ToxSci Advance Access originally published online on March 28, 2008
Toxicological Sciences 2008 104(1):74-85; doi:10.1093/toxsci/kfn062
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Species-Specific Kinetics and Zonation of Hepatic DNA Synthesis Induced by Ligands of PPAR
* School of Biology, University of Nottingham, University Park, Nottingham NG7 2RD, UK
Sanofi-Aventis, Willowburn Avenue, Alnwick, Northumberland NE66 2JH, UK
AstraZeneca Pharmaceuticals plc, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK
1 To whom correspondence should be addressed. Fax: +44-115-9513251. E-mail david.bell{at}nottingham.ac.uk.
Received February 13, 2008; accepted March 14, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Peroxisome proliferator–activated receptor
(PPAR) ligands evoke a profound mitogenic response in rodent liver, and the aim of this study was to characterize the kinetics of induction of DNA synthesis. The CAR ligand, 1,4-bis[2-(3,5-dichoropyridyloxy)]benzene, caused induction of hepatocyte DNA synthesis within 48 h in 129S4/SvJae mice, but the potent PPAR ligand, ciprofibrate, induced hepatocyte DNA synthesis only after 3 or 4 days dosing; higher or lower doses did not hasten the DNA synthesis response. This contrasted with the rapid induction (24 h) reported by Styles et al., 1988, Carcinogenesis 9, 1647–1655. C57BL/6 and DBA/2J mice showed significant induction of DNA synthesis after 4, but not 2, days ciprofibrate treatment. Alderley Park and 129S4/SvJae mice dosed with methylclofenapate induced hepatocyte DNA synthesis at 4, but not 2, days after dosing and proved that inconsistency with prior work was not due to a difference in mouse strain or PPAR ligand. Ciprofibrate-induced liver DNA synthesis and growth was absent in PPAR-null mice and are PPAR dependent. In the Fisher344 rat, hepatocyte DNA synthesis was induced at 24 h after dosing, with a second peak at 48 h. Lobular localization of hepatocyte DNA synthesis showed preferential periportal induction of DNA synthesis in rat but panlobular zonation of hepatocyte DNA synthesis in mouse. These results characterize a markedly later hepatic induction of panlobular DNA synthesis by PPAR ligands in mouse, compared to rapid induction of periportal DNA synthesis in rat. Key Words: liver growth; hepatocyte; PPAR; peroxisome proliferation; DNA synthesis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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The peroxisome proliferators were originally characterized as a class of structurally diverse compounds that caused liver cancer in rodents (Reddy et al., 1980
) and induced substantial changes in liver ultrastructure, including proliferation of endoplasmic reticulum and peroxisomes. This class of agents is now known to act through activation of the peroxisome proliferator–activated receptor (PPAR) (Issemann and Green, 1990), and it is clear that this class of carcinogen has a nongenotoxic mode of action (Ashby et al., 1994; Peters et al., 1997). The potent carcinogenicity of this class is shown by the fact that Wy-14,643 can induce 100% multifocal liver cancer in rodents after 1 year (Cattley et al., 1991). Ciprofibrate and methylclofenapate (MCP) are potent PPAR ligands (Mukherjee et al., 2002) that are also potent hepatocarcinogens (Meyer et al., 2003; Tucker and Orton, 1995).
The mechanism whereby PPAR ligands cause cancer is still largely unclear (Ashby et al., 1994; Klaunig et al., 2003; Peters et al., 2005). It is clear that DNA synthesis plays an important role in carcinogenesis and peroxisome proliferators induce hepatic DNA synthesis that is related to carcinogenesis (Marsman et al., 1988). It is therefore desirable to understand the mechanisms controlling the induction of DNA synthesis by ligands of the PPAR, particularly since these ligands augment normal liver size, in contrast to the regenerative growth pathways induced by partial hepatectomy (Mangnall et al., 2003). The mechanisms underlying PPAR ligand–induced augmentative liver growth are poorly understood (Menegazzi et al., 1997; Peters et al., 2005), beyond noting that DNA synthesis is dependent upon the PPAR (Peters et al., 1997).
