Altered Protein Expressions in Chronic PCB-153–Induced Human Liver (HepG2) Cells

Abstract

Polychlorinated biphenyls (PCBs) are a group of persistent and widely distributed environmental pollutants that have various deleterious effects, e.g., neurotoxic, endocrine disruption and reproductive abnormalities, including cancers. Chronic exposure to environmentally hazardous chemicals like PCBs is of great concern to human health. It has been reported earlier that apoptotic proteins change in rats under chronic PCB treatment. It is of importance to determine if chronically exposed human cells develop a different protein expression. In the present study, the authors chronically exposed metabolically competent human liver (HepG2) cells at 50 to 100 μM to examine the role of the well-known environmentally hazardous pollutant non-coplanar 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB-153) to study cell death. After 12 weeks of exposure these cells showed significant changes in apoptotic death in subsequent trypan blue growth assay, fluorescence microscopy, DNA fragmentation, and immunoblotting studies. Interestingly, chronically exposed cells showed marked differences in apoptotic and/or death-related proteins (e.g., Bcl2, Bak, and the pro and active forms of caspase-9, which were up-regulated), in contrast to acutely exposed (i.e., 48-h PCB-153 exposed) cells, which maintained linear growth despite repeated exposures. Similarly, tumor suppressor protein p53, proto-oncogene c-myc, and cell cycle regulator protein p21 were also up-regulated compared to nonchronically exposed HepG2 Cells. The results indicated that PCB-153–induced chronic exposure significantly altered different apoptotic (e.g., Bcl2, Bak, caspase-3) and tumor suppressor (e.g., p21, p53, and c-myc) proteins in the cellular model. These results suggest that chronic exposure to PCB-153 can induce cell survival by altering several apoptotic and tumor suppressor proteins.

Polychlorinated biphenyls (PCBs) are widespread xenobiotic environmental contaminants. Their physical properties, such as thermal stability, low reactivity, and low electrical conductance, which make them ideal for use as flame retardants, electrical insulators, lubricants, and liquid seals in industries, also allow them to persist in nature, leading to their accumulation in the environment and biological systems (McFarland and Clark 1989; Safe 1994). Although PCBs were banned from the USA in 1977, our biosphere contains approximately 750,000 tons of released PCBs (http://bio.nagaokaut.ac.jp). Despite an overall decrease of PCBs, there is great concern about the toxic effects of PCBs or like compounds. PCBs are known to cause reproductive (Den Hond et al. 2002), neurological (Schantz et al. 2003), endocrinal (Portigal et al. 2002), and other defects. PCBs also adversely affect fetal and infant development (Winneke, Walkowiak, and Lilienthal 2002) and are also immunotoxic (Lyche et al. 2004). The toxic effects and physical properties of PCBs are truly structure dependent. The biological effects of the coplanar PCBs have shown the induction of cytochrome P450 1A1 (CYP1A1, CYP1A2) in liver cells (Safe et al. 1985). There is a considerable body of evidence showing that different PCB congeners or their mixtures, known as archlors, can act as carcinogens in rodents (Silberhorn, Glauert, and Robertson 1990). There were positive associations between PCB exposure and cancer in the following organs: rectum, liver, biliary, pancreas, skin, prostate, kidney, brain, and lymphatic system (Glauert, Robertson, and Silberhorn 2001). However, PCBs do not appear to cause a consistent increase in the occurrence of one or more cancers in the occupational setting (Longnecker, Rogan, and Luicer 1997). Studies indicated that either a PCB mixture or individual congeners can act as tumor promoters (Silberhorn, Glauert, and Robertson 1990; Glauert, Robertson, and Silberhorn 2001). Several epidemiological studies have also shown a strong correlation between tumor promotion and carcinogenicity due to PCB exposure (Millikan et al. 2000; Dorgan et al. 1999; Gutters et al. 1998; Gustavsson and Hogstedt 1997). However, the carcinogenic effects of PCBs (Faroon, Jones, and de Rosa 2001; Laden et al. 2002) are controversial (Golden et al. 2003). Although PCBs clearly show tumor-promoting activity in the liver, their mechanism of action is not known and has been the subject of intensive investigation in recent years. A number of mechanisms have been proposed, including the induction of oxidative damage (Oakley et al. 1996), effects of vitamin A metabolism, and effects of intercellular communication (Glauert, Robertson, and Silberhorn 2001). Another mechanism by which oxidative stress from tumor promoters can influence carcinogenesis is by altering the gene expression in the cell (Tharappel et al. 2002). We have reported earlier the mechanism of congener-specific apoptotic cell death induced by PCB-153 and PCB-77 in human kidney cells in vitro (Chen et al. 2006).

