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J D Tugwood

Publications and source records attributed to J D Tugwood.

At least 19 recordsLinked to original sources
PubMed2002

Biology and toxicology of PPARgamma ligands.

The peroxisome proliferator activated receptor-gamma (PPARgamma) is an attractive target for therapeutic intervention, as modulation of PPARgamma-regulated pathways is potentially beneficial in a number of disease areas. This review provides an overview of what is known about the biology of PPARgamma, and an indication of what progress has been made towards drug development in several therapy areas. As well as efficacy, the safety of drugs is of course an important issue, and a substantial volume of preclinical and clinical information has already accumulated for PPARgamma agonists. Here we discuss some of the major toxicology issues with PPARgamma agonists, and give a perspective on likely issues concerning the development of PPARgamma modulators in the future.

DNA-Binding Proteins↗
PubMed1999

The peroxisome proliferator (PP) response element upstream of the human acyl CoA oxidase gene is inactive among a sample human population: significance for species differences in response to PPs.

Peroxisome proliferators (PP) cause peroxisome proliferation, associated with rodent hepatocyte growth perturbation and hepatocarcinogenesis. However, in humans this class of non-genotoxic carcinogens does not appear to have the same adverse effects. The peroxisome proliferator-activated receptor alpha (PPARalpha) mediates the effects of PPs in rodents via peroxisome proliferator response elements (PPREs) upstream of PP-responsive genes such as acyl coenzyme A oxidase (ACO). When the human ACO promoter was cloned previously, it was found to be active and to contain a consensus PPRE (-1918 AGGTCA C TGGTCA -1906). To confirm and extend those original findings, we isolated a 2 kb genomic fragment of the ACO gene promoter from a human liver biopsy and used it to create a beta-galactosidase reporter gene plasmid. The human ACO promoter reporter plasmid was added to both Hepalclc7 and NIH 3T3 cells together with a plasmid expressing mPPARa and assessed for its ability to drive PP-mediated gene transcription. The human ACO promoter fragment was inactive, unlike the equivalent rat ACO promoter fragment used as a positive control. The PPRE within our cloned fragment of the human ACO promoter differed at three positions (5'-AGGTCA G CTGTCA-3') from the previously published active human ACO promoter. Next, we studied the frequency of the inactive versus the active human PPRE within the human population. Using a PCR strategy, we isolated and analysed genomic DNA fragments from 22 unrelated human individuals and from the human hepatoma cell line HepG2. In each case, the PPRE contained the inactive sequence. These data show that the human ACO gene promoter found in a sample human population is inactive. This may explain at the genomic level the lack of response of humans to some of the adverse effects of the PP class of non-genotoxic hepatocarcinogens.

3T3 Cells↗
PubMed1999

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1997

Amino acid residues in both the DNA-binding and ligand-binding domains influence transcriptional activity of the human peroxisome proliferator-activated receptor alpha.

We have investigated the basis of the lack of activity of a natural variant human peroxisome proliferator-activated receptor alpha, hPPARalpha6/29. A subcloning approach was used to change the four variant amino acids in the hPPARalpha6/29 sequence, individually and in combination, to those found in an active human PPARalpha. Individual amino acid "back mutations" were unable to confer on hPPARalpha6/29 the ability to be activated by peroxisome proliferators in a transient transfection assay. Although hPPARalpha6/29 was able to bind specifically to DNA in the presence of the retinoid X receptor alpha (RXRalpha), the complete restoration of receptor transcriptional activity required two separate back mutations of the hPPARalpha6/29 sequence, namely amino acid 123 in the DNA binding domain, and amino acid 444 close to the C-terminus. This suggests that sequences in the PPARalpha DNA binding domain influence other receptor functions besides DNA binding.

Alanine↗
PubMed1996

Peroxisome proliferator-activated receptors: structures and function.

We have been attempting to elucidate the molecular mechanisms through which peroxisome proliferators exert their pleiotropic effects, with particular emphasis on understanding why humans appear unresponsive to these compounds. There is a wealth of data to implicate the peroxisome proliferator-activated receptor alpha (PPAR alpha) in mediating these effects in rodent species; PPAR alpha is expressed in tissues that show physiological changes in response to PPs, is transcriptionally activated in vitro by a variety of PPs, and it has been recently demonstrated that mice lacking this receptor are refractory to the effects of clofibrate and Wy-14,643, at least in the short term. It is conceivable that differences in PPAR alpha between responsive rodent and unresponsive human subjects may provide the key to understanding the basis of this species variation in response, and with this in mind we have been studying the biology of PPAR alpha in humans and looking at interindividual variation. There is already published evidence, albeit on only two sequences, for structural and functional polymorphism in human PPAR alphas. We have extended these findings, and shown that: There is considerable variation in hPPAR alpha cDNAs obtained from different individuals, both at the gross structural level (lack of a coding exon) and of a more subtle nature (single base changes leading to amino acid substitutions). One such cDNA, the sequence of which differs at only three amino acids from that published, encodes a receptor that is incapable of transcriptional activation by potent PPs. The degree to which hPPAR alpha transcripts are expressed in human livers can vary by up to an order of magnitude between individuals. The tissue-specific expression profile of PPAR alpha in humans is very different from that in rat and mouse. In particular, the human liver contains generally low levels of PPAR alpha in contrast to the responsive rodents, in which potent PPs cause liver tumors. Taken together, these data suggest first that human and rodent PPAR alphas differ according to a number of molecular and biochemical criteria, and secondly that there is a degree of interindividual variation in PPAR alpha structure and function. Studies are ongoing to clarify this further, but human polymorphism may go some way towards explaining the apparent paradox that active PPAR alpha receptors can be isolated from an "unresponsive" species.