The availability of mouse genetic tools (Ledda-Columbano et al., 2002; Lee et al., 1995), combined with the potent liver growth effect of PPAR ligands in the mouse (Peters et al., 1997), provides a compelling reason for using the mouse to characterize the kinetics and mechanisms of induction of hepatocyte DNA synthesis. It is important to characterize the kinetics of induction of DNA synthesis by PPAR ligands in the mouse as this information is essential for understanding the relationship with genes that regulate the induction of DNA synthesis. The early kinetics of induction of DNA synthesis by PPAR ligands were characterized using flow cytometry to characterize DNA synthesis in rodent liver (Styles et al., 1987). MCP induced high levels of DNA synthesis by as early as 24 h after dosing in the Alderley Park (AP) mouse (Styles et al., 1988), and the kinetics of induction were confirmed by another study in C57BL/6 mice (Styles et al., 1990). However, there is one report using immunohistochemical detection of bromodeoxyuridine (BrdU) incorporation in the CD-1 mouse that shows induction of DNA synthesis only after 3/4 days of dosing with the potent PPAR ligand, ciprofibrate, (Ledda-Columbano et al., 2003), and so this area is in dispute.
Characterization of the kinetics of induction of DNA synthesis is crucial for understanding the relationship with induced genes that might regulate this response, and so we have examined the time course of induction of hepatocyte DNA synthesis by PPAR ligands in several mouse strains and in the rat. In contrast to previous reports, we show that there are species-specific kinetics of induction of DNA synthesis and that the zonation of PPAR ligand–induced DNA synthesis is different between mouse and rat.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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All the chemicals were of the highest quality available. Ciprofibrate was a kind gift of Sanofi-Aventis and MCP, a kind gift of Dr C R Elcombe (CXR Biosciences, Dundee, UK), was synthesized by Lancaster synthesis Ltd, Morecambe, Lancs, UK. 1,4-Bis[2-(3,5-dichoropyridyloxy)]benzene (TCPOBOP) was obtained from Sigma (Poole, UK). 129S4/SvJae mice and their PPAR
-null congenic strain (129S4/Jae-Pparatm1Gonz/tm1Gonz) (Lee et al., 1995) were kind gifts of Frank Gonzalez (National Institutes of Health, MD) and were maintained as a colony in house. C57BL/6J:CRL and DBA/2J mice were obtained from Charles River Laboratories, AP mice from AstraZeneca Pharmaceuticals, and male Fischer 344 rats (F344/NHsd) were purchased from Harlan UK Ltd (Bicester, UK).
Chronic treatment protocol.
Mice (8–9 weeks old) or rats (14–15 weeks old) were randomized to treatment groups (normally n = 6) and then acclimatized to 10% orange juice in tap water as their sole source of drinking water for 1 week. BrdU was then added to the 10% orange juice (0.8 mg/ml final concentration), and after 1 day of exposure to the BrdU, animals were dosed by gavage with xenobiotic in corn oil (20 ml/kg body weight) and then dosed as indicated until killed by pentobarbital overdose. Body weight was determined daily throughout the procedure.
Acute rat treatment protocol.
Rats (14–15 weeks old) were randomized to treatment group and then dosed with peroxisome proliferator (in corn oil, 20 ml/kg body weight) by gavage. At the indicated time after treatment, animals were dosed ip with 100 mg/kg body weight of BrdU in sterile phosphate-buffered saline. Animals were killed by ip with pentobarbital overdose 2 h after BrdU administration.
Immunohistochemistry.
A blood sample was taken at necropsy, and serum prepared, followed by storage at – 80°C until determination of alanine aminotransferase (ALT) activity. The liver was weighed and a section of left lobe and small intestine was fixed in 10% neutral buffered formalin overnight at room temperature. Fixed tissues were used to prepare blocks and then heat-induced epitope retrieval was used with the Amersham mouse anti-BrdU labeling system to visualize BrdU; the specific nuclear staining required both primary and secondary antibodies and administration of BrdU (data not shown). Slides were counterstained with Harris' hemotoxylin and then mounted with distyrene, plasticizer, and xylene. For each animal, 2000 hepatocyte nuclei were scored in random fields, and the labeling index is (BrdU-labeled hepatocyte nuclei/total hepatocyte nuclei) x 100. Hepatocytes were identified morphologically. A sample of small intestine from each animal was tested as a positive control. For the determination of zonal distribution of labeled hepatocytes, the method was essentially as described (Barrass et al., 1993). Briefly, a field was defined as a radius of five to seven cells around either the portal space (the periportal region) or the central vein (the perivenous region), using small vessels of similar size. Five fields were counted for each of the periportal and centrilobular zones for each animal, and the total number of labeled nuclei was recorded.