Several other mechanistic studies have shown that cells exposed chronically to toxic chemicals do develop resistance to cell death, which is of great concern to human health. These drug-or chemical-resistant cells are also major concerns for any cancer chemotherapy, if the cells have developed resistance to particular drugs or chemicals (Brambila et al. 2002). In this regard, many tumor cells that may have developed a generalized resistance to cancer chemotherapeutics show cross-resistance to particular chemicals (Zaman et al. 1995; Borst et al. 2000) through an as yet unidentified mechanism. Similarly, humans are exposed to PCBs under chronic exposures in environmental settings and undergo irreversible translational changes of different proteins that lead to various diseases. The goal of the study reported here was to test the hypothesis that chronic exposures to PCB in human cells in vitro is also associated with alteration of proteins.

For these particular cellular protein expression studies, we have acclimatized a human liver (HepG2) cell line to PCB-153, a non-coplanar and para-substituted compound (detailed in Materials and Methods), in our laboratory following chronic exposure to PCB-153. We have evaluated the effects of PCB-153 on HepG2 cells using various methods, including measurement of cell viability, immunoblotting studies, DNA fragmentation, and fluorochrome staining of nuclei (fluorescence microscopy).

MATERIALS AND METHODS

PCB

2,2′,4,4′,5,5′-Hexachlorobiphenyl (PCB-153) (CAS no. 035065) was purchased from Ultra Scientific (RI, USA; product no. RPC-047). A 50 mM stock solution of the compound was prepared in DMSO and diluted further, to the working concentration, in the same diluents. The final concentration of DMSO in the culture medium was ≤0.1%.

Antibody and Reagents

Antibodies recognizing the following antigen were purchased as follows: Bcl2, bak, and caspase-9 from Oncogene Research Products (Boston, MA); caspase-3, cleaved caspase-3, p53, p21, and c-myc from Calbiochem (San Diego, CA); Suicide Track DNA Ladder Isolation Kit from Oncogene (La Jolla, CA; catalog no. AM41); and horseradish peroxidase–conjugated secondary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Line and Culture Condition

In this study, we selected a metabolically competent human hepatocellular carcinoma cell line (HepG2), which retains many of the functions of normal liver cells (Knowles, Howe, and Aden 1980) and expresses the activities of several phase I and II xenobiotic-metabolizing enzymes (Kansmuller et al. 1998). The liver is one of the target sites of PCBs and plays a role in the oxidative metabolism in this organ. Above all, several authors have used this cell line as a model system in the study of inhibition of cell proliferation and cell death in vitro (Michalakis et al. 2007), of the role of genes involved in transcriptional and translational processes (Castaneda, Rosin-Steiner, and Jung 2006), of apoptosis (Ho et al. 2007), and of genotoxic effects (An et al. 2006). These cells have also been selected to see the efficacies of antioxidant potential of many compounds (Goya, Mateos, and Bravo 2007). They have also been used as artificial livers in hepatic failure dogs (Wang et al. 2005).

Human hepatocellular carcinoma (HepG2; ATCC no. HB-8065) cells were grown in their respective culture medium as recommended by the American Type Culture Collection (ATCC; Manassas, VA). In particular, HepG2 cells were maintained in1 × Delbecco’s modified Eagle medium (DMEM) with low glucose and containing 100 units/ml penicillin G and 100 μg/ml streptomycin medium supplemented with 10% fetal bovine serum (FBS) (Invitrogen; heat inactivated) at 37°C in a atmosphere containing 5% CO₂.

