PPAR alpha and the regulation of cell division and apoptosis.

Peroxisome proliferators (PPs) such as the hypolipidaemic drug, nafenopin and the phthalate plasticiser 2-diethylhexylphthalate induce rodent hepatocyte cell proliferation and suppress apoptosis leading to tumours. PPs act via the nuclear hormone receptor peroxisome proliferator activated receptor alpha (PPAR alpha) which directly regulates genes implicated in the response to PPs such as the peroxisomal gene acyl CoA oxidase. As expected for xenobiotics that perturb proliferation, PPs alter expression of cell cycle regulatory proteins. However, the ability to alter expression of cyclins and cyclin-dependent kinases is shared by physiological hepatic mitogens such as epidermal growth factor and is thus unlikely to be specific to the PP-induced aberrant growth associated with hepatocarcinogenesis. Recent evidence suggests that the response of hepatocytes to PPs is not only dependent upon PPAR alpha but also on the trophic environment provided by nonparenchymal cells and by cytokines such as tumour necrosis factor alpha. Additionally, the ability of PPs to suppress apoptosis and induce proliferation depends upon survival signalling mediated by p38 mitogen activated protein kinase. The cross talk between PPAR alpha-mediated transcription, survival signalling and cell cycle will be discussed with particular emphasis on relevance to toxicology.

Role of cytokines in non-genotoxic hepatocarcinogenesis: cause or effect?

Chemicals with the potential to cause cancer through damaging DNA can be readily identified in a range of in vitro screens that detect genotoxicity. However, many carcinogens are non-genotoxic yet cause rodent tumours, particularly in the liver. Some non-genotoxic carcinogens such as the peroxisome proliferators (PPs) act directly to cause liver growth and proliferation, whereas others such as carbon tetrachloride cause liver damage, followed by regenerative hyperplasia. Current data support a role for cytokines such as tumour necrosis factor alpha (TNFalpha) and interleukin 1 (IL1) in hepatocarcinogenesis. However, these data give rise to conflicting hypotheses; in some experimental models, TNFalpha appears to mediate damage, whereas in others it is postulated to play a role in tissue repair. Recently, we have shown that TNFalpha acting via TNFalpha receptor 1 and p38 MAP kinase suppresses hepatocyte apoptosis. However, when new protein synthesis is disabled, TNFalpha becomes a death signal. An understanding of the role of cytokines in rodent hepatocarcinogenesis will allow the development of markers that can be used to identify, at an early stage, those chemicals with the potential to induce rodent tumours.

Identification of a possible association between carbon tetrachloride-induced hepatotoxicity and interleukin-8 expression.

Hepatotoxicants can elicit liver damage by various mechanisms that can result in cell necrosis and death. The changes induced by these compounds can vary from gross alterations in DNA repair mechanisms, protein synthesis, and apoptosis, to more discrete changes in oxidative damage and lipid peroxidation. However, little is known of the changes in gene expression that are fundamental to the mechanisms of hepatotoxicity. We have used DNA microarray technology to identify gene transcription associated with the toxicity caused by the hepatotoxicant carbon tetrachloride. Labeled poly A+ RNA from cultured human hepatoma cells (HepG2) exposed to carbon tetrachloride for 8 hours was hybridized to a human microarray filter. We found that 47 different genes were either upregulated or downregulated more than 2-fold by the hepatotoxicant compared with dimethyl formamide, a chemical that does not cause liver cell damage. The proinflammatory cytokine interleukin-8 (IL-8) was upregulated over 7-fold compared with control on the array, and this was subsequently confirmed at 1 hour and 8 hours by Northern blot analyses. We also found that carbon tetrachloride caused a time-dependent increase in interleukin-8 protein release in HepG2 cells, which was paralleled by a decrease in cell viability. These data demonstrate that carbon tetrachloride causes a rapid increase in IL-8 mRNA expression in HepG2 cells and that this increase correlates with a later and significant increase in the levels of interleukin-8 protein. These results illustrate the potential of microarray technology in the identification of novel gene changes associated with toxic processes.

A dominant negative form of IKK2 prevents suppression of apoptosis by the peroxisome proliferator nafenopin.

