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.

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.

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.

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.

Addition of peroxisome proliferator-activated receptor alpha to guinea pig hepatocytes confers increased responsiveness to peroxisome proliferators.

The fibrate drugs, such as nafenopin and fenofibrate, show efficacy in hyperlipidemias but cause peroxisome proliferation and liver tumors in rats and mice via nongenotoxic mechanisms. However, humans and guinea pigs appear refractory to these adverse effects. The peroxisome proliferator (PP)-activated receptor alpha (PPAR alpha) mediates the effects of PPs by heterodimerizing with the retinoid X receptor (RXR) to bind to DNA at PP response elements (PPREs) upstream of PP-regulated genes, such as acyl-CoA oxidase. Hepatic expression of PPAR alpha in guinea pigs and humans is low, suggesting that species differences in response to PPs may be due at least in part to quantity of PPAR alpha. To test this hypothesis, we introduced mouse PPAR alpha and its heterodimerization partner, RXR alpha, into guinea pig hepatocytes by transient transfection and determined responsiveness to the PP nafenopin by cyanide-insensitive palmitoyl-CoA oxidation (CIPCO). Expression of the mRNA for mouse PPAR alpha in transfected guinea pig hepatocytes was verified using species-specific PCR. In guinea pig hepatocytes transfected with control plasmids and treated with 50 microM nafenopin in the absence or presence of the RXR ligand, 9-cis-retinoic acid (5 microM) gave only a 1.7 +/- 1.5- or 3.3 +/- 1.5-fold induction in CIPCO, respectively. However, addition of ligands to hepatocytes co-transfected with both mPPAR alpha and RXR gave a strong induction of CIPCO (14.8 +/- 8.6-fold). Mouse, human, and guinea pig PPAR alpha showed equivalent function in the CIPCO assays. Thus, quantity of PPAR alpha plays a significant role in the lack of response to PPs in guinea pigs. In humans, however, lack of PPAR alpha may be only one factor dictating lack of response because recent data show that the human acyl-CoA oxidase gene lacks a functional PP response element.

Peroxisome proliferator-activated receptor alpha: role in rodent liver cancer and species differences.

Peroxisome proliferators (PPs) are chemicals of industrial and pharmaceutical importance that elicit liver carcinogenesis by a non-genotoxic mechanism. One of the intriguing properties of PPs is that the pleiotropic effects of these compounds (including increased DNA synthesis and peroxisome proliferation) are seen in rats and mice only, but not humans. It is important to determine the risks to humans of environmental and therapeutic exposure to these compounds by understanding the mechanisms of non-genotoxic hepatocarcinogenesis in rodents. To understand this apparent lack of human susceptibility, attention has focused on the peroxisome proliferator-activated receptor alpha (PPARalpha), which appears to mediate the effects of PPs in rodents. It is also known to mediate the hypolipidaemic effects that fibrate drugs exert on humans with elevated plasma cholesterol and triglyceride levels. Human PPARalphas share many functional characteristics with the rodent receptors, in that they can be transcriptionally activated by PPs and regulate specific gene expression. However, one key difference is that PPARalpha is less abundant in human than in rodent liver, which has led to the suggestion that species differences result from quantitative differences in gene expression. In this review we describe the effects of PPs and what is known of the molecular mechanisms of action and species differences with respect to rodents and man. Attention will be given to differences in the amounts of PPARalpha between species as well as the 'qualitative' aspects of PPARalpha-mediated gene regulation which might also explain the activation of some genes and not of others in human liver by PPs.

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.

Immediate-early gene expression during regenerative and mitogen-induced liver growth in the rat.