In Vitro Cytotoxicity and Cell Growth Assay

HepG2 cells were cultured in a 100-mm tissue culture dish with an initial cell number at which 40% to 50% confluency could be achieved after 24 h. These exponentially growing cells were exposed to different concentrations of PCB-153 for 48 h. The adherent cells were harvested with trypsin, washed with phosphate-buffered saline (PBS) twice, and collected by centrifugation at 1500 rpm for 5 min. Viable cell counts were enumerated by hemocytometer using 0.2% trypan blue (Freshney 1987). The experiment was repeated three or more times. The representative data shown in this paper were reproducible in three independent experiments.

PCB-153 Chronic Exposures of Liver (HepG2) Cells

Liver (HepG2) cells were plated in 75-cm² Nalgene filter cap flasks with a cell number at which 20% to 40% confluency would be achieved within 24 h. To mimic chronic exposure in these exponentially growing cells, we started acclimatizing those cells with a sublethal concentration of PCB-153 (20 μM), and cells were allowed to grow up to the next passage. The constant exposure and gradual increase in PCB-153 (in DMSO) concentration was maintained in subsequent passages, until the desired linear growth (trend of the doubling time similar to that of the normal HepG2 cells) with 50, 75, and 100 μM concentrations was reached; the cells were then considered as chronically exposed cells (R). Control cell lines were allowed to grow with DMSO only (≤0.1% of the total medium v/v) to ensure that the changes seen were not due to DMSO exposure. Each of these PCB-153–sensitized cells was exposed to PCB-153 in their respective chronic exposure level for a further 12 weeks in subsequent passages into new flasks following trypsinization, in which cells maintained their exponential growth. To define the stability of the acquired tolerance of PCB-153 in HepG2 cells, these cells were passaged in PCB-free medium for 2 weeks and then treated with PCB-153 of their respective (50 to 100 μM) concentration (Romach et al. 2000). The cellular viability study represents a similar exponential growth compared to control cells (Figure 2C1–E1 compared to A1).

Cell Lysis and Immunoblot Analysis

Following trypsinization and washing twice with PBS, cells were centrifuged at 100 × g (1500 rpm) and lysed with a lysis buffer consisting of 50 mM HEPES (pH 7.4), 20 mM EDTA, 0.5 mM sodium orthovanadate, 10 mM sodium glycerophosphate, 1 mM sodium fluoride, 10% glycerol, 0.5% Nondiet P40 (NP-40), 5 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (ABSF). 20 μg of total proteins (in each lane) were resolved by 4% to 20% Tris-glycine sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to a polyvinylidene fluoride (PVF) membrane (Immobilon; Millipore, Billerich, MA) in a Hoffer transblotter using 25 mM Tris, 192 mM glycine, and 20% methanol. After the transfer was completed (150 V, 2 to 3 h), the blots were blocked for an hour in a blocking buffer containing 5 (w/v) blotto (Santa Cruz Biotechnology, Santa Cruz, CA) in TTBS (10 mM Tris-HCl, 140 mM NaCl [pH 7.4], and 1% [v/v] Tween 20). The membranes were extensively washed three times with TTBS. The blots were placed in their respective primary antibodies at optimal conditions for 1 h. After three washes with PBS, the horseradish peroxidase–conjugated specific secondary antibody was added and further incubated in the presence of 5% (w/v) nonfat dry milk (blotto; Santa Cruz Biotechnology, CA) in TBS buffer containing 2.5% Tween 20 (T-TBS). The membranes were washed extensively in T-TBS, and detection was performed with an enhanced chemoluminescence reagent (Amersham Pharmacia Biotech, USA) according to the manufacturer’s instruction.

