An evaluation of a low-density DNA microarray using cytochrome P450 inducers.

The aim of this study was to validate a low-density DNA microarray "Rat HepatoChip", which contains 59 genes from a range of potential toxic markers and drug metabolism-related genes. Liver mRNA was isolated from rats dosed with six different chemicals, dexamethasone, troleandomycin, miconazole, clotrimazole, and methylclofanapate, which are all known to induce different cytochrome P450 genes, and isoniazid, which does not cause histopathological changes. Replicate microarrays were used to measure the variability in the chips and in the process. The average variability in signal between different chips observed in triplicate experiments was 33% ranging from 21 to 39% depending on genes. We also demonstrated a strong correlation between the liver histopathology and the gene expression profiles indicating that the gene expression profile reflects histopathological changes. These results suggest that the Rat HepatoChip microarray may provide a fast and effective tool for assessing the toxicity profile of developmental drug candidates during the drug discovery process.

Potential role for peroxisome proliferator activated receptor (PPAR) in preventing colon cancer.

BACKGROUND: Peroxisome proliferator activated receptors (PPARs) are nuclear hormone receptors involved in genetic control of many cellular processes. PPAR and PPAR have been implicated in colonic malignancy. Here we provide three lines of evidence suggesting an inhibitory role for PPAR in colorectal cancer development. METHODS: Levels of PPAR mRNA and protein in human colorectal cancers were compared with matched non-malignant mucosa using RNAse protection and western blotting. APC(Min)/+ mice were randomised to receive the PPAR activator methylclofenapate 25 mg/kg or vehicle for up to 16 weeks, and small and large intestinal polyps were quantified by image analysis. The effect of methylclofenapate on serum stimulated mitogenesis (thymidine incorporation), linear cell growth, and annexin V and propidium iodide staining were assessed in human colonic epithelial cells. RESULTS: PPAR (mRNA and protein) expression levels were significantly depressed in colorectal cancer compared with matched non-malignant tissue. Methylclofenapate reduced polyp area in the small intestine from 18.7 mm(2) (median (interquartile range 11.1, 26.8)) to 9.90 (4.88, 13.21) mm(2) (p=0.003) and in the colon from 9.15 (6.31, 10.5) mm(2) to 3.71 (2.71, 5.99) mm(2) (p=0.009). Methylclofenapate significantly reduced thymidine incorporation and linear cell growth with no effect on annexin V or propidium iodide staining. CONCLUSIONS: PPAR may inhibit colorectal tumour progression, possibly via inhibition of proliferation, and may be an important therapeutic target.

Studies on the induction of rat hepatic CYP1A, CYP2B, CYP3A and CYP4A subfamily form mRNAs in vivo and in vitro using precision-cut rat liver slices.