Peroxisome proliferators (PPs) are a class of non-genotoxic chemicals that cause rodent liver enlargement and hepatocarcinogenesis. In primary rat hepatocyte cultures, PPs suppress spontaneous apoptosis and that induced by a number of pro-apoptotic stimuli such as transforming growth factor-beta(1). Tumour necrosis factor alpha (TNF-alpha) and the transcription factor NFkappaB have been implicated in the mode of action of PPs. TNF-alpha signalling to NFkappaB is thought to be responsible for many of the effects elicited by this cytokine. NFkappaB regulates gene expression in immunity, stress responses and the inhibition of apoptosis. Activation of NFkappaB requires the successive action of NFkappaB-inducing kinase and the phosphorylation of NFkappaB inhibitory proteins (IkappaB) by an IkappaB kinase (IKK) complex. The IKK2 subunit of IkappaB kinase is thought to be essential for NFkappaB activation and prevention of apoptosis. To determine whether IKK2 plays a role in the suppression of apoptosis by PPs, we expressed a dominant negative form of IKK2 (IKK2dn) in primary rat hepatocyte cultures. Infection with an adenovirus construct expressing IKK2dn caused apoptosis in control primary rat hepatocytes in the absence of exogenous TNF-alpha. Moreover, IKK2dn-induced apoptosis could not be rescued by addition of TNF-alpha or the peroxisome proliferator nafenopin. These results demonstrate a requirement for intracellular signalling pathways mediated by IKK2 in the suppression of apoptosis by the PP class of hepatocarcinogens.

Species differences in response to diethylhexylphthalate: suppression of apoptosis, induction of DNA synthesis and peroxisome proliferator activated receptor alpha-mediated gene expression.

Diethylhexylphthalate (DEHP) is a phthalate plasticizer that belongs to the peroxisome proliferator (PP) class of rodent nongenotoxic hepatocarcinogens. Previously, we have shown that MEHP (a principal metabolite of DEHP and the proximal PP) induced DNA synthesis and suppressed apoptosis in rat but not in human hepatocytes in vitro. Here, we present further studies of species differences in response to DEHP. In rats, 4 days of exposure to DEHP (950 mg/kg per day by gavage) induced peroxisomal beta-oxidation, DNA synthesis and suppressed apoptosis. In contrast, there was no response of guinea pig liver to DEHP. In rat hepatocytes in vitro, MEHP (250, 500 and 750 microM) induced peroxisomal beta-oxidation, DNA synthesis and suppressed apoptosis. In contrast to the pleiotropic response noted in rat hepatocytes, there was no response of human hepatocytes to 250, 500 or 750 microM MEHP. PPs activate the peroxisome proliferator activated receptor alpha (PPARalpha) that binds to DNA at peroxisome proliferator response elements (PPREs) within the promoters of PP-responsive genes such as rat acyl CoA oxidase (ACO). However, the human ACO gene promoter differs at three bases within the PPRE from the rat ACO promoter and appears refractory to PPs. To address species differences in response to DEHP at the molecular level, we used promoter-reporter gene assays to compare the ability of MEHP to induce gene expression from the rat or the human ACO promoter. MEHP gave a concentration-dependent increase in reporter gene expression from the rat ACO gene promoter with either mouse or human PPARalpha. In contrast, the human ACO promoter was unable to drive MEHP-induced gene transcription irrespective of the species origin of PPARalpha. These data provide further weight of evidence at the cellular and molecular levels for a lack of risk to human health from the phthalate DEHP.

Apoptosis and proliferation in nongenotoxic carcinogenesis: species differences and role of PPARalpha.

Peroxisome proliferators (PPs) are nongenotoxic rodent hepatocarcinogens that cause liver enlargement and hepatocarcinogenesis associated with peroxisome proliferation, induction of hepatocyte DNA synthesis and suppression of apoptosis. Acyl CoA oxidase (ACO) is a key enzyme of peroxisomal beta-oxidation and its transcriptional activation by PPs is often used as marker for the rodent response. PPs activate the peroxisome proliferator activated receptor-alpha, PPARalpha. Recent data suggest a role for tumour necrosis factor alpha (TNFalpha). This cytokine appears to be permissive for a PPARalpha-dependent growth response to PPs. Humans and guinea pigs appear to be nonresponsive to the adverse effects of PPs noted in rodents. These species differences can be attributed to reduced quantity of full length functional PPARalpha in human liver and evidence supports the presence of a truncated form of PPARalpha, hPPARalpha8/14 in human liver. In addition, species differences could be attributed to qualitative differences in the PPARalpha-mediated response because the promoter for human ACO differs in sequence and activity from the rat equivalent. These data contribute to our understanding of how chemicals may cause tumours in rodents and how this response may differ in humans.

Suppression of mouse hepatocyte apoptosis by peroxisome proliferators: role of PPARalpha and TNFalpha.