DNA Fragmentation Studies

The primary objective of this study was to see the effects of chronic exposure of PCB-153 in human livers cells. A simplified and fast protocol for the analysis of DNA fragmentation during apoptosis was adapted as previously described (Kratzmeier et al. 1999) after slight modification. Briefly, the HepG2 cells were grown in 100-mm tissue culture dishes (less than 10⁶ cells) and treated with 70 μM of PCB-153. All the plates were washed three times with PBS. Eight hundred microliters of lysis solution (0.2% Triton X, 10 mM Tris, 10 mM EDTA) was added to the plates and incubated for 5 min at 4°C. The plates were scraped thereafter to transfer the cells to a 2-ml microcentrifuge tube. The tubes were then centrifuged at 9400 × g for 10 min, and supernatant was transferred to a fresh tube, discarding the pellets. The supernatant containing the DNA was purified with a polymerase chain reaction (PCR) purification kit (Qiagen MiniElute PCR Purification Kit; catalog no. 28004). For regular DNA precipitation, to each 400 μl of supernatant, 8 μl of 5 M NaCl was added and vortexed for 5 s. Eight hundred microliters of ethanol was then added, again vortexed for 5 s, and the tubes were incubated on ice for 10 min. Tubes were then centrifuged at 13000 rpm for 5 min. The supernatant was discarded, and the ethanol was air dried. The precipitated DNA was resuspended in 30 μl of Rnase-free water. DNA concentration was determined by means of the absorbance at 260 nm, with 1 absorbance unit at 260 nm corresponding to 50 μg/ml of double-stranded DNA. DNA samples (5 μg/lane) were elctrophoresed in 1.5% agarose gel in a buffer containing 89 mM Tris (pH 8.0), 89 mM boric acid, and 2 mM EDTA, and were run for 1.5 h at 100 V. We use DNA molecular marker Suicide Track DNA Ladder Isolation Kit from Oncogene. After electrophoresis, the gel was stained in 0.5 μg/ml ethidium bromide solution, and DNA was visualized using ultraviolet lights. Lanes with positive control (cells treated with 0.5 μg/ml actinomycin D, provided in the kit) and negative control (cells without treatment of PCB or actinomycin D) were included.

Nuclear Morphology/Fluorescence Microscopic Studies

The biochemical hallmark of apoptosis is intranucleosomal DNA cleavage. The DNA-binding flurochrome Hoechst 33342 was used to assess the effect of PCB-153 on the extent of apoptosis in the HepG2 cell line. HepG2 cells were grown in a monolayer to 40% to 50% confluency in a LAB-TEK chamber slide (2-well; Paranox slide no. 177429) and treated with 70 μM of PCB-153 over 24 h with untreated cells as a control. After the treatment schedule (0 to 24 h), the cells were washed with 1 × PBS to remove the medium. Cells were then fixed with 100% cold methanol for 20 min, washed with PBS, and stained with 3 M Hoechst 33342 dye in PBS for 15 min. Cells were again washed with PBS. Excess PBS was blotted off from the slide, and the slide was mounted with the antifade mounting medium Vectra Mount (H-5000). Fluorescent nuclei were seen under a microscope using a green filter as described. Similarly, PCB-153–resistant HepG2 cells grown under chronic exposure were also plated in the same LAB-TEK chamber slide and exposed to the respective concentrations of PCBs (50, 75, 100 μM) on the same time schedule as the primary HepG2 cell culture. The remainder of the process was the same as described before.

Statistical Analysis

All cell viability results are expressed as mean ± SE of the three biological replicates. The percentage of living cells was calculated and a nonlinear regression was performed (dose-response curves). The x-axis has a linear scale rather than a logarithmic scale (see Figure 1). For quantification of the immunoblots, the background was subtracted from the scanned image and the band’s integrated density was quantified three times with ImageJ software (v1.34s from NIH). A nonlinear regression (second-order polynomial) curve was used to analyze the protein expression data of triplicate immunoblots, using Prism 4 software.

RESULTS

Cytotoxicity

The effect of PCB-153 as a single agent on the survival of HepG2 cells was first assessed for 24 h. At this point, PCB-153 inhibits HepG2 cell with 50% survival (LC₅₀) at 67 ± 2.5 (SE) μM (Figure 1). Significant decreases in cell viability were noted, with the increased concentration of PCB-153. For experimental purposes, we chose the 70 μM concentration of PCB-153 for treatment on HepG2 cells for the ease of experimental design. When the cytotoxicity of PCB-153 after chronic exposure to HepG2 cells was evaluated for the respective exposure levels (50, 75, 100 μM), there was no decrease in cellular viability, even after 12 weeks of continuous PCB-153 exposures. PCB-153 chronically exposed cells showed linear growth (Figure 2C1–E1 ) whenever their survival rates were checked compared to control HepG2 cells (Figure 2A1 ).