1. Real-time quantitative reverse transcription-polymerase chain reaction methodology (TaqMan(R)) was used to examine the induction of some selected rat hepatic cyto-chrome P450 (CYP) forms in vivo and in vitro using cultured precision-cut liver slices. 2. TaqMan primers and probe sets were developed for rat CYP1A1, CYP1A2, CYP2B1, CYP2B1/2, CYP3A1, CYP3A2 and CYP4A1 mRNAs. 3. To characterize the responsiveness of the rat CYP mRNA TaqMan primers and probe sets, rats were treated in vivo with a single intraperitoneal dose of 500 mg kg(-1) Aroclor 1254 (ARO) and with four daily oral doses of either 50 mg kg(-1) day(-1) dexamethasone (DEX) or 75 mg kg(-1) day(-1) methylclofenapate (MCP). Treatment with ARO produced 22 600-, 5480-, 648-, 52-, 47- and 9-fold increases in levels of CYP1A1, CYP2B1, CYP2B1/2, CYP1A2, CYP3A1 and CYP3A2 mRNA, respectively. DEX treatment produced 97-, 24-, 8- and 4-fold increases, respectively, in CYP3A1, CYP2B1, CYP2B1/2 and CYP3A2 mRNA levels, and MCP produced 339-, 126- and 25-fold increases, respectively, in CYP4A1, CYP2B1 and CYP2B1/2 mRNA levels. All three CYP inducers also increased microsomal CYP content and produced corresponding increases in CYP1A, CYP2B, CYP3A and CYP4A form marker enzyme activities. 4. Rat liver slices were cultured for 6 and 24 h in medium containing 0.1 micro M insulin and 0.1 micro M DEX, and also for 24 h in medium containing only 0.1 micro M insulin (DEX-free medium). Liver slices were cultured in control medium or in medium containing either 10 micro M beta-naphthoflavone (BNF), 10 micro g ml(-1) ARO, 500 micro M sodium phenobarbitone (NaPB), 20 micro M pregnenolone-16alpha -carbonitrile (PCN), 50 micro M Wy-14,643 (WY) or 50 micro M MCP. 5. With the exception of the effect of BNF on CYP1A1 mRNA levels, the induction of all the CYP mRNAs studied was greater after 24- than after 6-h treatment. Generally, the magnitude of induction of CYP mRNA levels was greater after 24 h in liver slices cultured in DEX-free than in DEX-supplemented medium. 6. Treatment of liver slices with BNF and ARO for 24 h in DEX-free medium produced 21- and 35-fold increases, respectively, and 38- and 37-fold increases, respectively, in CYP1A1 and CYP1A2 mRNA levels. NaPB, PCN, WY and MCP did not increase either CYP1A1 or CYP1A2 mRNA levels. 7. After 24 h, levels of CYP2B1/2 mRNA were increased 18-, 20-, 9-, 16- and 13-fold by treatment with ARO, NaPB, PCN, WY and MCP, respectively. PCN also produced 56- and 4-fold increases, respectively, in CYP3A1 and CYP3A2 mRNA levels. 8. Treatment with WY and MCP for 24 h produced 437- and 186-fold increases, respectively, in levels of CYP4A1 mRNA. None of the other CYP inducers studied had any effect on CYP4A1 mRNA levels. 9. The results demonstrate the utility of cultured precision-cut liver slices as an in vitro model system to evaluate the effects of xenobiotics on rat CYP1A, CYP2B, CYP3A and CYP4A form mRNA levels.

CYP isoform induction screening in 96-well plates: use of 7-benzyloxy-4-trifluoromethylcoumarin as a substrate for studies with rat hepatocytes.

1. In this study, 7-benzyloxy-4-trifluoromethylcoumarin (BFC) was evaluated as a substrate to assess the induction of cytochrome P450 (CYP) isoform enzyme activities in rat hepatocytes using a 96-well plate format. 2. BFC was metabolized by both untreated and sodium phenobarbitone (NaPB)-treated rat hepatocytes in a time- and concentration-dependent manner to the highly fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC). 3. HFC was extensively conjugated with D-glucuronic acid and/or sulphate in both untreated and NaPB-treated rat hepatocytes, thus necessitating the inclusion of an enzymatic deconjugation step in the assay procedure. 4. The time-course of induction of 7-ethoxyresorufin metabolism by the CYP1A inducer beta-naphthoflavone (BNF), 7-benzyloxyresorufin metabolism by the CYP2B inducer NaPB and BFC metabolism b both BNF and NaPB was studied in rat hepatocytes treated for 24-96 h. The optimal time for induction of metabolism of all three substrates was 72 h, with no medium changes being necessary during this period. 5. The effect of treatment with 0.5-20 microM BNF, 50-2000 microM NaPB, 2-20 microM dexamethasone (DEX), 20-100 microM methylclofenapate (MCP), and 50 and 200 microM isoniazid (ISN) for 72 h on BFC metabolism in cultured rat hepatocytes was studied. BFC metabolism was induced by treatment with BNF, NaPB and MCP, but not with either DEX or ISN. 6. The metabolism of BFC in liver microsomes from the control rat and rat treated with CYP isoform inducers was also studied. BFC metabolism was induced by treatment with NaPB, BNF and DEX. 7. The metabolism of BFC was also studied using microsomes from baculovirus-infected insect cells containing rat cDNA-expressed CYP1A, CYP2B, CYP2C and CYP3A isoforms. Whereas BFC was metabolized to some extent by all the rat cDNA-expressed CYP isoforms examined, at a substrate concentration of 2.5 microM the greatest rates of BFC metabolism were observed with the CYP1A1, CYP1A2 and CYP2B1 preparations. 8. In summary, the results demonstrate that BFC is a good substrate for assessing the induction of CYP1A and CYP2B isoforms in rat hepatocytes in a 96-well plate format.