The nongenotoxic carcinogens phenobarbitone (PB) and methyl clofenapate (MCP) and the hepatomitogen pregnenolone 16 alpha carbonitrile (PCN) are direct inducers of hepatic S-phase in rats, whereas the S-phase seen after partial hepatectomy is regenerative. We have investigated S-phase and immediate-early gene expression (c-myc and c-jun) in rat liver following these treatments to study the differences in gene expression associated with direct vs. regenerative responses. Both partial hepatectomy (one- and two-thirds) and mitogen treatment caused an increase in hepatic S-phase that peaked around 36 hours. Two-thirds partial hepatectomy caused the greatest increase in S-phase followed by one-third partial hepatectomy, then the mitogens PCN, MCP, and PB in that order. This order of response was also seen with c-jun and to a lesser degree with c-myc expression, suggesting that immediate-early gene expression might be linked not only to regenerative S-phase but also to direct mitogen-induced responses.

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.

Changes in gap junctional intercellular communication in mouse skin carcinogenesis.

Gap junction intercellular communication (GJIC) has been measured in cell lines that represent different stages of chemically induced mouse skin carcinogenesis. No significant difference in GJIC, as measured by dye spread, was found in cultures of normal keratinocyte, papilloma or squamous carcinoma cell lines. There was no correlation, in this system, between the presence of a mutant Ha-ras gene and down-regulation of communication. There was, however, a marked decrease in GJIC (80-90%) on progression from squamous to spindle carcinoma cells. Measurement of GJIC in somatic cell hybrids shows that the genetic defect responsible for this down-regulation is recessive and is common to two independently isolated spindle cell lines. No abnormalities were found in the spindle cells in expression of connexin 43, a cell component involved in gap junction formation and permeability. However, expression of E-cadherin, a cell-cell adhesion molecule implicated in the process of gap junction formation, was missing in the spindle carcinoma cells. Introduction of an E-cadherin cDNA into the spindle cells partially restored junctional communication without causing any noticeable alterations in cell morphology. During the study a non-tumourigenic keratinocyte line, a sub-clone of a normal keratinocyte line, was also found to have a low level of GJIC. However, the defect in this line was shown, by genetic complementation in somatic cell hybrids, to be different from that in the spindle carcinoma cell lines. Consistent with these data, analysis by immunofluorescence shows an abnormal distribution of connexin 43 in these cells.

Evidence for a Wolbachia symbiont in Drosophila melanogaster.

The bacterial cell division gene, ftsZ, was used as a specific probe to show the presence of a symbiotic bacterium in two wild type strains of Drosophila melanogaster. Under stringent hybridization conditions we have shown that the bacterium is transferred to the progeny of these strains from infected mothers and can be eradicated by treatment with the antibiotic tetracycline. We have characterized this bacterium, by amplifying and sequencing its 16S rRNA gene, as being a member of the genus Wolbachia, an organism that is known to parasitize a range of insects including Drosophila simulans. In a series of reciprocal crosses no evidence was found that the symbiont causes cytoplasmic incompatibility (CI) which is known to occur in infected strains of D. simulans. The implications of these findings are discussed.

Cloning and characterization of an ftsZ homologue from a bacterial symbiont of Drosophila melanogaster.

A 1194 bp open reading frame that codes for a 398 amino acid peptide was cloned from a lambda gt11 library of Drosophila melanogaster genomic DNA. The predicted peptide sequence is very similar to three previously characterized protein sequences that are encoded by the ftsZ genes in Escherichia coli, Bacillus subtilis and Rhizobium meliloti. The FtsZ protein has a major role in the initiation of cell division in prokaryotic cells. Using a tetracycline treatment that eradicates bacterial parasites from insects, the ftsZ homologue has been found to be derived from a bacterium that lives within the D. melanogaster strain. However, polymerase chain reaction (PCR) amplification of the gene from treated embryos suggests that it is not derived from a gut bacterium. Nevertheless, by amplifying and characterizing part of the 16S rRNA from this bacterium we have been able to demonstrate that it is a member of the genus Wolbachia, a parasitic organism that infects, and disturbs the sexual cycle of various strains of Drosophila simulans. We suggest that this ftsZ homologue is implicated in the cell division of Wolbachia, an organism that fails to grow outside the host organism. Sequence and alignment analysis of this ftsZ homologue show the presence of a potential GTP-binding motif indicating that it may function as a GTPase. The consequences of this function particularly with respect to its role in cell division are discussed.