 0.05) (Fig. 6a). CAT and GST activities tended to be similar to that of SOD. These increased significantly (P < 0.05 and P < 0.01) with increasing DEHP concentration, peaked at 0.6 mg/L DEHP, and then decreased 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, and reached a peak at 0.6 mg/L DEHP (Fig. 6d). The decrease in the activities of the antioxidant enzymes post-DEHP exposure was in line with the findings in mouse and human endometrial stromal cells (You et al. 2014; Cho et al. 2015). Meanwhile, this result revealed that inhibited antioxidant defence activity was observed at doses above 0.6 mg/L DEHP, which is also supported by the formation of ROS. 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 0.27-, 0.56-, 0.63-, and 0.69-fold, respectively, relative to the control group (P < 0.01) (Fig. 6e). The result of changes in the activities of antioxidants suggested that lower concentrations of DEHP (0.2 and 0.6 mg/L) were able to improve the antioxidant ability of liver, while impaired antioxidant functions were induced by higher concentrations (1.8 and 5.4 mg/L). MDA content plays an important role in reflecting the degree of oxidative damage (Zou et al. 2015). As shown in Fig. 6f, at 0.2, 1.8, and 5.4 mg/L DEHP, the MDA content increased with increasing dose (P < 0.05). However, the MDA content decreased slightly upon treatment with 0.6 mg/L DEHP, because the high levels of antioxidant functions removed excessive ROS and maximised protection against oxidative stress (Zhao et al. 2014).
Excessive ROS, imbalance in antioxidants, and MDA content are used as major biomarkers of oxidative stress in organisms (Kobayashi et al. 2006; Marrocco et al. 2017). Our study findings suggested that DEHP induced oxidative stress in the liver. In addition, oxidative stress is strongly related to the toxic effects of DEHP on the liver, especially in terms of the histopathological biomarkers (Ben Ameur et al. 2012; Zhang et al. 2019). Therefore, oxidative stress might have played a significant role in mediating the liver structural damage in the previous histopathological analyses.
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 in the liver to 0.30-, 0.31-, 0.33-, and 0.39-fold, respectively, of that in the control treatment (P < 0.01) (Fig. 7a). 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). Upon exposure to 0.2, 0.6, 1.8, and 5.4 mg/L DEHP, the mRNA expression of sod increased with 1.24-, 1.06-, 0.30-, and 0.28-fold, respectively, of that in the control treatment (Fig. 7b). The transcription of cat was obviously increased in the 0.6 and 1.8 mg/L DEHP-exposed groups, but slightly inhibited in 5.4 mg/L DEHP-exposed group, as compared to that in the control group (P < 0.01) (Fig. 7c). The 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). Downstream antioxidant enzyme genes (cat, sod, gpx, and gst), regulated by nrf2, also have an outstanding protective effect to oxidative stress (Cavin et al. 2008). From these results, it can be seen that the expression patterns of antioxidant genes correspond to those of the antioxidant enzymes. In addition, 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, activating genes related to oxidative stress, and highly stimulating the antioxidant function of the organism, to prevent oxidative damage. However, the antioxidant function was suppressed because of the decreased antioxidant genes at higher concentrations. Our outcome was in contrast with the suppressed mRNA transcriptional expression of oxidative stress genes (sod, cat, and gst) observed in case of Oryzias latipes exposed to DEHP at environmentally relevant concentrations, although the concentrations used in that study were ten times lower than those used in the present study, and the variations were due to different exposure times to DEHP and the different resistance systems of the organisms (Yang and Li 2018). Many studies have revealed that the activation of the nrf2 signalling pathway and downstream antioxidant enzyme genes prevents lipid peroxidation, structural damage, and disease in the livers (Gong and Cederbaum 2006; Gan et al. 2012). Therefore, the histopathological and biochemical findings in the current study presented evidence that the oxidative stress mediated by the suppression of antioxidant genes contributed to the production of lipid peroxidation products and damaged structures in the liver.
Based on the histopathological and ultrastructural findings of the study, it can be speculated that lipid accumulation occurred upon 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 genes related to lipid metabolism were 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 0.16-, 0.21-, 0.27-, and 0.40-fold, respectively, as compared to the control treatment (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 the environmentally relevant 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 ultrastructural and histological results of our study, it can be determined that the mechanism of disordered fatty acid β-oxidation finally resulted in lipid accumulation and oxidative stress in the livers exposed to DEHP.