Acyl-CoA Oxidase↗
PubMed1996

A human peroxisome-proliferator-activated receptor-gamma is activated by inducers of adipogenesis, including thiazolidinedione drugs.

We have cloned a human cognate of the mouse peroxisome-proliferator-activated receptor-gamma (hPPAR gamma) from a human placenta cDNA library. Sequence analysis reveals a high degree of similarity with the mouse receptor and, like other PPAR, hPPAR gamma forms heterodimers with the retinoid X receptor alpha (RXR alpha) and binds in vitro to DNA elements containing direct repeats of the sequence TGACCT. In common with mouse PPAR gamma, hPPAR gamma is expressed strongly in adipose tissue, but significant levels also are detectable in placenta, lung and ovary. In vitro trans-activation data suggest hPPAR gamma is only poorly activated by xenobiotic peroxisome proliferators, although certain fatty acids and eicosanoids are potent activators of this receptor. Both mouse and human PPAR gamma are capable of being activated by thiazolidinedione drugs, although the two receptors appear to differ in their sensitivity to these compounds. Taken together, these data suggest a high degree of structural and functional similarity between mouse and human PPAR gamma, and provide evidence for variation in human receptor structure which may result in differential sensitivity to activators.

Adipose Tissue↗
PubMed1996

The alpha1-fetoprotein locus is activated by a nuclear receptor of the Drosophila FTZ-F1 family.

The alpha1-fetoprotein (AFP) gene is located between the albumin and alpha-albumin genes and is activated by transcription factor FTF (fetoprotein transcription factor), presumed to transduce early developmental signals to the albumin gene cluster. We have identified FTF as an orphan nuclear receptor of the Drosophila FTZ-F1 family. FTF recognizes the DNA sequence 5'-TCAAGGTCA-3', the canonical recognition motif for FTZ-F1 receptors. cDNA sequence homologies indicate that rat FTF is the ortholog of mouse LRH-1 and Xenopus xFF1rA. Rodent FTF is encoded by a single-copy gene, related to the gene encoding steroidogenic factor 1 (SF-1). The 5.2-kb FTF transcript is translated from several in-frame initiator codons into FTF isoforms (54 to 64 kDa) which appear to bind DNA as monomers, with no need for a specific ligand, similar KdS (approximately equal 3 x 10(-10) M), and similar transcriptional effects. FTF activates the AFP promoter without the use of an amino-terminal activation domain; carboxy-terminus-truncated FTF exerts strong dominant negative effects. In the AFP promoter, FTF recruits an accessory trans-activator which imparts glucocorticoid reactivity upon the AFP gene. FTF binding sites are found in the promoters of other liver-expressed genes, some encoding liver transcription factors; FTF, liver alpha1-antitrypsin promoter factor LFB2, and HNF-3beta promoter factor UF2-H3beta are probably the same factor. FTF is also abundantly expressed in the pancreas and may exert differentiation functions in endodermal sublineages, similar to SF-1 in steroidogenic tissues. HepG2 hepatoma cells seem to express a mutated form of FTF.

Amino Acid Sequence↗
PubMed1995

Identification and characterization of DNA elements implicated in the regulation of CYP4A1 transcription.

We have identified a peroxisome proliferator response element (PPRE) approx. 4300 nucleotide upstream of the rat cytochrome P-450 CYP4A1 gene. Two members of the steroid-hormone-receptor superfamily, the peroxisome proliferator-activated receptor-alpha (PPAR alpha) and the retinoid X receptor-alpha (RXR alpha), bind specifically to this element as a heterodimer, and this element confers responsiveness to the peroxisome proliferator Wyeth-14,643 when tested in co-transfection assays. A second element, located 35 nucleotides further upstream, fails to bind PPAR alpha/RXR alpha heterodimers and is unresponsive to Wy-14,643 in co-transfection assays. Both elements are, however, responsive to 9-cis-retinoic acid in the presence of RXR alpha, when tested in the co-transfection assay. As RXR alpha fails to bind to either element as a homodimer, we suggest that RXR alpha interacts with PPAR alpha to regulate transcription via the proximal element, and interacts with some other cellular factor to regulate transcription via the more distal element. This is consistent with previous reports that a number of peroxisome proliferator-regulated genes contain PPRE-like elements as part of their regulatory sequences, which may be recognized by several receptor combinations. This provides further evidence that PPARs and their co-factors are important in mediating the pleiotropic action of peroxisome proliferators.