The nongenotoxic hepatocarcinogens diethylhexylphthalate and methylclofenapate induce DNA synthesis preferentially in octoploid rat hepatocytes.

Diethylhexylphthalate (DEHP), a rodent carcinogen, and 1,4-dichlorobenzene (DCB), a noncarcinogen in rat liver, are potent hepatomitogens. We have reported previously that 7-day dosing with DEHP induced a higher bromodeoxyuridine labeling index (LI) in binuclear octoploid (2x4N) rat hepatocytes than did DCB, suggesting that induction of DNA synthesis in 2x4N hepatocytes might represent a more substantial carcinogenic risk. We compared 2 additional rodent hepatocarcinogens, methylclofenapate (MCP) and phenobarbitone, with ethylene thiourea (ETU), a noncarcinogenic hepatomitogen in rat. All 3 chemicals increased hepatic LI; the 8N population had the highest LI, but only the carcinogens increased LI in the 2x4N and 4N populations. To identify the target population for induction of DNA synthesis, we used a 1-hour pulse label at the peak of induction. The results were consistent with the 7-day data, and again the highest LI was in the 8N population. The nongenotoxic rodent carcinogens MCP and DEHP induced a significant increase in the LI in the 2x4N population, whereas ETU and DCB did not. These data support the hypothesis that increased DNA synthesis within the minority 2x4N population may be more significant for subsequent hepatocarcinogenesis.

The genetic toxicity of the peroxisome proliferator class of rodent hepatocarcinogen.

Peroxisome proliferators comprise a structurally diverse class of chemicals. Some of the members of this class show evidence of genetic toxicity (most evidently the in vitro clastogen Wyeth 14,643, WY), while others do not (most evidently methyl clofenapate, MCP). When attempting to understand the mechanism of rodent hepatocarcinogenesis of this class of chemicals the possible role of genetic toxicity should be assessed on a class-wide basis, i.e., if just one peroxisome proliferator is shown to be unequivocally inactive as a genetic toxin, genetic toxicity cannot be implicated in the carcinogenic activity of peroxisome proliferators as a class. In an earlier paper, we established MCP as inactive in a range of in vitro and in vivo genetic toxicity assays. However, the top dose level of MCP that could be tested for induction of chromosome aberrations (clastogenicity) in human lymphocytes and CHO cells was limited by the relative insolubility of the test agent in the assay medium. Methyl clofenapate was not toxic up to a dose that produced precipitate, so cannot be directly compared with WY, which induced aberrations only at toxic dose levels. In the present paper, we have evaluated the clastogenicity of the carcinogenic peroxisome proliferator nafenopin (NAF) at dose levels up to those that are toxic to CHO cells, and found no evidence of chromosome aberration induction. These data isolate further the genetic toxicity of WY from other peroxisome proliferators, and increase confidence in the proposal that genetic toxicity does not play a critical role in the hepatocarcinogenicity of peroxisome proliferators.

Species differences in peroxisome proliferation; mechanisms and relevance.

