Inhibition of the promotion of hepatocarcinogenesis by 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB-153) by the deletion of the p50 subunit of NF-κB in mice

Abstract

Polychlorinated biphenyls (PCBs) are persistent and ubiquitous environmental chemicals that bioaccumulate and have hepatic tumor promoting activity in rodents. The present study examined the effect of deleting the p50 subunit of NF-κB on the hepatic tumor promoting activity of 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB-153) in mice. Both wild-type and p50−/− male mice were injected i.p. with diethylnitrosamine (DEN, 90 mg/kg) and then subsequently injected biweekly with 20 i.p. injections of PCB-153 (300 μmol/kg/injection). p50 deletion decreased the tumor incidence in both PCB- and vehicle-treated mice, whereas PCB-153 slightly (P = 0.09) increased the tumor incidence in wild-type and p50−/− mice. PCB-153 increased the total tumor volume in both wild-type and p50−/− mice, but the total tumor volume was not affected by p50 deletion in either PCB- or vehicle-treated mice. The volume of tumors that were positive for glutamine synthetase (GS), which is indicative of mutations in the beta-catenin gene, was increased in both wild-type and p50−/− mice administered PCB-153 compared to vehicle controls, and inhibited in p50−/− mice compared to wild-type mice (in both PCB- and vehicle-treated mice). The volume of tumors that were negative for GS was increased in p50−/− mice compared to wild-type mice but was not affected by PCB-153. PCB-153 increased cell proliferation in normal hepatocytes in wild-type but not p50−/− mice; this increase was inhibited in p50−/− mice. In hepatic tumors, the rate of cell proliferation was much higher than in normal hepatocytes, but was not affected by PCB treatment or p50 deletion. The rate of apoptosis, as measured by the TUNEL assay, was not affected by PCB-153 or p50 deletion in normal hepatocytes. In hepatic tumors, the rate of apoptosis was lower than in normal hepatocytes; PCB-153 slightly (P = 0.10) increased apoptosis in p50−/− but not wild-type mice; p50 deletion had no effect. Taken together, these data indicate that the absence of the NF-κB p50 subunit inhibits the promoting activity of PCB-153 and alters the proliferative and apoptotic changes in mouse liver in the response to PCBs.

Introduction

Polychlorinated biphenyls (PCBs) were commercially manufactured in the United States from 1930 to 1970 for use as dielectrics in transformers and capacitors, and as cooling fluids in hydraulic systems. PCBs have also been used in the formulation of lubricating and cutting oils, in pesticides and flame retardants, and as plasticizers in paints, copying paper, adhesives, sealants and plastics (World Health Organization, 1976). The stability of these compounds, one of their commercial attributes, has led to their worldwide distribution in the environment, an observation which was first reported by Jensen (1966). The production of PCBs peaked in 1970 and has steadily declined thereafter as many countries throughout the world have banned certain uses or limited their production. Nevertheless these compounds remain in use and in our environment today and represent a potential human health hazard (Robertson and Hansen, 2001).

Although most PCBs are not particularly acutely toxic, their persistence and lipophilicity as well as their propensity to accumulate in fatty tissues raise concerns over their long-term effects. In animals and humans, chronic exposure to PCBs produces a variety of effects including decreased body weight (wasting syndrome), chloracne, edema, liver hypertrophy, porphyria, estrogenic activity, immunosuppression and neurotoxicity (National Research Council, 1979, Robertson and Hansen, 2001). The carcinogenicity of PCBs in humans has been examined in several epidemiological studies, and associations have been noted in some studies between PCB exposure and cancers of the liver, biliary tract, and intestines, as well as malignant melanoma (Faroon et al., 2001). In the Yusho poisoning incident, a statistically significant increase in the mortality from liver cancer wasobserved in males (but not females) exposed to PCBs (Kuratsume et al., 1996).

The evidence to date indicates that mixtures of PCBs can induce preneoplastic lesions and hepatocellular carcinomas in animals when given at appropriate doses for extended periods of time (Silberhorn et al., 1990, Mayes et al., 1998). Although their potency varies, mixtures of halogenated biphenyls as well as many individual congeners have been reported to be promoters of carcinogenesis in various liver tumor models (Glauert et al., 2001). Generally those compounds which are inducers of cytochrome P-450 (such as the higher halogenated biphenyls) were more potent as promoters. This includes PCB congeners that activate the Ah receptor, the constitutive androstane receptor (CAR), and the pregnane X receptor (PXR) (Ludewig et al., 2007).

Although PCBs clearly have promoting activity in the liver, their mechanism of action is not known. A number of mechanisms have been proposed, including direct effects on signal transduction pathways, induction of oxidative stress, effects on vitamin A metabolism, and effects on intercellular communication (Glauert et al., 2001). One mechanism by which PCBs may promote hepatic tumors is by inducing oxidative damage in the liver. Forms of oxidative damage that may be important are the induction of lipid peroxidation, the induction of oxidative DNA damage, and the alteration of gene expression. The majority of studies have found that PCBs increase hepatic lipid peroxidation (Kamohara et al., 1984, Oda et al., 1987, Dogra et al., 1988, Pelissier et al., 1990, Saito, 1990, Fadhel et al., 2002). Certain congeneric PCBs administered for short time periods were found to increase the levels of oxidative DNA damage, using 8-hydroxyguanosine as the endpoint (Oakley et al., 1996).