Statistics.
Student's t-test was used for comparison of two groups and a paired t-test for examining body weight loss. ANOVA followed by a post hoc test (Dunnett's, Newman-Keuls) was used for multiple comparisons. For time-course studies, the control group time points were tested to determine if they were significantly different from each other. If not, the control group values were pooled, and the pooled values were used for comparisons against the treated groups (Figs. 2A and 2C, 3, 4, and 5A).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Experiments were undertaken to determine whether PPAR
ligands induce hepatic DNA synthesis in mice at 24 h after dosing (Styles et al., 1988, 1990). Male 129S4/SvJae mice were administered the potent PPAR ligand, ciprofibrate, and hepatocytes labeled with BrdU, essentially as described for the rat acute dosing protocol (Materials and Methods), except with ip administration of ciprofibrate. This protocol yielded satisfactory staining of intestinal nuclei for BrdU as a positive control for labeling but showed no significant induction of hepatocyte DNA synthesis over 18–42 h, over a dose range of 25–100 mg ciprofibrate/kg body weight (Brett Jeffer, unpublished data). Administration of BrdU at 2 h before death limits the window for detection of DNA synthesis to 2 h, and so BrdU was administered continuously in the drinking water to ensure that there was continuous exposure to BrdU. Continuous exposure to BrdU is known to be more sensitive for detecting low levels of DNA synthesis or where DNA synthesis occurs over a period of days (Eldridge et al., 1990). Although BrdU has been administered in the drinking water at doses of 1 mg/ml, e.g. (Ledda-Columbano et al., 2003), administration of BrdU at 0.8 mg/ml in the drinking water led to significant body weight loss in Balb/c or 129S4/SvJae mice (data not shown); several studies show body weight loss with BrdU in the drinking water (Jecker et al., 1997; Reome et al., 2000), attributed to the bitter taste of the BrdU. Therefore, 129S4/SvJae mice were acclimatized to 10% orange juice as the sole source of drinking water for 1 week, before adding the BrdU (0.8 mg/ml) to the 10% orange juice; the hypothesis was that the orange juice would mask the taste of the BrdU. This protocol yields stable mouse body weights (Fig. 1A) and effective labeling of a positive control tissue, the small intestine (Supplementary Fig. 1): note that the nuclear labeling of intestinal cells is uniform throughout the length of villus stained, showing that there is consistent BrdU staining, and suggesting consistent bioavailability of BrdU, throughout the period of dosing with BrdU. A single dose of 100–400 mg/kg ciprofibrate did not increase serum ALT levels, demonstrating that these doses of ciprofibrate did not cause hepatotoxicity and regenerative regrowth in the liver (Fig. 1A). These doses did increase liver weight at 48 h after dosing, which was statistically significant (Fig. 1A), but failed to cause a statistically significant increase in labeling of hepatocyte labeling index (Fig. 1A).
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In order to verify that the chronic BrdU labeling protocol leads to efficient incorporation of BrdU label in liver, as well as intestine, animals were treated with the CAR agonist, TCPOBOP, at 3 mg/kg, which is characterized to cause rapid induction of DNA synthesis in CD-1 mice (Ledda-Columbano et al., 2003
). There was a statistically significant threefold increase in ALT in male, but not female, mice (data not shown), showing a small effect on liver cell damage. The liver to body weight ratio was significantly increased in both male and female mice (Fig. 1B), and the hepatocyte labeling index was induced by TCPOBOP treatment to 25–50% (Figs. 1B–D). Thus, the BrdU dosing methodology leads to effective labeling of hepatocyte DNA synthesis that has been induced within 48 h of xenobiotic (TCPOBOP) treatment in 129S4/SvJae mice; consequently, an artifact of dosing methodology can be excluded as a reason for the failure to detect induction of hepatic DNA synthesis in mouse by PPAR ligands (Fig. 1).