Peroxisome proliferators (PPs) are a diverse group of nongenotoxic chemicals that in rodents cause hepatic peroxisome proliferation, liver enlargement, increased replicative DNA synthesis and suppression of apoptosis. The effects of PPs in vivo can be reproduced in vitro where PPs can induce mouse hepatocyte DNA synthesis and suppress both spontaneous apoptosis and that induced by transforming growth factor beta (TGFbeta). In vitro, high concentrations (>500 U/ml) of exogenous tumour necrosis factor (TNFalpha) [M. Rolfe, N.H. James, R.A. Roberts, TNF suppresses apoptosis and induces S-phase in rodent hepatocytes: a mediator of the hepatocarcinogenicity of peroxisome proliferators?, Carcinogenesis 18 (1997) 2277-2280] are also able to stimulate hepatocyte DNA synthesis and suppress apoptosis, implicating TNFalpha in mediating or permitting the liver growth response to PPs. Here, using cultured mouse hepatocytes isolated from PPARalpha null mice, we have examined the role of the peroxisome proliferator activated receptor alpha (PPARalpha) in mediating the suppression of apoptosis caused by PPs. In addition we have investigated further the role of TNFalpha in mediating the rodent response to PPs. The PP nafenopin (50 microM) was unable to stimulate DNA synthesis measured by bromodeoxyuridine incorporation in these PPARalpha null mouse hepatocytes (96% of control), unlike epidermal growth factor, a growth factor used as a positive control. In assays of apoptosis using H33258 staining of chromatin condensation, nafenopin was unable to suppress either spontaneous or TGFbeta1-induced apoptosis. In contrast, high concentrations of TNFalpha (>500 U/ml) were able to both stimulate DNA synthesis (204% of control) and suppress apoptosis in PPARalpha null hepatocytes (40% and 38% of control for spontaneous and TGFbeta1-induced apoptosis respectively). However, TNFalpha could not stimulate beta-oxidation of palmitoyl CoA in either PPARalpha null mouse or B6C3F1 (PPARalpha wild type) mouse hepatocytes. These data confirm the dependence of the response to PPs on PPARalpha by demonstrating that PPARalpha mediates the suppression of hepatocyte apoptosis in response to PPs. In addition, the data provide evidence that high concentrations of TNFalpha can modulate DNA synthesis and apoptosis in the absence of PPs and PPARalpha. Thus, in vivo, physiological levels of TNFalpha may be permissive for a PPARalpha-dependent growth response to PPs.

Tumour necrosis factor alpha (TNFalpha): role in suppression of apoptosis by the peroxisome proliferator nafenopin.

The peroxisome proliferator (PPs) class of non-genotoxic rodent hepatocarcinogens induce mouse hepatocyte DNA synthesis and suppress apoptosis. This phenotype can be reproduced in vitro using exogenous tumour necrosis factor alpha (TNFalpha), suggesting a role for TNFalpha in mediating the liver growth response to PPs. In hepatocytes isolated from the peroxisome proliferator activated receptor alpha (PPARalpha) null mouse, PPs are unable to stimulate DNA synthesis or to suppress either spontaneous or TGFbeta1-induced apoptosis. However, the ability of TNFalpha to modulate hepatocyte survival and growth is unaltered, suggesting that TNFalpha acts independently or downstream of PPARalpha to mediate the growth changes associated with PPARalpha activation. Since PPARalpha is a ligand activated transcription factor, we determined if TNFalpha gene expression was altered by PP treatment during an early time window preceding PP-induced growth changes. However there was no induction of TNFalpha expression by nafenopin over the constitutive levels noted in control cultured cells. In summary, TNFalpha acts downstream or independently of PPARalpha to mediate the suppression of apoptosis and induction of DNA synthesis by PPs. In this in vitro model, the PP nafenopin do not appear to mediate de novo TNFalpha gene expression suggesting that the response to nafenopin may be mediated by bioactivation or release of pre-existing TNFalpha protein from Kupffer cells.

The role of protein kinase B and mitogen-activated protein kinase in epidermal growth factor and tumor necrosis factor alpha-mediated rat hepatocyte survival and apoptosis.

Perturbation of hepatocyte growth regulation is associated with a number of liver diseases such as fibrosis and cancer. These diseases are mediated by a network of growth factors and cytokines that regulate the induction of hepatocyte proliferation and apoptosis. In this study, we have investigated the role of signaling pathways activated by tumor necrosis factor alpha (TNF-alpha) and epidermal growth factor (EGF) in the regulation of apoptosis induced by transforming growth factor beta(1) (TGF-beta(1)), because this physiological factor is believed to regulate spontaneous apoptosis in the liver. We show that pretreatment with (10 ng/mL) EGF or (25 ng/mL) TNF-alpha can suppress TGF-beta(1)-induced apoptosis by 73% and 50%, respectively, in isolated rat hepatocytes. However, suppression of TGF-beta(1)-induced apoptosis by EGF and TNF-alpha occurs via different protein kinase signaling pathways. Using specific inhibitors, we show that suppression of apoptosis by EGF is dependent on activation of phosphoinositide 3-kinase (PI 3-kinase) and the extracellular signal regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathways, but not p38 MAP kinase. In contrast, suppression of TGF-beta(1)-induced apoptosis by TNF-alpha does not require PI 3-kinase and protein kinase B (PKB or Akt)-mediated pathways, but is dependent on ERK and p38 MAP kinase activity. These data contribute to our understanding of the intracellular survival signals that play a role in normal liver homeostasis and in diverse pathological conditions.