Nuclear Morphology

In order to test the relationship between PCB-153 congener treatment and apoptosis induction, HepG2 cells were cultured with a 70 μM (IC₅₀ level) concentration of PCB for different periods of time and analyzed for typical morphological features of apoptosis using Hoechst dye. Nuclear changes were observed by fluorescence microscopy. Florescence staining of apoptotic cells revealed changes in nuclear morphology. The result shown in Figure 2 shows apoptotic bodies (cell shrinkage, nuclear membrane blebbing, chromatin condensation). The apoptotic death is also supported by the results of the cell surviviality assay.

DNA Fragmentation

The qualitative apoptotic death was also determined by a DNA fragmentation assay, which follows the same time response curve profile. In fact, no changes were observed in DNA fragmentation at any treatment of PCB up to 8 h of exposure. At later time points (12 and 24 h) of PCB-153 exposure (70 μM), more DNA fragmentation from 12 h of exposure time (Figure 3) was observed than in control HepG2 cells. Therefore there was a correlation between the results obtained by the Hoechst dye technique and by the DNA fragmentation assay (Figures 2 and 3). In contrast, no changes were observed in DNA fragmentation in any of the HepG2 cells that were under chronic exposure (Figure 3), which also corroborates their linear growth (Figure 2C1–E1 ) compared to unexposed control cells (C). No significant differences in apoptotic induction from 0 to 24 h of exposure time were noted in control cells (C) incubated with 0.1% DMSO used as vehicle for PCB, including both positive and negative controls.

Immunoblotting Studies

Our study was designed to examine the basic differences for the important apoptotic inducer and tumor suppressor protein levels in chronic and time-dependent action of PCB-153 on HepG2 cells. We chose Bcl2, Bak, caspase-9, cleaved caspase-9, p21, p53, and c-myc for their relevance and importance in apoptosis and cancer.

When HepG2 cells were treated with PCB-153, several proteins such as Bcl2 and caspase-9 (active form), responsible for inducing apoptosis, were found to be significantly elevated in chronically treated HepG2 cells over a period of 12 weeks (Figure 4A and C ). Bak was found to be decreased (Figure 4A and B ). A nonlinear regression (second-order polynomial) was used to analyze the expression of data in triplicate immunoblots. Bcl2 showed a complete down-regulation over time, indicating mitochondrial damage (Figure 4A and B ).

In separate experiments, highly significant differences in protein expressions were observed when the effect of PCB on HepG2 cells (exposure up to 48 h) was compared with the cells chronically exposed to PCB over their respective concentrations (50, 75, 100 μM). c-myc and Bak were completely down-regulated over 48 h of short-term exposure (Figure 4A and B ). Bak was significantly up-regulated in PCB-153 chronically exposed human liver cells (Figure 4A and C ), whereas it shows down-regulation in higher exposures (100 μM), when compared to 50 μM chronically exposed cells (Figure 4A and C ). c-myc showed a down-regulation trend in chronically exposed groups with increasing concentrations (Figure 4C ).

The expression of Bcl2 also showed reverse expression, which was up-regulated in the chronically exposed human liver cell line (HepG2), and complemented the antiapoptotic nature, thereby maintaining continuous growth. Caspase-9 and cleaved caspase-9 showed altered protein expression when these two exposure levels were compared (Figure 4B and C ). Significant changes were found in the p21 and p53 tumor suppressor protein levels. They were completely down-regulated in short-term exposures, up to 48 h, of PCB-153 to HepG2 cells (Figure 4A and B ), whereas these proteins (p21 and p53) levels were up-regulated at their respective levels of exposure over 12 weeks’ time in PCB-153 chronically exposed human liver cells (R) (Figure 4A and C ). However, there is no such significant variation of the other protein at this level of exposure targeted so far in chronically PCB-exposed HepG2 cell groups at 50, 70, and 100 μM of PCB-153.