Peroxisome proliferators are a class of structurally diverse chemicals, which induce liver carcinogenesis in rodents through interaction and activation of the Peroxisome Proliferator-Activated Receptor alpha (PPARalpha). PPARalpha agonists elicit a powerful pleiotropic response, which include hypolipidaemia. We have examined the response of species that are classically unresponsive to peroxisome proliferators. Whereas hamster responds to PPARalpha agonists by hepatomegaly and induction of marker genes, the guinea pig does not undergo hepatomegaly or induction of marker genes, such as CYP4A13. Both the hamster and the guinea pig have PPARalpha, and the guinea pig receptor has been characterised to be fully functional, as demonstrated in reporter gene expression assays. However, the guinea pig PPARalpha is expressed at low levels in liver, and the currently favoured hypothesis to explain species differences in hepatic peroxisome proliferation invokes the low level of PPARalpha as the principal determinant of species responsiveness. However, the demonstration that guinea pigs and humans undergo hypolipidaemia induced by PPARalpha-agonists calls into question the mode of action of PPARalpha agonists in "non-responsive" species.

Species differences in hepatic peroxisome proliferation, cell replication and transforming growth factor-beta1 gene expression in the rat, Syrian hamster and guinea pig.

The objective of this study was to evaluate species differences in the hepatic effects of three potent rodent peroxisome proliferators, namely methylclofenapate (MCP), ciprofibrate (CIP) and Wy-14,643 (WY), particularly with respect to effects on replicative DNA synthesis and transforming growth factor-beta1 (TGF-beta1) gene expression. Male Sprague-Dawley rats, Syrian hamsters and Dunkin-Hartley guinea pigs were given daily oral doses of 0 (corn oil) and 75 mg/kg MCP for periods of 6 and 21 days. Syrian hamsters and guinea pigs were also treated with 25 mg/kg CIP and 25 mg/kg WY. Relative liver weights were significantly increased in peroxisome proliferator-treated rats and Syrian hamsters, but not in guinea pigs. Hepatic peroxisomal (palmitoyl-CoA oxidation) and microsomal (lauric acid 12-hydroxylase) fatty acid oxidising enzyme activities and CYP4A isoform mRNA levels were significantly increased in rats and Syrian hamsters, whereas only minor effects were observed in the guinea pig. Replicative DNA synthesis was studied by implanting 7-day osmotic pumps containing 5-bromo-2'-deoxyuridine during study days -1 to 6 and 14 to 21. Hepatocyte labelling index values were increased by MCP in the rat, but neither MCP, CIP nor WY produced any significant effect on replicative DNA synthesis in the Syrian hamster and guinea pig. MCP treatment increased TGF-beta1 and insulin-like growth factor II/mannose-6-phosphate (IGFII/Man6P) receptor gene expression in the rat. In the Syrian hamster, effects on TGF-beta1 and IGFII/Man6P receptor gene expression were also observed in some instances, whereas TGF-beta1 mRNA levels were essentially unchanged in the guinea pig. These results provide further evidence for marked species differences in response to rodent peroxisome proliferators. While peroxisome proliferators produce a wide spectrum of effects in rat liver, other species such as the Syrian hamster and guinea pig are less responsive and in the case of some endpoints (e.g., cell replication) may be refractory.

High throughput ribonuclease protection assay for the determination of CYP3A mRNA induction in cultured rat hepatocytes.

