Effects of DMP on blood antioxidant capacity
After DMP treatment, the ROS content and activity of plasma SOD changed conversely along with increasing DMP dose (Fig. S1), demonstrating DMP inhibited the activity of SOD and are toxic to the antioxidant defense system. It should be noted that the ROS production is not significant at doses of 50 and 250 mg/Kg, probably ascribed to the fact that enzymatic antioxidants or lipids have consumed excess ROS. The unpaired electron of radicals may damage the lipids of cellular membranes, which is initiated by a process known as LP. To testify to the assumption, we measured the content of MDA, which is the product of LP. The MDA level developed conversely with SOD, showing that DMP induced ROS targeted at the fatty acids in membrane phospholipids and destructed the cellular membranes. These findings align with our (Chi et al., 2021; Li et al., 2019) and Zhang’s in vitro results, the latter of whom firstly discovered oxidative damage of bisphenol S(Zhang et al., 2016b). Compared with heavy metals(Matović et al., 2015) and other industrial additives like sodium fluoride(Umarani et al., 2015), DMP is much less toxic to the antioxidative system. In contrast, dibutyl phthalate(Wang et al., 2020), also a phthalate ester, has apparent less toxicity than DMP.
Haemocytes, antibody, and pro-inflammatory cytokines
Having shown that DMP invokes oxidative stress, we questioned whether it triggers immunohematological responses. As illustrated in Fig. 1, the numbers of RBC, WBC, and LYM without antioxidants are lower than that in the control group, demonstrating a downward tendency with increasing DMP concentration. Therefore, DMP is bio-active enough to inhibit the proliferation of hemocytes and initiate an inflammatory response. In quest of the DMP immunohematological toxicity mechanism, we used two important in vivo non-enzymatic antioxidants (vitamin C and E) as antagonists. Apparently, these antioxidants are effective stimulators of hemocyte proliferation. ROS likely participates in a conglomerate of steps that lead to innate immune activation.
The pro-inflammatory cytokine and immunoglobulin levels are usually tested to predict the immunomodulatory effects of exogenous substances and the possibility of inflammation-mediated toxicity. Alterations in antibody and cytokine expression were observed, suggesting inflammation effects of DMP (Fig. 1). Compared to the untreated, IgG, IL-4, -6, and IFN-γ are negatively associated with the exposure dose; however, IL-2 experiences hormesis and stays unchanged at the highest dose. In addition, antibody IgG production is elevated at light concentration and then declined compared to the untreated. All the phenomena suggest suppressed immune defense and destructed inflammatory regulation. What is surprising is that the antioxidants generate a significant change in IL-2 and -6 production with and suppression. The administration of antioxidants may polarize the balance between the Th1 and Th2 cytokines towards one specific pathway(Elsabahy and Wooley, 2013; Liu et al., 2009). Together, it is concluded that DMP is reactive enough to initiate an inflammatory response, and antioxidants may worsen immune balance.
We determined induction of apoptosis/necrosis in lymphocytes using flow cytometry analysis. The treatment of rats with DMP resulted in lymphocyte apoptosis/necrosis. As shown in Fig. 2A, treatment with DMP generated a population shift from normal cells to apoptotic/necrotic ones. There is a significant increase in Annexin V and PI staining, with the percentages going up from 1.79-7.07-9.62 to 18.93% as the exposure dose rises.
We further investigated the T helper cell subset CD4 to determine if the single subpopulation was biased toward the production of ROS. It seems that DMP is a potent inhibitor of CD3+CD4+ T cell proliferation or differentiation. Zone Q2 in Fig. 2B shows the fluorescence level of CD3+CD4+ T cell. Analysis of the CD3+CD4+ population demonstrated a remarkable diminution in treated rats and an approximate 2.5-fold decrease. It is thus suspected that the apoptotic/necrotic lymphocytes are mainly CD3+CD4+ cells. Therefore, the decrease of CD3+CD4+ T cell content makes a cytokine decline, which indirectly leads to a deterioration in immunoglobulin secretion and immune function.