The time course of the hepatic response to ciprofibrate was examined, to determine if the DNA synthesis response occurs at a later stage than in the first 2 days after dosing. Figure 2 shows that liver weight is significantly increased as early as 2 days after dosing and that liver weight has increased by 
100% at 6 days after dosing commenced. The DNA synthesis response was later than the liver growth, with significant induction after 3 days of administration of ciprofibrate (although with large variation) and subsequently (Fig. 2A). In order to ensure that the kinetics of induction of DNA synthesis was not an artifact of too high or low a dose, the hepatocyte DNA synthesis response was examined after 3 or 4 days of dosing with ciprofibrate (Fig. 2B). As little as 10 mg/kg/day of ciprofibrate caused a significant increase in liver to body weight ratio, and there was a tendency for the increase in weight to be larger after 4 days, compared to 3 days (Fig. 2B). In this experiment, the induction of DNA synthesis was lower at 3 days than in the previous experiment (Figs. 2A and 2B), but the induction of DNA synthesis was statistically significant. At 4 days after dosing, there was a robust induction of hepatocyte DNA synthesis at 30 or 100 mg/kg/day of ciprofibrate (Fig. 2B), and this latter dose was demonstrated to give the highest induction of DNA synthesis. The hepatic effects of ciprofibrate were examined in female mice to determine if there was a sex difference in response. The liver to body weight ratio was significantly induced after 3 or 4 days of dosing (Fig. 2C), but hepatocyte DNA synthesis was only significantly increased after 4 (but not 3) days of dosing (Fig. 2C), comparable with the male, showing that there is no marked sex difference in induction of hepatocyte DNA synthesis by PPAR ligands. Thus, ciprofibrate induced hepatocyte DNA synthesis only after 3/4 days of dosing, and the dose giving the highest induction of DNA synthesis has been defined.
These studies had used the 129S4/SvJae mouse, and strain differences were one explanation for the fact that the response seen in Figure 2 is much later than that reported by Styles (Styles et al., 1988, 1990). Therefore, we investigated the induction of hepatocyte DNA synthesis in C57BL/6J mice; however, the results are essentially similar to those seen in 129S4/SvJae mice, with significant induction of hepatocyte DNA synthesis after 4 (but not 2) days of dosing with ciprofibrate (Fig. 3A). DBA/2J mice were also dosed using a similar protocol (Fig. 3B); liver to body weight ratio is significantly induced at 2 days after dosing, but the hepatic labeling index is not significantly increased at 2 days after dosing but is significantly increased at 4 and 6 days after dosing commenced. It is therefore less likely that the discrepancy between this study and the work of Styles (Styles et al., 1988, 1990) is simply due to an idiosyncracy of the 129S4/SvJae mouse strain since these workers had shown that MCP induces hepatocyte DNA synthesis by 24 h in C57BL/6 mice (Styles et al., 1990). In order to exclude the possibility that ciprofibrate may have some effect that is not mediated by the PPAR, the effects of ciprofibrate were tested in congenic 129S4/SvJae mice that are nullizygous for the PPAR (Abdullah Al Kholaifi, unpublished data). These data showed that ciprofibrate at 100 mg/kg/day had no significant effect on liver to body weight ratio or on the hepatocyte labeling index, or ALT (data not shown), consistent with the work of Peters et al. (1997). This proves that the induction of liver growth and hepatic DNA synthesis by ciprofibrate requires PPAR and given that ciprofibrate is a known PPAR ligand and peroxisome proliferator (Meyer et al., 2003; Mukherjee et al., 2002), this constitutes proof that these effects are caused by a direct action of ciprofibrate on the PPAR.