Role for tumor necrosis factor alpha receptor 1 and interleukin-1 receptor in the suppression of mouse hepatocyte apoptosis by the peroxisome proliferator nafenopin.

Peroxisome proliferators (PPs) cause rodent liver enlargement and tumors. In vitro, PPs induce rat and mouse hepatocyte DNA synthesis and suppress apoptosis, a response mimicked by exogenous tumor necrosis factor alpha (TNFalpha). Here, we determine the role of TNF receptor 1 (TNFR1), TNF receptor 2 (TNFR2), and nuclear factor kappa beta (NFkappaB) in the response of mouse hepatocytes to the PP, nafenopin. Nafenopin (50 micromol/L) induced DNA synthesis as measured by bromodeoxyuridine (BrdU) incorporation, suppressed cell death as measured by Hoechst 33258 staining, induced peroxisomal beta-oxidation as measured by cyanide insensitive palmitoyl CoA oxidation (PCO) and caused activation of nuclear factor kappa beta (NFkappaB) as determined by electrophoretic mobility gel shift assay (EMSA). The induction of DNA synthesis and the suppression of apoptosis in response to nafenopin was abrogated completely by blocking antibodies to TNFR1 but not to TNFR2. In contrast, the induction of peroxisomal beta-oxidation by nafenopin was not blocked by the anti-TNFR1 antibody. Next, we evaluated the response of hepatocytes to interleukin-1 (IL-1), another proinflammatory cytokine. IL-1alpha (2.5 ng/mL) and, to a lesser extent, IL-1beta (5 ng/mL), shared the ability of TNFalpha to induce DNA synthesis and suppress apoptosis. In addition, anti-IL-1 receptor, type 1/p80 (IL-1R) antibodies were able to abrogate the response to nafenopin. IL-1alpha was still able to perturb hepatocyte growth in the presence of the anti-TNFR1 antibody suggesting that IL-1alpha acts independently rather than by elaborating TNFalpha. In summary, these data provide additional evidence for a role for hepatic cytokines in the perturbation of hepatocyte growth by PPs such as nafenopin.

Suppression of apoptosis and induction of DNA synthesis in vitro by the phthalate plasticizers monoethylhexylphthalate (MEHP) and diisononylphthalate (DINP): a comparison of rat and human hepatocytes in vitro.

Diethylhexylphthalate (DEHP) and diisononylphthalate (DINP) are plasticizers with many important commercial, industrial and medical applications. However, both DEHP and DINP are rodent peroxisome proliferators (PPs), a class of compounds that cause rodent liver tumours associated with peroxisome proliferation, induction of hepatic DNA synthesis and the suppression of apoptosis. Despite these effects in the rodent, humans appear to be nonresponsive to the adverse effects of PPs. Previously, we have shown that the fibrate hypolipidaemic peroxisome proliferator, nafenopin, induced DNA synthesis and suppressed apoptosis in rat but not in human hepatocytes. In this work, we have examined species differences in the response of rat and human hepatocytes to DEHP and DINP in vitro. In rat hepatocytes in vitro, both DINP and MEHP (a principle metabolite of DEHP and the proximal peroxisome proliferator) caused a concentration-dependent induction of DNA synthesis and suppression of both spontaneous and transforming growth factor beta1 (TGFbeta1)-induced apoptosis. Similarly, both MEHP and DINP caused a concentration-dependent induction of peroxisomal beta-oxidation although the response to DINP was less robust. In contrast to the pleiotropic response noted in rat hepatocytes, neither DINP nor MEHP caused an induction of beta-oxidation, stimulation of DNA synthesis and suppression of apoptosis in human hepatocytes cultured from three separate donors. These data provide evidence for species differences in the hepatic response to the phthalates DEHP and DINP, confirming that human hepatocytes appear to be refractory to the hepatocarcinogenic effects of PPs first noted in rodents.

The non-genotoxic hepatocarcinogen nafenopin suppresses rodent hepatocyte apoptosis induced by TGFbeta1, DNA damage and Fas.

