Numerous studies have reported that BaP reduced cell viability in various cell lines [29–31]. In agreement with these studies, we found that BaP at concentrations ranging from 16 to 64 M signif-icantly reduced the viability of Hep3B cells at the time points of 12 and 24 h. This result was further confirmed by flow cytometry, which demonstrated that BaP induced necrosis after 12 h treatment and apoptosis after 24 h treatment in Hep3B cells. BaP induced both apoptosis and necrosis in Hep3B cells, which was in consistence with those found in other cell lines [3,15,32], and the shift of the mode of cell death from necrosis to apoptosis have been reported before [33,34].
BaP is not only a substrate for CYP1A1 and CYP1B1, but also an inducer of its protein expression and enzyme activity [35–37]. CYP1A1 was found to play a pivotal role in BaP-induced apopto-sis, and inhibition of CYP1A1 reversed BaP-induced apoptosis in Hepa1c1c7 cells [38]. In the present study, we observed an increase in EROD activity, which mainly reflects CYP1A1 activity in Hep3B cells at 6 and 12 h after BaP treatment. The enzymatic induction would transform more BaP into its toxic intermediates and ulti-mately lead to cell death. Notably, a previous study suggested that pifithrin alpha (p53 inhibitor) caused a potent inhibition of CYP1-related activity [39]. In this study, the lower fold induction in EROD activity may link to the characteristics of the cell line (p53-null). However, we did not investigate CYP1B1 induction. Moreover, induction of CYP1B1 induction in BaP-treated Hep3B cells could not be excluded.
The intermediates generated during BaP metabolism could enter the redox cycling and generate excessive ROS (eactive oxygen species) through the ortho-quinone pathway [40]. Both apoptotic and necrotic cell death can be induced by ROS [41]. In this study, DCF fluorescence, MDA (malondialdehyde) and T-AOC (total antioxidative capability) as indexes of oxidative status of BaP-treated cells were used. We found increased oxidative stress in the cells, presenting increase in ROS production at 6 and 12 h, and in MDA forma-tion at 6, 12 and 24 h in BaP-treated cells. At 6 and 12 h after the treatment, increased T-AOC in the cells further confirmed the induction of oxidative stress by BaP. These results are consistent with our previous findings and other studies [4,42,43], implicating ROS generation may play a role in BaP-induced cell death.
Mitochondria are the major source of ROS generation [7]. However, excessive ROS generation can cause mitochondrial dys-function and induce apoptotic and necrotic cell death [6]. In the present study, we demonstrated that BaP disrupted MMP (mitochondrial membrane potential), ATP generation (as indicated by lactic acid (LD) level) and the activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase, indicating BaP induced mitochondrial toxicity. The time course of these changes were neg-atively correlated with that of ROS formation, with the observed reduction at 6 and 12 h and the detected recovery at 24 h after BaP treatment, suggesting an association between ROS generation and mitochondrial dysfunction. The increased LD level at early time points (6 and 12 h) implicated reduction of ATP level. The cells with ATP depletion and mitochondrial dysfunction were reduced to undergo necrotic cell death. Instead, the decreased LD level at later time point (24 h) implicated an elevated ATP level. The cells surviving in the early necrotic phase recovered mitochondrial potential and energy to undergo ATP-dependent apoptotic cell death. These results indicated a link between BaP-induced apoptotic and necrotic cell death with mitochondrial dysfunction.
JNK (c-Jun N-terminal kinase) is activated in response to BaP treatment, and regulates BaP-induced apoptosis [44,45]. Experimental data suggests that JNK activation induces mitochondrial dysfunction and subsequent cell death [46]. In the present study, we found JNK activation only after 24 h of BaP treatment. However, mitochondria dysfunction was detected at 12 and 24 h. Our results suggests other pathways, in addition to JNK activation, are responsible for the mitochondrial dysfunction induced by BaP. JNK activation also induces Bax-dependent apoptosis through stabilizing Bax protein at post-transcriptional level and promoting Bax translocation to the mitochondria [47,48]. Activation of JNK and Bax at 24 h after BaP treatment indicated that BaP possibly induced apoptosis via JNK/Bax dependent mitochondrial pathway. However, inactivation of JNK in BaP-treated cells at the 12 h time point may indicate that JNK activation is not required for BaP-induced neuroblastoma and other human cancers, Cell 90 (1997) 809–819. necrosis.
Our previous study has revealed that the expression level of p73 mRNA (but not the protein level) was elevated by BaP in MRC-5 and H1299 cell lines, but it was not involved in BaP-induced necrosis [4]. In accordance with the previous study, we showed that p73 protein level was not affected by BaP in Hep3B cells, and BaP-induced apoptosis is probably independent of p73.
In conclusion, our study demonstrates for the first time that BaP induces early necrosis and later apoptosis in Hep3B cells. The BaP-induced cell death is associated with mitochondrial dysfunction and probably independent of p73. Further studies need to be conducted to fully understand the role of mitochondrial dysfunction in BaP-induced cell death and the underlying mechanisms.