Styles showed rapid induction of hepatic DNA synthesis in AP mice, using MCP, and given the importance of this report, we undertook a direct comparison between 129S4/SvJae and AP mice using the same dose of the same peroxisome proliferator (MCP) used by Styles (Styles et al., 1988). MCP caused a significant induction of liver growth at days 2–4 after dosing commenced (Fig. 4A) in 129S4/SvJae mice and showed significant induction of hepatocyte DNA synthesis after 3 or 4 (but not 2) days of dosing (Fig. 4A); this is consistent with previous data using the PPAR ligand, ciprofibrate (Figs. 1–3
). When this experiment was repeated using AP mice, similar results were obtained (Fig. 4B): hepatic growth was statistically significant after 2–4 days of dosing, but labeling index was not significantly increased after 1, 2, or 3 days dosing, and was only statistically significantly increased after 4 days of dosing with MCP. These data exclude mouse strain or peroxisome proliferator as a cause of the delayed hepatic DNA synthesis response seen with our data, as compared to the results of Styles (Styles et al., 1988, 1990), and provide strong evidence that the induction of hepatic DNA synthesis by PPAR ligands is delayed until after 3/4 days in the mouse.
A direct comparison was made with the induction of hepatic DNA synthesis by ciprofibrate in the Fisher344 rat. Hepatocyte DNA synthesis was significantly increased at 24, 48, and 96 h after dosing when using the chronic BrdU dosing protocol, attaining a 38% labeling index (Fig. 5A). The labeling index at the 24-h time point was significantly different from control (control 1.33 ± 0.37% [mean and SD] vs. ciprofibrate 4.4 ± 2.3%) on a t-test. Chronic dosing with BrdU involves exposing animals to the labeling agent for a period of days, thereby leading to a high background value for DNA synthesis in the control group as a result of DNA synthesis over the whole of this period. Therefore, the experiment was repeated using an acute ip dose of BrdU at 2 h before killing the animals, to characterize the DNA synthesis response within the shorter time frame of the first 48 h after dosing. The liver weight was slightly, but significantly, increased at 30 and 36 h after dosing, with a larger increase at 48 h after dosing with ciprofibrate (Fig. 5A). In contrast to the mouse (Figs. 1–4), ciprofibrate significantly induced hepatocyte DNA synthesis as early as 24 h after dosing, with levels falling back to background before a second wave of DNA synthesis at 48 h after the first dose (Fig. 5B). The induction of hepatocyte DNA synthesis at 24 h was examined by varying the dose of ciprofibrate, confirming an early induction of hepatocyte DNA synthesis at 24 h and further showing that 50–200 mg ciprofibrate/kg body weight are optimal doses for inducing hepatocyte DNA synthesis (Fig. 5C). At 300 mg ciprofibrate/kg body weight, there is no significant induction of liver to body weight ratio and no induction of hepatocyte DNA synthesis; the diminished nature of these responses, compared with lower doses, suggests that 300 mg/kg body weight is overtly toxic and is suppressing the liver growth response. These results demonstrate that there is a species difference between mouse and rat in the kinetics of induction of hepatocyte DNA synthesis by PPAR ligands.