1. A rapid 96-well plate based method for the determination of CYP3A mRNA induction in primary rat hepatocytes has been developed which has substantial advantages over current technologies including the ability to test the effect of relatively large numbers of new chemical entities on the expression of CYP3A mRNA in hepatocytes. 2. The ribonuclease protection assay detects changes in mRNA levels in small numbers of hepatocytes by the utilization of a radiolabelled antisense riboprobe that will hybridize CYP3A1 and CYP3A23. Using in situ hybridization techniques in conjunction with Amersham 96-well Cytostar-T scintillating microplates, there is no need for isolation of mRNA. A simple ribonuclease digestion step allows quantitative data to be generated easily within 1 week of hepatocyte isolation. 3. Rat hepatocytes were cultured for 48 h post-isolation on the Cytostar plates coated with a basal matrix of Matrigel. Prototypical CYP3A inducers (dexamethasone and pregnenolone 16alpha-carbonitrile) have been studied using various treatment periods from 0.5 to 24 h. Methylclofenapate and beta-naphthoflavone, prototypical inducers of CYP4A and CYP1A respectively, have been used as controls to show specificity of the [33P]-labelled riboprobe for the CYP3A family. 4. Time-dependent increases in CYP3A mRNA were demonstrated following exposure of hepatocytes to prototypical CYP3A inducers, but not for methylclofenapate or beta-naphthoflavone, so demonstrating specificity for CYP3A mRNA over CYP1A and CYP4A. Analysis of the 24-h induction data demonstrates that significant differences from controls can be determined and that induction potential can be assessed. The system has the potential to screen for overall CYP3A mRNA induction in response to compounds at an early stage in drug research.

The coordinate regulation of DNA synthesis and suppression of apoptosis is differentially regulated by the liver growth agents, phenobarbital and methylclofenapate.

The coordinate regulation of DNA synthesis and suppression of apoptosis was investigated in a rat hepatocyte cell culture system which supports high level induction of DNA synthesis by the peroxisome proliferator, methylclofenapate (MCP) (Plant, N.J. et al., 1998, Carcinogenesis, 19, 925-931). The peroxisome proliferators are hepatocyte mitogens in chemically defined media: glucocorticoid-induced PPARalpha is linked to peroxisome proliferator mitogenesis (Plant, N.J. et al., 1998, Carcinogenesis, 19, 925-932). Phenobarbital (PB) induced moderate induction of DNA synthesis (200-300% of control), but the peak of induction was 40 h after treatment. In hepatocytes that had undergone DNA synthesis, PB increased the proportion of binucleates by 200-300%. Both PB and MCP were able to suppress apoptosis in a dose-dependent manner, while the endogenous mitogen epidermal growth factor failed to suppress apoptosis. The suppression of apoptosis by MCP was reversible; withdrawal of MCP led to rapid induction of apoptosis. The presence of hydrocortisone is required for suppression of apoptosis by peroxisome proliferators, but not for PB. MCP failed to suppress apoptosis in primary cultures of guinea-pig hepatocytes. Comparison of the stability of hepatocytes labelled with bromodeoxyuridine (BrdUrd) and [3H]thymidine revealed that approximately 40% of cells labelled with BrdUrd were lost over a period of 14 days, whereas cells labelled with thymidine remained stable over this period. Hepatocytes were therefore treated with MCP, labelled with [3H]thymidine, maintained for 14 days, and peroxisome proliferator withdrawn. While the apoptotic index in unlabelled cells was 1.7%, no apoptosis was detected in labelled cells. In order to compare the mechanism of suppression of apoptosis, hepatocytes were cultured in the presence of either PB or MCP for 14 days. When MCP was substituted for PB in cells cultured in the presence of PB, the monolayer was maintained, but when PB was used to replace MCP in cells cultured in the presence of MCP, the monolayer of hepatocytes degenerated rapidly. The results demonstrate mechanistic differences in the coordinate regulation of cell growth and apoptosis in hepatocytes by PB and MCP.

The peroxisome proliferators are hepatocyte mitogens in chemically-defined media: glucocorticoid-induced PPAR alpha is linked to peroxisome proliferator mitogenesis.