The suppression of apoptosis may contribute to the carcinogenicity of the peroxisome proliferators (PPs), a class of non-genotoxic rodent hepatocarcinogens. Our previous work demonstrated that the PP nafenopin suppressed both spontaneous and transforming growth factor beta1 (TGFbeta1)-induced hepatocyte apoptosis both in vivo and in vitro. Here, we extend these observations by demonstrating the ability of nafenopin to suppress apoptosis induced by other major candidates for the signalling of cell death in the liver. Treatment of rat or mouse hepatocyte monolayers with TGFbeta1 or the DNA damaging drugs etoposide or hydroxyurea induced high levels of apoptosis. Western blot analysis did not support a role for either p53 or p21waf1 in etoposide-induced apoptosis in rat hepatocytes. Treatment of mouse hepatocytes with an agonistic anti-Fas antibody also resulted in an induction of high levels of apoptosis. Pre-addition and continued exposure to nafenopin suppressed apoptosis induced by all three stimuli. Overall, our studies demonstrate that the ability of nafenopin to protect hepatocytes from apoptosis is not restricted to species or apoptotic stimulus. It is possible, therefore, that the PPs may suppress apoptosis by acting on diverse signalling pathways. However, it seems more likely that nafenopin suppresses hepatocyte apoptosis elicited by each death stimulus by impinging on a core apoptotic mechanism.

A peroxisome proliferator-activated receptor-alpha (PPARalpha) cDNA cloned from guinea-pig liver encodes a protein with similar properties to the mouse PPARalpha: implications for species differences in responses to peroxisome proliferators.

The peroxisome proliferator class of non-genotoxic rodent hepatocarcinogens cause hepatocyte DNA synthesis, peroxisome proliferation and liver tumours when administered to rats and mice, but fail to induce S-phase or peroxisome proliferation in hepatocytes from other species including guinea-pigs, dogs, and primates including humans. There are compelling data that implicate a nuclear receptor, the peroxisome proliferator-activated receptor-alpha (PPARalpha) as an important mediator of the toxic and carcinogenic effects of peroxisome proliferators (PPs). We were interested to consider the guinea-pig as a possible model for human responses to these compounds. This manuscript describes the isolation of a full-length cDNA encoding PPARalpha from guinea-pig liver that is closely related to receptors identified previously in mouse, rat and human. RNA hybridisation experiments suggested that the livers of the PP-responsive rat and mouse contained relatively high levels of PPARalpha transcripts, whereas in human and guinea-pig liver PPARalpha mRNA was much less abundant. Functional analyses suggested that the guinea-pig PPARalpha was able to be activated by PPs. DNA binding studies using in vitro translated proteins showed that the guinea-pig receptor was able to bind specifically to DNA in the presence of the retinoid X receptor (RXR), and transient transfection assays showed that the guinea-pig PPARalpha was capable of being transcriptionally activated in a concentration-dependent fashion by the PPs Wy-14,643 and nafenopin. Also, in guinea-pig primary hepatocyte cultures, a dominant negative repressor of PPARalpha ablated the suppression of spontaneous apoptosis by PPs. Taken together, these data show that the 'non-responsive' guinea-pig expresses active PPARalpha in the liver at reduced levels, and may be a useful model for exploring the mechanisms underlying the human response to PPs.

Evidence for the suppression of apoptosis by the peroxisome proliferator activated receptor alpha (PPAR alpha).

Peroxisome proliferators (PPs) are a class of nongenotoxic rodent hepatocarcinogens. We have demonstrated previously that PPs suppress both spontaneous rat hepatocyte apoptosis and that induced by exogenous stimuli such as transforming growth factor-beta1 (TGFbeta1). PPs transcriptionally activate the peroxisome proliferator activated receptor-alpha (PPAR alpha), a member of the nuclear hormone receptor superfamily. Here, we investigate whether activation of PPAR alpha mediates the suppression of rat hepatocyte apoptosis induced by PPs. We isolated a naturally occurring variant form of PPAR alpha (hPPAR alpha-6/29) from human liver by PCR cloning. Electrophoretic mobility shift assays (EMSA) demonstrated that hPPAR alpha-6/29 shared the ability of mPPAR alpha to heterodimerise with the retinoid X receptor (RXR) and bind to DNA. When hPPAR alpha-6/29 was transfected into Hepa1c1c7 cells together with a reporter plasmid containing a PPAR response element (PPRE), hPPAR alpha-6/29, unlike mPPAR alpha, could not be activated by PPs. Furthermore, hPPAR alpha-6/29 could act as a dominant negative regulator of PPAR-mediated gene transcription since increasing concentrations of hPPAR alpha-6/29 abrogated the activation of co-transfected mPPAR alpha. When introduced into primary rat liver cell cultures by transient transfection, hPPAR alpha-6/29 prevented the suppression of hepatocyte apoptosis by the PP nafenopin, but not that seen in response to phenobarbitone (PB), a nongenotoxic carcinogen whose action does not involve PPAR alpha. The suppression of hepatocyte apoptosis was abrogated completely even though only 30% of hepatocytes were transfected, suggesting the involvement of a soluble factor. These data indicate that activation of rat liver PPAR alpha provides a survival signal for hepatocytes, preventing their death in response to apoptotic stimuli.