Feature, GO functional, and KEGG pathway analysis of transcriptomic responses
Exposure to DMP caused changes in global transcript expression (Fig. S2). Of all the transcripts, 570 overlapped across all treatment groups, either upregulated or downregulated. Interestingly, the medium and low dose treatments have the lowest and highest dysregulated transcripts, respectively. This phenomenon is consistent with the recent study revealing hormesis of dysregulated genes in 17α-Ethinylestradiol-treated rainbow trouts(Schultz et al., 2021) and is also consistent with the above toxicity features.
We further analyzed the 570 DEGs using GO and KEGG pathway enrichment analysis. The results of GO analysis show that exposure to DMP dysregulated a high number of genes in biological process, cell component, and molecular function. Among all the enriched genes, organonitrogen compound metabolic process, cytoplasm, intracellular part, cytoplasmic part, intracellular, and protein binding take percentages above 35% (Fig. 3A).
It’s known that intracellular components, especially mitochondria and cytoplasm, are the primary sources of ROS production(Zhang et al., 2016a). One of the most important cellular responses to ROS is protein (organonitrogen compound) oxidation, which changes in thiol/disulfide pairs affect protein function. The ROS-dependent signaling processes, such as cytokine production, are also likely to be affected. Therefore, it’s pretty evident that DMP would pose a significant risk on the blood immunology of rats.
KEGG pathway enrichment (Fig. 3B) as well as mechanism prediction (Fig. S3) analysis manifests that signal pathway alterations are closely involved in the metabolism following DMP exposure. Considering the hints from KEGG and GO analysis, it is likely that intracellular and cytoplasmic ROS is the cornerstone that contributes to metabolic dysfunction and inflammatory signaling. Cytosolic ROS are high possibly formed through NOX (nicotinamide adenine dinucleotide phosphate oxidase) activities, which influence metabolic processes like pentose phosphate pathway activity, glycolysis, and downstream oxidative phosphorylation, and autophagy(Forrester et al., 2018; Panieri and Santoro, 2016). Moreover, these disorders are also likely to be associated with lipid metabolism, a biological process where estrogen and xenoestrogen (like PAEs) exert regulatory control(Mankidy et al., 2013; Palmisano et al., 2017). The metabolomic disorders, intricately intertwined with the inflammation effects, result in the apoptosis/necrotic of immunocytes.
Expression level of genes related to ROS and immunocyte apoptosis
To further explore the potential relationship between ROS and the immunotoxic as well as the potential apoptosis pathways, the expression of Bcl-2 family genes (Bcl-2, Bax, Bid, and Bak-1) was analyzed. As shown in Fig. 4A, the expression levels of all the markers increase in folds with increasing dose, demonstrating an overexpression in blood cells. It should be noted that although all the pro-apoptotic genes overexpressed after exposure, the expression of the anti-apoptotic gene Bcl-2 also increased. Therefore, DMP may cause apoptosis through the endoplasmic reticulum pathway and meantime, the anti-apoptotic gene also over-expressed as antagonism.
Apoptosis can be initiated through overexpression of death receptors (Fig. 4B) which are categorized as the TNF family members. Fas is a known death receptor and participates in the process of apoptosis with the involvement of FasL (Fas ligand) and FADD (Fas-associated death domain protein), which is also highly related to ROS and cytokines (Lenzi et al., 2018; Matés et al., 2012). Besides, previous observations have shown that FasL plays a regulatory role on the during the environmental toxicant-induced cell apoptosis(Wang and Su, 2018), which is in agreement with our findings.
Among the pro-apoptotic caspases, caspase-2, -8, and -9 are the activators of apoptosis, and caspase-3, -6, and -7 are the executors(Li and Yuan, 2008). Activation of caspases (Fig. 4C) may lead to disorder of cytoskeleton, resulting in chromatin concentration, cell structure disintegration, and apoptosis. When these enzymes are activated, DNA enzymes are subsequently activated to degrade DNA and induce apoptosis.