Peroxisome proliferator-induced mitogenesis is believed to play a role in hepatocarcinogenesis, but it has not been possible to demonstrate high level induction of DNA synthesis by peroxisome proliferators in cultured hepatocytes. We now show that four structurally dissimilar peroxisome proliferators (methylclofenapate, Wy-14 643, tetradecyl-3-thia acetic acid and clofibrate) cause high level induction of DNA synthesis in primary cultures of rat hepatocytes, routinely 7-9 fold above control, with up to 29% of cells undergoing S-phase. Peroxisome proliferators induce DNA synthesis rapidly, with maximal response 24 h after dosing [compared with 48 h for epidermal growth factor (EGF)]; indeed, peroxisome proliferators were mitogenic in a chemically defined medium, i.e. with no added exogenous growth factors. EGF-treated hepatocytes that had undergone DNA synthesis comprised 23% binucleated cells, whereas hepatocytes induced into S-phase by peroxisome proliferators contained only 3% binucleated cells, demonstrating a distinct response of hepatocytes to peroxisome proliferators and EGF. The presence of a glucocorticoid was essential for peroxisome proliferator-induced DNA synthesis, but not for EGF-induced DNA synthesis, demonstrating that the requirement for glucocorticoids is selective for peroxisome proliferators. Hydrocortisone was shown to induce the expression of peroxisome proliferator activated receptor-alpha (PPAR alpha), and we propose that it is the glucocorticoid-induced expression of PPAR alpha that is essential for peroxisome proliferator mitogenesis. This in vitro system provides a powerful tool for investigating the mechanism and role of peroxisome proliferator-induced mitogenesis in liver growth and carcinogenesis.

Molecular basis of non-responsiveness to peroxisome proliferators: the guinea-pig PPARalpha is functional and mediates peroxisome proliferator-induced hypolipidaemia.

The guinea pig does not undergo peroxisome proliferation in response to peroxisome proliferators, in contrast with other rodents. To understand the molecular basis of this phenotype, the peroxisome proliferator activated receptor alpha (PPARalpha) from guinea-pig liver was cloned; it encodes a protein of 467 amino acid residues that is similar to rodent and human PPARalpha. The guinea-pig PPARalpha showed a high substitution rate: maximum likelihood analysis was consistent with rodent monophyly, but could not exclude rodent polyphyly (P approximately 0.06). The guinea-pig PPARalpha cDNA was expressed in 293 cells and mediated the induction of the luciferase reporter gene by the peroxisome proliferator, Wy-14,643, dependent on the presence of a peroxisome proliferator response element. Moreover the PPARalpha RNA and protein were expressed in guinea-pig liver, although at lower levels than in a species which is responsive to peroxisome proliferators, the mouse. To determine whether the guinea-pig PPARalpha mediated any physiological effects, guinea pigs were exposed to two selective PPARalpha agonists, Wy-14, 643 and methylclofenapate; both compounds induced hypolipidaemia. Thus the guinea pig is a useful model for human responses to peroxisome proliferators.

Effects of peroxisome proliferators and 12-O-tetradecanoyl phorbol-13-acetate on intercellular communication and connexin43 in two hamster fibroblast systems.

Effects of 12-O-tetradecanoyl phorbol 13-acetate (TPA) and the hepatic peroxisome proliferators (HPPs) clofibrate, methyl clofenapate (MCP), di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) were studied in 2 gap junctional intercellular communication (GJIC) systems, metabolic cooperation in V79 cells and microinjection/dye transfer in Syrian hamster embryo (SHE) cells and V79 cells. TPA inhibited GJIC in both systems but was considerably more potent in V79 cells. SHE cells showed a rapid and transient inhibition of GJIC after exposure to HPPs, with maximal inhibition occurring at 5-15 min. The transient inhibition could be caused by metabolization of the compounds. Clofibrate and MEHP produced strong inhibition of metabolic cooperation in V79 cells at high concentrations, while the effect of MCP and DEHP was lower. However, DEHP, MEHP and clofibrate strongly inhibited dye transfer in V79 cells after a 30 min exposure. Clofibrate also showed a dose- and time-dependent effect on dye transfer in V79 cells. The phosphorylation status of the gap junction protein connexin43 (Cx43) changed minimally in SHE cells after exposure to TPA or HPPs. Cx43 from V79 cells was strongly affected by TPA, but not by HPPs. Immunofluorescence of Cx43 disappeared in both cell types when they were exposed to TPA and MEHP, but not to the other HPPs. Thus, there is no direct correlation between the inhibition of GJIC and changes in the phosphorylation status of Cx43 or the appearance of Cx43 in immunofluorescence experiments. The discrepancies may partly be explained by binding of accessory proteins to Cx43. We point out sequences that may be involved in such binding.

