5.2 Effects of PVC MPs in Hepatic System of Mice
5.3.1 PVC MPs Induce Inflammation in Liver
By referring to Table 4.3 and Figure 4.2 in Section 4.2.2, the results demonstrated that the liver weight increased significantly in high dosage group but no remarkable increment in the low and medium dosage group. Overall, the liver weight and liver index (the ratio of liver weight to body weight) of the PVC MPs-treated mice were elevated as compared to control but the mice liver weights are still within the normal range which is 3 to 5 % of the body weight.
Similar findings were identified before. Studies have reported that PE MPs ingestion can result in a significant increase in the liver weight and index in mice which exceed the normal range (Zhao et al., 2021). This suggests that MPs could induce inflammation in liver and thereby impair the liver function.
According to Prata et al. (2020), chronic inflammation could be induced by the inability of immune system to eliminate synthetic particles such as MPs. This is supported by a study which showed that PS MPs can induce inflammation in human lung epithelial BEAS-2B cells via ROS production (Dong et al., 2020).
According to El-Mihi et al. (2017), ROS can induce the inflammatory mediators to release which lead to incorporation of immune cells to secrete pro- inflammatory cytokines and growth factors to enhance inflammatory response.
Histopathological studies and detection of proinflammatory cytokines are needed to justify the inflammation in PVC MPs -treated mice’s liver in this present study.
56 5.3.2 Putative Mechanism Actions of PVC Plastics Additives in Disrupting Mitochondrial Apoptotic Pathway in Mice Liver
Up to date, there are no studies done on the effects of PVC MPs to mitochondrial apoptosis pathway in mice liver by examining both the gene expressions of BCL-2 and caspase-3. In a related study, DEHP has been reported to cause oxidative effects in rat insulinoma cells by inducing apoptosis via regulation of BCL-2 signalling pathway. The results revealed that gene expression of BCL-2 was down-regulated whereas caspase-3 was up-regulated in the rat insulinoma cells treated with DEHP, which showed consistent result obtained in this present study (Li et al., 2021). This finding further indicates that DEHP incorporated PVC MPs could induce apoptosis in hepatocytes by activating the mitochondrial apoptotic. Furthermore, Li et al. (2021) reported that DEHP significantly reduced the expression levels of PI3K and p-Akt proteins which showed that ROS generated by DEHP result in inhibition of PI3K/Akt signalling pathway.
Inhibition of the PI3K/Akt regulatory pathway remarkably decreased the expression level of BCL-2 as BCL-2 anti-apoptotic protein is the downstream molecule of Akt (Trejo-Solís et al., 2018). The decreased BCL-2 expression induced an increase in the mitochondrial membrane permeability thereby lead to releasing of cytochrome c to the cytoplasm. Moreover, cytochrome c in turn bound to Aparf-1, and activated caspase-9 by cleaving pro-caspase-9 into active dimer, which subsequently results in activation of effector caspase-3 and increased expression of caspase-3, thus promoting apoptosis (Xiao et al., 2018).
Therefore, this indicates DEHP incorporated PVC MPs could reduce the BCL- 2 expression and increase caspase-3 expression via inhibition of PI3K/Akt signalling pathway by ROS production.
57 In addition, DEHP and BPA can leach out from the PVC MPs into the environment which could be ingested or inhaled by many living organisms.
DEHP and BPA has been reported to generate ROS and lead to DNA damage which in turn activates the p53-mediated mitochondrial apoptotic pathway (Gao et al., 2017; Huang et al., 2018). In response to severe DNA damage, p53 can reduce anti-apoptotic proteins and elevate pro-apoptotic proteins expression to regulate apoptosis (Yoshida and Miki, 2010). Previous study reported that DEHP elevated the p53 level which in turn reduced the expression of BCL-2 and thereby increased the release of cytochrome c that led to activation of the downstream caspase-9 and caspase-3 in rat testes tissues (Fu et al., 2020).
Another previous study demonstrated that BPA activated p53 via phosphorylation and induced the mitochondrial disruption by translocation into mitochondria and the interaction between p53 and BCL-2 family proteins subsequently reduced the BCL-2 expression and resulted in overexpression of caspase-3 (Huang et al., 2018). These results were similar to the findings of this present study. This suggests that PVC MPs could activate the p53-mediated mitochondrial apoptotic pathway via induction of severe DNA damage in mice liver which in turn alter the gene expression levels of BCL-2 and caspase-3.
Overall, all the discussed effects were dose-dependent and only observed clearly in high dose group of mice. The fold change of gene expression of BCL- 2 and caspase-3 was less remarkable in low and medium dose group of mice exposing to the same PVC MPs chemical substances. This suggests that the effects on alteration of gene expression could be due to the actions of epigenetically toxic chemical additives incorporated in PVC MPs. According to
58 Singh and Li (2012), epigenetic alterations such as DNA methylation, histone modification, and micro RNAs (miRNAs) are the most common target of toxic chemical exposure to cause deleterious effects on health. These changes lead to alteration of gene expression without altering the DNA sequence. A previous study reported that DEHP exposure changed the expression profile of miRNAs which target on mRNAs involved in the PI3K/Akt signalling pathway by reducing the miR-19a-3p (Zhang et al., 2019). Lowered miR-19a-3p by DEHP exposure raises the level of PTEN which in turn reduces the phosphorylation of Akt and eventually leads to down-regulation of the downstream BCL-2 expression. Moreover, another previous study revealed that mono (2-ethylhexyl) phthalate (MEHP) exposure changed the expression levels of a miRNA called miR-16 which targets the regulation of BCL-2 mRNA as it is complementary to the bases 3287 to 3279 in the BCL-2 cDNA 3’-end (Meruvu et al., 2016).
MEHP exposure increased the expression of miR-16 which negatively regulates the BCL-2 expression by down-regulation of BCL-2 and promotes apoptosis through increasing expression of caspase-3. In addition, Santangeli et al. (2017) reported that BPA exposure could lead to epigenetic changes by DNA methylation through addition of methyl group to the carbon cytosine specifically the 5th ring. The putative epigenetic effects of PVC plastic additives on BCL-2 are summarized in Figure 5.1. However, the epigenetic effects of BPA on apoptosis-regulation related genes are not yet well studied. Therefore, the PVC MPs incorporated with phthalates and BPA in this study possesses potential epigenetic toxicity in altering the gene expression of BCL-2 and caspase-3 in mice liver.
59 Figure 5.1: Putative epigenetic effects of PVC plastic additives on BCL-2.