Animals↗
PubMed1994

Dexamethasone and insulin demonstrate marked and opposite regulation of the steady-state mRNA level of the peroxisomal proliferator-activated receptor (PPAR) in hepatic cells. Hormonal modulation of fatty-acid-induced transcription.

Fatty acids and the peroxisomal proliferator, 3-tetradecylthioacetic acid (TTA) stimulate transcription of peroxisomal beta-oxidation enzymes. Recently, we have shown that their actions are markedly modulated by dexamethasone and insulin which show synergistic and inhibitory effects, respectively. In this study, we describe the regulation of the peroxisomal proliferator-activated receptor (PPAR), a member of the steroid-hormone-receptor superfamily, in a similar manner by hormones and fatty acids, supporting the hypothesis that PPAR may act as a ligand-activated transcription factor. Northern-blot analysis of steady-state mRNA levels revealed three different specific transcripts for PPAR of 10.2, 4.6 and 1.8 kb, and the former two being regulated in hepatic tissue, hepatocytes and hepatoma cells. Dexamethasone produced a pronounced overall stimulatory effect (15.3-fold) in rat hepatocytes, while insulin blocked this action completely. Minor inductions of PPAR mRNA (up to twofold induction) were observed when different fatty acids were administrated alone. However, in combination with dexamethasone, additive or synergistic actions, mounting to 24-fold stimulation, were observed, while insulin always exerted an over-riding down-regulatory effect. In non-fasting rats receiving dexamethasone, elevation of serum insulin, a slight increase in serum free fatty acids accompanied by PPAR mRNA level increases of 2.4-fold and stimulation of liver peroxisomal acyl-CoA oxidase mRNA were observed. Our results suggest that PPAR mRNA expression is under strict hormonal control and that the fatty acids and hormones affect PPAR mRNA levels in a manner analogous to the regulation of the peroxisomal beta-oxidation enzymes. The PPAR gene-regulating unit apparently contains hormone-response elements (HRE) for dexamethasone and insulin, which are thus functionally important for PPAR transcription in liver cells, making a significant enhancement or inhibition of the physiological actions of fatty acids possible.

Acyl-CoA Oxidase↗
PubMed1994

Mechanistically-based human hazard assessment of peroxisome proliferator-induced hepatocarcinogenesis.

In this review we have evaluated the relationship between peroxisome proliferation and hepatocarcinogenesis. To do so, we identified all chemicals known to produce peroxisome proliferation and selected those for which there are data (on peroxisome proliferation and hepatocarcinogenesis) which meet certain criteria chosen to facilitate comparison of these phenomena. The summarised data and definition of the methodology used has been collected in appendices. These comparisons enabled us to evaluate the relationship between these phenomena using reliable data. As there is a good correlation between them, we further explored the mechanisms of action that have been proposed (direct genotoxic activity, production of hydrogen peroxide, cell proliferation and receptor activation). The relationship between these events in other species, including humans, was also reviewed and finally an overview of the assessment of human hazard is presented in section IX. Some of the first chemicals which were shown to produce peroxisome proliferation were also hepatocarcinogens whose carcinogenicity could not be readily explained by genotoxic activity. This raised the suggestion that the unusual phenomenon of peroxisome proliferation was intricately linked to the carcinogenic activity of these agents. Three questions have exercised the attention of regulatory, industrial and academic toxicology since then; are chemicals which elicit peroxisome proliferation in the liver actually a coherent class of chemical carcinogens?; does the early biological phenomenon of peroxisome proliferation have real predictive value for and mechanistic association with rodent carcinogenesis?; and what hazard/risk do these agents pose to humans that may be exposed to them? Whether peroxisome proliferators are indeed a discrete class of rodent carcinogens would appear to be the single, most important question. If so, then the assumptions and procedures relevant to human hazard and risk assessment should be applied to the class and should be essentially generic; if not, each chemical should be considered independently. Our critical analysis of the published data for over 70 agents which have been shown to possess intrinsic ability to induce peroxisome proliferation in the livers of rodents has led to the conclusion that there exists a strong correlation between peroxisome proliferation as n early effect in the liver and hepatocarcinogenicity in chronic exposure studies. An almost perfect correlation was observed between the induction of peroxisomes in the rodent liver and the eventual appearance of tumours following chronic exposure The few exceptions to this were largely explainable (section II).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