Comparative effects of clofibrate and methyl clofenapate on morphological transformation and intercellular communication of Syrian hamster embryo cells.

The Syrian hamster embryo (SHE) cell system was used to evaluate the ability of two hepatocarcinogenic structurally related peroxisome proliferators (PPs) to induce morphological transformation (MT) of SHE colonies and to inhibit gap junctional intercellular communication (GJIC). Clofibrate and methyl clofenapate (MCP), which was shown to be a more active PP and a more potent carcinogen in vivo than clofibrate, were compared. MCP appeared slightly more active in vitro than clofibrate in affecting MT and GJIC of SHE cells. The morphological transformation of SHE colonies was induced by 50 microM MCP, against 100 microM clofibrate. Moreover, 50 microM MCP potentiated the transforming effects of both benzo[a]pyrene and 12-O-tetradecanoylphorbol-13-acetate. The inhibition of GJIC, measured by transfer of lucifer yellow, was transient and occurred at concentrations inducing morphological transformation. MCP inhibited dye transfer at 50 microM and the inhibition lasted up to 24 h at 100 microM. Inhibition of communication lasted only 4 h with clofibrate and occurred at a higher concentration (175 microM). This study showed that both the SHE cell transformation and dye transfer assays were able to display the different activities of the two PPs, even though the difference in potency observed was smaller than in vivo. It also revealed interactions between non-genotoxic carcinogens and the ability of the SHE cell transformation assay to detect these combined effects.

Changes in protein and mRNA levels of growth factor/growth factor receptors in rat livers after administration of phenobarbitone or methylclofenapate.

The effects of phenobarbitone and methylclofenapate were studied on the expression of growth factor and growth factor receptors in livers of male Wistar rats. The major findings were: (1) a significant reduction in epidermal growth factor receptor (EGFR) protein observed with both treatments, and (2) levels of EGFR transcripts were only slightly decreased with both compounds. The reduction in the receptor level therefore does not occur via regulation of transcription. Mannose-6-phosphate receptors (M6PR, also called insulin-like growth factor II receptor) and M6PR transcripts remained unchanged in both experimental groups. Hepatocyte growth factor receptor (HGFR) transcripts were also unchanged in both experimental groups. Transcript levels of transforming growth factor-beta 1 (TGF-beta 1) were lower in both treatment groups compared with the control; the reduction was significant in the methylclofenapate group. This may have relevance to the finding by others that nafenopin, another peroxisome proliferator, suppresses rat hepatocyte apoptosis. Another finding of general interest was that the three "housekeeping genes", namely albumin, actin and glyceraldehyde-3-phosphate dehydrogenase, were influenced by both treatments thus limiting their use as controls for gel loading. The adaptation of a growth regulatory mechanism via EGFR and its ligands may provide conditions such that cells with aberrant growth control have a selective growth advantage over normal cells thus promoting tumorigenesis.

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.

The peroxisome proliferations WY-14,643 and methylclofenapate induce hepatocyte ploidy alterations and ploidy-specific DNA synthesis in F344 rats.