DISCUSSION

Most of the studies on the mechanism of toxicity of PCBs have been focused on coplanar, dioxin-like congeners mediating toxicological effects of these compounds (Safe 1994). However, it is increasingly clear that certain ortho-chlorine-substituted, non-coplanar PCB (PCB-153 here) congeners also exhibit important biological activities (Fisher et al. 1998). There is a continuing controversy about the carcinogenic effects of PCBs (Golden et al. 2003). The Agency for Toxic Substance and Disease Registry (ASTDR), in its 1999 report, found that the carcinogenicity of PCBs was not concluded, which was then reversed in 2000 by a report stating the carcinogenicity of PCBs (ASTDR 1999; ASTDR 2000). The reasons for these substantial changes in conclusions concerning the human carcinogenicity of PCBs are unknown, and it is still a controversial subject. The aim of this short study on cellular basis was to determine and compare the contribution of an ortho-substituted PCB congener (PCB-153) under acute and chronic exposures to the induction of apoptosis and alteration of related protein expression. We have evaluated the relevance of apoptosis in HepG2 cell death induced by PCB-153 in comparison to chronically PCB-153 exposed cells.

We have been reporting here for the first time, to our knowledge, on the protein expression changes of human liver cells (HepG2) under chronic PCB exposure. This has been a significant tool to demonstrate the marked differences between chronically exposed HepG2 cells and upon acute PCB exposure. Treatment of HepG2 cell cultures with PCB-153 resulted in the loss of cell viability in a time- and concentration-dependent manner in our experimental studies. The toxicity has been suggested to involve apoptosis (Lowe and Lin 2000). Thus, in the present study, we have combined morphological and biochemical techniques to establish the relevance of apoptosis in the death of HepG2 cells induced by PCB and the significant differences in the tumor suppressor p53 protein. In the time range utilized, the loss of cell viability originated by the treatment of HepG2 cell cultures with PCB-153 is fundamentally due to the induction of apoptosis. This has been reversed in the chronically exposed cells where the cellular integrity and nonapoptotic nature is pronounced by their linear growth and cellular morphology studies. The apoptosis begins and reaches higher values when the cells are incubated with PCB-153, using any of the techniques described in Materials and Methods. This is perfectly correlated with the loss of cell viability because PCB-153 is more toxic, arriving at 35% to 50% cellular death after 24 h of incubation with 70 μM, along with DNA fragmentation. There are several reports that confirm the induction of apoptosis by non-coplanar PCB congeners in a variety of biological systems (Shin et al. 2000; Hwang et al. 2001).

The activation of caspase during apoptosis results in the dramatic morphologic changes (Cohen 1997). Specifically, it seems that a number of death stimuli target mitochondria, stimulating the release of several proteins like Bcl2, Bak, and Bcl2L1, including cytochrome c. Once released into the cytosol, cytochrome c binds to its adapter molecule, Apaf-1, which then activates procaspase-9. Caspase-9 can signal downstream and activates procaspase-3 and -7 (Robertson and Orrenius 2000), and caspase-3 activation is an early marker and an irreversible point in the development of apoptosis (Saraste and Pulkki 2000). Our study demonstrates that di-ortho-substituted nonplanar PCB-153 congener may significantly induce apoptosis in a caspase-dependant manner (Chen et al. 2006), but the counter action of Bcl2 and Bak maintains and increases cellular viability in the chronically exposed PCB-resistant cell lines, which corroborates the earlier studies (Sanchez-Alonso et al. 2003). It shows that different molecular mechanisms may operate in the induction of apoptosis, depending on the planarity or nonplanarity of the PCB congeners as was evident in our earlier studies (Chen et al. 2006) and by others (Santiago et al. 2006).

Evidence suggests that activation of c-myc expression has contributed to genetic alterations of the c-myc locus in various malignancies, and the ability of c-myc to transform cultured cells and induce tumors in transgenic animals attests to its central role in many neoplasms (Dang 1991). The recent efforts directed toward understanding the function of c-myc protein in cancer biology are excellently discussed in a number of reviews (Bissonette, Echeverri, and Mahboubi 1992; Henricksson and Luscher 1996). In our study, the PCB-153 chronically exposed cells have shown overexpression of c-myc protein when compared to short-term exposures where no significant expressions were observed. This may be due to an indirect target gene of c-myc, whose expression is altered as a consequence of expression of the direct Myc target genes and whose expression is connected to c-myc–dependent phenotypes such as cellular proliferation, transformation, or apoptosis (Dang 1999). c-myc overexpression in the cells was also found to induce extensive apoptosis that is p53 independent and may be linked to lactate dehydrogenase (LDH)-A expression (Shin et al. 1998) which may also be inhibited by Bcl2 (Bissonette, Echeverri, and Mahboubi 1992).