The peroxisome proliferator nafenopin does not suppress hepatocyte apoptosis in guinea-pig liver in vivo nor in human hepatocytes in vitro.

In rats and mice, nafenopin is a nongenotoxic hepatocarcinogen, which induces hepatic DNA synthesis and enzyme induction both in vivo and in hepatocyte cultures in vitro. However, humans and guinea-pigs are considered to be non-responsive to the liver growth effects of peroxisome proliferators (PPs). The ability to stimulate cell replication coupled with the ability to suppress apoptosis is thought to underpin the carcinogenicity of nongenotoxic carcinogens such as PPs. Previous studies in this laboratory have shown that in rats in vivo and in vitro nafenopin suppressed spontaneous hepatocyte apoptosis and that induced by the physiological negative growth regulator transforming growth factors beta1 (TGFbeta1). In addition nafenopin suppressed apoptosis in cultured hepatocytes from guinea-pig and hamster. The effects of PPs on apoptosis in human hepatocyte cultures is not known. To correlate these previous in vitro findings to the known species differences in hepatocarcinogenicity of PPs we have investigated the effects of nafenopin on guinea-pig liver growth in vivo. Also, we have examined the effects of nafenopin on apoptosis in cultures of human hepatocytes, a valuable model for human risk assessment. Nafenopin did not inhibit either spontaneous or TGFbeta1 induced apoptosis in human hepatocytes in vitro. Administration of nafenopin to guinea-pigs in vivo produced none of the changes seen previously in responsive species, such as rats and mice. There was no change in liver/body weight ratio, peroxisomal volume of hepatocytes or DNA synthesis as determined by incorporation of bromodeoxyuridine and there was no suppression of apoptosis. The lack of response to nafenopin in guinea-pigs in vivo and human hepatocytes in vitro provides further evidence that these species may be refractory to the liver growth effects of PPs despite the ability of guinea-pigs and humans to respond to PPs by alterations in lipid metabolism. The data presented add to our overall understanding of species differences in response to the PP class of rodent nongenotoxic carcinogens.

Suppression of hepatocyte apoptosis and induction of DNA synthesis by the rat and mouse hepatocarcinogen diethylhexylphlathate (DEHP) and the mouse hepatocarcinogen 1,4-dichlorobenzene (DCB).

Nongenotoxic rodent hepatocarcinogens do not damage DNA but cause liver tumours in the rat and mouse, associated with the induction of hepatic DNA synthesis. Previously, we have demonstrated that nongenotoxic hepatocarcinogens such as phenobarbitone and the peroxisome proliferator (PP), nafenopin, also suppress rat hepatocyte apoptosis. The nongenotoxic chemicals 1,4-dichlorobenzene (DCB) and the PP, diethylhexyl phthalate (DEHP), both induce high levels of DNA synthesis in rat liver in vivo, but only DEHP is hepatocarcinogenic in this species. Here, we investigate whether the difference in rat carcinogenicity of these two hepatic mitogens may be due to differences in their ability to suppress hepatocyte apoptosis. In rat hepatocytes in vitro, MEHP (the active metabolite of DEHP) induced DNA synthesis 2.5-fold (P = 0.001) and suppressed 10- and 4-fold, respectively both spontaneous (P = 0.0008) and transforming growth factor beta1 (TGFbeta1)-induced (P = 0.0001) apoptosis. DCB gave a small (1.7-fold) increase in DNA synthesis (P = 0.03) and a small (1.7- to 2-fold) suppression of both spontaneous (P = 0.022) and TGFbeta1-induced (P = 0.015) apoptosis. We next analysed the induction of DNA synthesis and the suppression of apoptosis in rat liver in vivo. Both DEHP and DCB were able to induce DNA synthesis although, as seen in vitro, the induction by DCB (4.2-fold; P = 0.023) was less marked than that with DEHP (13.4-fold; P = 0.007). Similarly, DEHP and DCB were both able to suppress rat hepatocyte apoptosis in vivo but the magnitude of the suppression was comparable; apoptosis was reduced to undetectable levels in four out of five animals with DCB and three out of five with DEHP. Since both chemicals suppressed apoptosis and induced DNA synthesis in rat liver but, overall, DCB was less potent, the disparate hepatocarcinogenic potential of these two chemicals could arise from differences in the magnitude of growth perturbation. To test this hypothesis, we repeated the studies in mouse, a species where both DCB and DEHP are hepatocarcinogenic. Both in vitro and in vivo, DCB and DEHP/MEHP were able to suppress apoptosis and induce hepatocyte DNA synthesis in the mouse with comparable potencies. The data support the hypothesis that the carcinogenicity of nongenotoxic hepatocarcinogens is associated strongly with the ability to perturb hepatocyte growth regulation. However, the ability to effect such changes is not unique to nongenotoxic carcinogens and is common to some noncarcinogenic chemicals, such as DCB, suggesting that the growth perturbation may need to exceed a threshold for carcinogenesis.