Nuclear factor-κB (NF-κB) is a eukaryotic transcription factor family consisting of dimers of the following proteins: p50 (NF-κB1), p65 (RelA), p52 (NF-κB2), c-Rel, and RelB. It is normally found in the cytoplasm as an inactive dimer bound to an inhibitory subunit, IκB, which also has several family members, including IκBα, IκBβ, IκBγ, and IκBɛ (Karin and Lin, 2002). Upon activation, NF-κB is released from IκB and translocates to the nucleus, where it increases the transcription of specific genes. There are two main pathways: the classical pathway, in which the p50:p65 heterodimer is predominate; and an alternative pathway, in which the p52:RelB dimer is activated (Senftleben et al., 2001, Karin and Lin, 2002). These processes require the phosphorylation of IκB, followed by the subsequent degradation via an ubiquitin-mediated 26S proteosome pathway (Karin and Lin, 2002). A 900 kDa complex, termed the IκB kinase (IKK) complex has been identified and consists of two kinase subunits of IKK, IKKα and IKKβ, and a regulatory subunit, IKKγ (Zandi et al., 1997, Karin and Delhase, 2000). These two kinase subunits form homo- or hetero-dimers that phosphorylate IκB molecules, leading to their degradation. NF-κB has been shown to be important in the activation of genes that regulate cell proliferation and apoptosis (Beg et al., 1995, Fitzgerald et al., 1995).

Several studies have used genetically modified mice to examine the role of NF-κB subunits on cell proliferation and apoptosis in the liver and other tissues. A clear role for NF-κB in inhibiting apoptosis by TNF-α or other apoptosis inducers has been demonstrated in several cell types, in studies in which NF-κB activity has been inhibited by the deletion of one of its subunits, the inhibition of its translocation, or the expression of a dominant negative form of IκB (Beg and Baltimore, 1996, Vanantwerp et al., 1996, Wang et al., 1996, Xu et al., 1998a, Schoemaker et al., 2002). However, DNA synthesis and liver regeneration were not affected by the absence of the p50 subunit following partial hepatectomy or carbon tetrachloride treatment (Deangelis et al., 2001). Similarly, the hepatic-specific expression of a truncated IκBα super-repressor did not affect DNA synthesis, apoptosis, or liver regeneration following partial hepatectomy, but led to increased apoptosis after treatment with TNF-α (Chaisson et al., 2002). Also, the hepatic inflammatory response after ischemia/reperfusion was not altered in p50−/− mice (Kato et al., 2002). In addition, B cells lacking p50, RelB, or c-Rel (but not p52 or p65) have decreased proliferation in response to LPS (Kontgen et al., 1995, Sha et al., 1995, Snapper et al., 1996a, Snapper et al., 1996b, Horwitz et al., 1999). Overall, whether specific NF-κB subunits are essential for cell proliferation depends on the tissue and the stimulus for DNA synthesis.

One mechanism by which NF-κB may be activated is by increased oxidative stress. NF-κB can be activated in vitro by H₂O₂, and its activation can be inhibited by antioxidants, such as vitamin E or N-acetyl cysteine (NAC), or by increased expression of antioxidant enzymes (Staal et al., 1990, Schreck et al., 1991, Schreck et al., 1992, Meyer et al., 1993, Nilakantan et al., 1998, Li et al., 2000, Calfee-Mason et al., 2004). In addition, agents that activate NF-κB frequently also increase oxidative stress (Schmidt et al., 1995). However, Hayakawa et al. (2003) found that NAC inhibits NF-κB activation independently of its antioxidant function.

PCBs also can activate NF-κB. PCB-77 was found to activate NF-κB in rats after long-term treatment but not after a single dose (Tharappel et al., 2002, Lu et al., 2003, Glauert et al., 2005). PCB-153, in contrast, was found to activate NF-κB in rats after a single dose (Lu et al., 2003), but only activated NF-κB after multiple doses in one of three studies (Tharappel et al., 2002, Lu et al., 2004, Glauert et al., 2005). We subsequently found that cell proliferation induced by PCB-153 could be inhibited in mice lacking the p50 subunit of NF-κB (p50−/− mice) (Lu et al., 2004).

In this study we have examined if the deletion of the p50 subunit of NF-κB would inhibit the tumor promoting activity of PCB-153. Wild-type and p50−/− mice were first administered diethylnitrosamine (DEN) as a tumor initiator and then were exposed to PCB-153. The induction of hepatic tumors as well as effects on cell proliferation and apoptosis were quantified.