In the last decade, significant advances have been made in the discovery of apoptosis and the genes that control it. Apoptosis, also known as programmed cell death, is a physiological and irreversible process in tissue homeostasis that leads to DNA fragmentation, which can be initiated not only by physiological stimuli but also various chemical substances (Piechotta et al. 1999). Now it is clear that some oncogene mutations disrupt apoptosis, leading to tumor initiation, progression, and metastasis (Lowe and Lin 2000). p53 was the first tumor suppressor gene linked to apoptosis, in which mutations occur in the majority of human tumors and are often associated with an advanced tumor stage (Wallace-Brodeur and Lowe 1999). However, p53 could induce apoptosis when overexpressed in a myeloid leukemia cell line, suggesting that p53 might also regulate cell survival (Yonish-Rouach et al. 1991). Although initial studies on Bcl2 and p53 established the importance of apoptosis in carcinogenesis, it is now clear that mutation in many cancer-related genes can disrupt apoptosis (Lowe and Lin 2000). In our study, p53 was completely down-regulated in short-term exposure over time (48 h) in HepG2 cells, showing a loss of cell viability and apoptosis (Figure 3). In contrast, p53 is highly overexpressed in the chronically exposed cells. The corresponding p21 levels was also up-regulated over long-term exposures (Figure 4). That p53 proteins overexpressed in PCB-resistant cells pose a greater puzzle. Disruption of several p53 effectors in apoptosis (e.g., Bax and caspase-9) can also promote oncogenic transformation and tumor development (McCurrach et al. 1997). In contrast, p21, a cell cycle kinase, which was elevated in long-term exposures essential for p53-mediated arrest, is rarely mutated in human tumors (Biggs and Kraft 1995). Nevertheless, these data do not imply that the other p53 activities are dispensable for tumor suppression; rather, they simply argue that its apoptotic activity is important.

Disruption of apoptosis may also contribute to tumor metastasis. To metastasize, a tumor cell must acquire the ability to survive in the bloodstream and invade foreign tissue. Some studies also argue that p53 and Bcl2 can also influence cell death in suspension (Nikoforov et al. 1996); others observe enrichment for p53 or Bcl2 overexpression in metastases (Popescu et al. 1998). Earlier, several researchers have shown that ‘non-dioxin-like’ PCBs suppress apoptosis in rat hepatocytes (Bohnenberger et al. 2001), which corroborates with our results in single-dose and time-dependent studies with PCB-153, but differs in the results of the chronically exposed human cell line.

In summary, our results show that the increase of the tumor suppressor protein p53, a hallmark of cellular response to genotoxic stress, is highly up-regulated (Figure 4A and C ) in PCB-153 chronically exposed cells. It is believed that this activity is responsible for tumor suppressive function (Brown and Wouters 1999) in chronically exposed cells, as well as inducing downstream p21 (Figure 4A and C ), despite significant activation of proto-oncogene c-myc. In our experimental condition, p53 acts as “guardian of the genome,” controlling the cell cycle and apoptosis. In short-term exposure of PCB-153 to HepG2 cells, the p53 protein level is low. Chemical inducers of DNA damage (Jessen-Eller et al. 2002) and other stresses (Oakley et al. 1996) normally increase p53 levels to induce apoptosis and might have caused the increase of p53 proteins in chronically PCB-exposed HepG2 cells. The counter action of Bcl2 and Bak increases cellular viability in the PCB chronically exposed HepG2 cells. These findings also agree with the DNA fragmentation (laddering) and nuclear deformation by fluorescence studies. This study could be a useful tool to validate the efficacies of PCBs to explore how these chemical agents impact living cells and to understand how inappropriate responses to environmental molecules and internal cellular cues can lead to the development of different diseases, on which further studies are warranted, and are in progress.