Peroxisome proliferator-activated receptor (PPAR) alpha-regulated growth responses and their importance to hepatocarcinogenesis.

Peroxisome proliferators (PPs) are a class of non-genotoxic rodent hepatocarcinogens that act by perturbing liver growth regulation. We have demonstrated previously that PPs suppress both spontaneous rat hepatocyte apoptosis and that induced by exogenous stimuli such as transforming growth factor-beta1 (TGF beta1). More recently, we have demonstrated that PPs can suppress apoptosis induced by more diverse stimuli such as DNA damage or ligation of Fas, a receptor related to the tumour necrosis factor alpha (TNF alpha) family of cell surface receptors. PPs transcriptionally activate the peroxisome proliferator activated receptor-alpha, PPAR alpha, a member of the nuclear hormone receptor superfamily. We investigated whether activation of PPAR alpha mediates the suppression of rat hepatocyte apoptosis induced by PPs. We isolated a naturally occurring variant form of PPAR alpha (hPPAR alpha-6/29) from human liver by PCR cloning. hPPAR alpha-6/29 shared the ability of mPPAR alpha to bind to DNA but, unlike mPPAR alpha, could not be activated by PPs. Furthermore, hPPAR alpha-6/29 could act as a dominant negative regulator of PPAR-mediated gene transcription. When introduced into primary rat liver cell cultures by transient transfection, hPPAR alpha-6/29 prevented the suppression of hepatocyte apoptosis by the PP nafenopin, but not that seen in response to phenobarbitone (PB), a non-genotoxic carcinogen whose action does not involve PPAR alpha. The suppression of hepatocyte apoptosis was abrogated completely even though only 30% of hepatocytes were transfected, suggesting the involvement of a soluble factor. Recent data have suggested that TNF alpha, perhaps released by liver Kupffer cells in response to PPs, may play a key role in mediating the effects of PPs on hepatocyte growth regulation.

Tumour necrosis factor alpha (TNF alpha) suppresses apoptosis and induces DNA synthesis in rodent hepatocytes: a mediator of the hepatocarcinogenicity of peroxisome proliferators?

Peroxisome proliferators (PPs) are a class of non-genotoxic rodent hepatocarcinogens that cause increased hepatocyte DNA synthesis, peroxisome proliferation and liver enlargement. We have demonstrated previously that PPs suppress both spontaneous rat hepatocyte apoptosis and that induced by the physiological negative regulator of liver growth, transforming growth factor beta (TGF beta1). Evidence suggests that the suppression of apoptosis by PPs is mediated via activation of the peroxisome proliferator activated receptor-alpha (PPAR alpha), a member of the nuclear hormone receptor superfamily. Here, we investigate the effects of tumour necrosis factor alpha (TNF alpha) on cultured rat or mouse hepatocytes to determine whether TNF alpha influences hepatocyte growth in a manner analogous to that seen with PPs. Rat recombinant TNF alpha was found to stimulate DNA synthesis and suppress apoptosis in isolated rat hepatocyte monolayers (P < or = 0.01). These effects were seen in the range of 500-5000 U/ml with a maximum effect at 5000 U/ml. Similarly, mouse recombinant TNF alpha was able to stimulate DNA synthesis in mouse hepatocyte monolayers (P < or = 0.01) with a maximal effect at 1000 U/ml. Suppression of mouse hepatocyte apoptosis by TNF alpha was not detected, possibly because of the low levels of apoptosis under control conditions. However, when the levels of mouse hepatocyte apoptosis were augmented using TGF beta1, TNF alpha caused a significant suppression (P < or = 0.01). The neutralization of TNF alpha using anti-TNF alpha antibodies abrogated significantly (P < or = 0.01) the suppression of apoptosis by the PP, nafenopin. These data that suggest TNF alpha may mediate, at least in part, the growth perturbation, liver enlargement and hepatocarcinogenesis seen in response to the PP class of non-genotoxic hepatocarcinogens.

Species differences in response to peroxisome proliferators correlate in vitro with induction of DNA synthesis rather than suppression of apoptosis.