WY-14,643 (WY) and methylclofenapate (MCP) are peroxisome proliferators (PP) and hepatocarcinogens in rats. MCP causes hepatic polyploidization and preferentially induces replicative DNA synthesis in binucleate tetraploid hepatocytes (2 X 2N) in young Alpk:AP rats. To compare the effect of WY and MCP on hepatocyte ploidy and ploidy-specific DNA synthesis, male F344 rats were fed WY (0.1% in diet) or gavaged with MCP (25 mg/kg/day in corn oil) for 2, 5, or 10 days. Four rats per treatment group (including corn oil and diet control groups) were euthanized and the livers perfused at each time point. To identify cells undergoing DNA synthesis, all animals received BrdU by continuous infusion for 2 or 5 days prior to euthanasia. Hepatocyte ploidy and DNA synthesis were determined using one- or two-parameter flow cytometry. Averages +/- SEM for adult male F344 rats as a percentage of total hepatocytes for each ploidy subclass are 2N = 3.4 +/- 0.7%, 4N = 69.9 +/- 1.9%, 2 X 2N = 14.4 +/- 2.4%, 8N = 2.2 +/- 0.4%, and 2 X 4N = 9.6 +/- 0.9%. Significant alterations were not induced in the proportions of 2 X 2N or 4N ploidy subclasses by WY or MCP at any time point. However, WY caused increases in 8N hepatocytes at 2, 5, and 10 days (2 days, 5.2% vs 2.2% for controls; 5 days, 7.0% vs 3.1% for controls; 10 days, 6.4% vs 3.6% for controls) as did MCP at 5 and 10 days (5 days, 6.3% vs 2.5% for controls; 10 days, 5.3% vs 2.9% for controls). In addition, a majority of BrdU-containing hepatocytes were 4N following 5 and 10 days of WY and MCP [34.3% (WY) and 16.8% (MCP) vs 1.8% and 1.1% for controls, respectively, for 2 X 2N (5 days) as a percentage of total hepatocytes]. Hepatocytes with intermediary DNA content (between tetraploid and octaploid) from MCP- and WY-treated rats were predominantly mononuclear, the percentage of binucleate hepatocytes being similar to or less than the percentage of binucleate cells within the total tetraploid hepatocyte population. These data suggest that polyploidization is induced by PP and induction of S-phase by WY and MCP occurs primarily in 4N hepatocytes in mature F344 rats and not within 2 X 2N hepatocytes. Identification of a ploidy subpopulation at risk for tumor development in rodents is essential for clarifying the role of cell replication in risk assessment studies of PP.

Induction of cytochrome P450 isoenzymes in cultured precision-cut rat and human liver slices.

1. The effect of some xenobiotics on levels of selected cytochrome P450 (CYP) isoenzymes determined by Western immunoblotting and associated enzyme activities has been studied in 72-h cultured rat and human precision-cut liver slices. 2. In cultured rat liver slices, 0.5 mM sodium phenobarbitone (PB), 25 microM beta-naphthoflavone (BNF), and 20 micrograms/ml Aroclor 1254 (ARO) induced mixed-function oxidase enzyme activities. Western immunoblotting of liver slice microsomes was performed with antibodies to rat CYP1A2, 2B1/2 and 3A. Compared with 72-h control (dimethyl sulphoxide only treated) rat liver slice microsomes, PB induced CYP2B1/2 and 3A, BNF induced CYP1A2, and ARO induced CYP1A2, 2B1/2, and 3A. 3. The peroxisome proliferators methylclofenapate (MCP), ciprofibrate (CIP) and Wy-14,643 (WY) induced palmitoyl-CoA oxidation in 72-h cultured rat liver slices. Compared with 72-h control rat liver slice microsomes, MCP, CIP, and WY all induced levels of CYP4A. 4. In cultured human liver slices, 20 micrograms/ml ARO, but not 0.5 mM MCP, induced 7-ethoxyresorufin O-deethylase activity. Neither ARO nor MCP had any effect on homogenate palmitoyl-CoA oxidation and microsomal lauric acid 11- and 12-hydroxylase activities. Compared with 72-h control human liver slice microsomes, ARO induced CYP1A2, and MCP appeared to induce CYP4A. Further studies would be required to confirm that CYP4A isoenzymes could be induced by xenobiotics in human liver slices. 5. These results demonstrate that cultured liver slices may be used in evaluating the effect of xenobiotics on both rat and human CYP isoenzymes.