Tumorigenesis caused by the peroxisome proliferator (PP) class of non-genotoxic hepatocarcinogens is species restricted; rat and mouse are considered responsive whereas the available evidence suggests that humans, non-human primates, dogs, hamsters and guinea pigs are non-responsive. We have demonstrated previously that the PP, nafenopin can suppress rat hepatocyte apoptosis both in vitro and in vivo. Here we describe the ability of nafenopin to suppress apoptosis in mouse, hamster, guinea pig and rat hepatocytes and induce S-phase in mouse and rat hepatocytes. Hepatocyte monolayers from all species examined degenerated rapidly in culture. However, nafenopin (50 microM) reversibly maintained the viability of both rat and mouse hepatocytes. This maintenance was associated with a decrease (P < or = 0.01) in the number of hepatocytes displaying chromatin condensation patterns characteristic of apoptosis. Treatment of rat and mouse monolayers with 5 ng/ml transforming growth factor-beta 1 (TGF beta 1) induced high levels of apoptosis (P < or = 0.01); co-addition of nafenopin suppressed this induced apoptosis (P < or = 0.01). TGF beta 1 also induced apoptosis in hamster and guinea pig hepatocytes (P < or = 0.01) and unexpectedly nafenopin was able to suppress this induced apoptosis (P < or = 0.01) as well as reversibly maintaining the viability of hamster and guinea pig hepatocyte monolayers. Thus, all the species examined responded to nafenopin by a suppression of both spontaneous and TGF beta 1-induced apoptosis. In contrast, only rat and mouse hepatocytes showed an induction of S-phase in response to nafenopin (P < or = 0.01). Certain key experiments were repeated using the PPs methyl clofenapate (MCP) (100 microM) and Wy-14, 643 (10 microM). Both were able to suppress spontaneous and TGF beta 1-induced apoptosis in rat and guinea pig hepatocytes although the effects of MCP were weak (P < or = 0.05) compared with nafenopin or Wy-14 643 (P < or = 0.01). The rat and mouse liver tumour promoter, phenobarbitone (PB) was assessed also. Rat hepatocytes responded to PB with a suppression of apoptosis and an induction of S-phase (P < or = 0.01). Hamster and guinea pig cells gave no response in the S-phase assay and exhibited no suppression of either spontaneous or TGF beta 1-induced apoptosis. Interestingly, nafenopin suppressed the apoptosis induced by the DNA damaging drugs, etoposide and hydroxyurea (P < or = 0.01) suggesting that PPs can impact on diverse apoptosis signalling pathways. Overall, species differences in response to the non-genotoxic hepatocarcinogens studied, correlate with induction of DNA synthesis rather than with suppression of apoptosis. The data extend our knowledge of the mechanisms of species differences in non-genotoxic hepatocarcinogenesis, posing interesting questions on the relative roles of apoptosis and DNA synthesis in carcinogenesis.

Dosing-induced stress causes hepatocyte apoptosis in rats primed by the rodent nongenotoxic hepatocarcinogen cyproterone acetate.

It has been proposed that several nongenotoxic compounds act as hepatocarcinogens by suppressing the apoptosis that would normally act to remove damaged or potentially initiated cells from the liver. During our investigations of this hypothesis using a widely applied protocol, we have found that the stress induced by the process of gavage dosing can induce massive apoptosis in livers uniquely primed by withdrawal of the hepatomitogen cyproterone acetate from the hyperplastic rat liver. This effect of gavage dosing was not seen in livers of naive animals. Apoptosis was measured by both in situ end labeling (ISEL) of the DNA damage associated with programmed cell death and conventional hematoxylin and eosin (H&E) staining of apoptotic morphology. Apoptotic rates measured by H&E increased significantly from 0.005 +/- 0.010% on Day 11 to 0.657 +/- 0.315% of hepatocytes on Day 15, 4 days after cessation of 10 days dosing with CPA (120 mg/kg). The readministration of CPA suppressed > 89% of this Day 15 apoptosis. However, the readministration of vehicle alone (corn oil) caused a 390% increase in apoptosis to 2.56 +/- 1.31% of hepatocytes. Similar results were obtained using ISEL. Measurements of liver to body weight ratios and total DNA per liver reflected these changes in cell loss by apoptosis. In a second experiment, CPA was administered for 10 days as before then animals were subjected to readministration of CPA in corn oil, CPA in saline, corn oil, saline, or sham dosed. Again, apoptosis was dramatically suppressed by the readministration of CPA in either vehicle but was dramatically increased to around 2% of hepatocytes in all other groups, including the sham dosed group. Data on food consumption provided no evidence for a reduction in food intake as a causative agent but rather pointed to a less efficient usage of food in the stressed animals. The ability of stress to induce liver apoptosis should be borne in mind in the design and interpretation of future toxicological studies aimed at understanding the putative suppression of apoptosis by liver nongenotoxic carcinogens and other toxicants.