Given these distinct results in mouse and rat, the lobular zonation of induction of hepatocyte DNA synthesis in liver was examined, as previously described (Barrass et al., 1993). Figure 6A shows that there is marked periportal distribution of labeled hepatocyte nuclei in the rat, whereas the mouse shows a panlobular distribution of labeled cells. Quantification of these data (Fig. 6B) shows that there is a difference in the zonation of induced hepatocyte DNA synthesis between the rat and mouse, with statistically significant preferential periportal induction of DNA synthesis by PPAR ligands in the Fisher344 rat and no significant difference between periportal and centrilobular induction of DNA synthesis in the 129S4/SvJae mouse. The data in Figure 6B are typical of data from 129S4/SvJae mice treated with 30–100 mg ciprofibrate/kg/day for 3 or 4 days, 100 mg ciprofibrate/kg/day for 3–6 days, 25 mg/kg/day MCP for 3 or 4 days, or C57BL/6 mice treated with 100 mg ciprofibrate/kg/day for 4 days (data not shown). Likewise, the preferential periportal distribution of induced DNA synthesis in rat hepatocytes is consistent over a dose range of 50–200 mg ciprofibrate/kg and when using a chronic BrdU administration regime (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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The mouse and rat show differential kinetics of induction of hepatocyte DNA synthesis after exposure to PPAR
ligands, with the mouse response being delayed to 3 days after dosing. Given that these results differ from previous work (Styles et al., 1988, 1990), it was necessary to undertake extensive controls. Immunohistochemical detection of incorporated BrdU has been extensively validated for detection of replicating hepatocytes (e.g., Eldridge et al., 1990; Ledda-Columbano et al., 2003) and positive controls include labeling of intestine and labeling in liver from mice treated with the CAR ligand, TCPOBOP, and that results obtained using acute and chronic BrdU dosing protocols were comparable. Hence, the labeling regime and immunohistochemical detection system were robustly validated for detecting early induction of DNA synthesis. BrdU treatment can decrease body weight (Abdullah Al Kholaifi, unpublished data), and so mouse body weight was measured, although this elementary control is not often described in the literature; mouse body weight was found to be unaffected by BrdU in all experiments shown. PPAR ligands can cause (focal) necrosis (e.g., Woods et al., 2007), thereby leading to regenerative growth; therefore, serum ALT (a marker of liver cell damage) was shown to be unaffected by the doses of PPAR ligands used in this study, and there was no evidence of necrosis detected by examination of histological sections (not shown). These control experiments prove unambiguously that ciprofibrate fails to induce hepatocyte DNA synthesis within 2 days after dosing.
Styles (Styles et al., 1988) found rapid induction of hepatocyte DNA synthesis by PPAR ligands in AP and heterozygous Snell dwarf mice on C57BL/6 (Styles et al., 1990), whereas our results were obtained in 129S4/SvJae mice, suggesting a strain difference in response. Mouse strain can affect liver function (Akiyama et al., 2001; Manenti et al., 1994), but published evidence that strain differences affect peroxisome proliferation is inconclusive (Budroe et al., 1992; Dwivedi et al., 1989; Jones et al., 1995). C57BL/6 and DBA/2J mice were treated with ciprofibrate, showing that the kinetics of induction of liver growth and DNA synthesis were similar to that in 129S4/SvJae mice. AP mice are an outbred stock of Swiss origin (Beck et al., 2000; Chia et al., 2005) and so we directly tested in AP mice using the same dose of the same PPAR ligand described in (Styles et al., 1988). The induction of hepatocyte DNA synthesis response was delayed to 3/4 days after administration of MCP in 129S4/SvJae and in AP mice, thus showing remarkably little effect of strain difference in the response of the mouse to PPAR ligands in three different mouse strains. Thus, we have been unable to replicate the results of Styles in both mouse strains; while it is possible that the AP mice, being outbred, may have undergone strain drift, the C57BL/6 mice are inbred and are unlikely to show significant strain drift. Moreover, the concordance between the results obtained in 129S4/SvJae, AP, C57BL/6J, and DBA/2J mice in our hands excludes the possibility that the discordance between our data, and those of Styles (Styles et al., 1988, 1990), arises from mouse strain differences.
The possibility that ciprofibrate had off-target (i.e., non-PPAR mediated) effects that confounded its liver growth function was also considered. Ciprofibrate is a potent PPAR ligand (Mukherjee et al., 2002), has similar effects to another potent (Bell and Elcombe, 1991; Bell et al., 1991) PPAR ligand, MCP, and the use of PPAR nullizygous mice provides compelling evidence that the liver growth and DNA synthesis effects of ciprofibrate are mediated by the PPAR.
It is difficult to explain the difference in kinetics of induction of DNA synthesis reported by Styles (Styles et al., 1988, 1990) and in this study. Table 1 compares the methodology used by Styles et al. and in this paper, and the principal remaining variable is that this study used immunohistochemical detection of incorporated BrdU, whereas Styles detected incorporated BrdU by isolation of hepatocytes and flow cytometry of the isolated hepatocytes (Styles et al., 1987). PPAR ligands cause an increase in liver size, and the consequent increase in hepatocyte size (Abdullah Al Kholaifi, Abeer Amer, unpublished data) could cause differential recovery of liver cells during the hepatocyte isolation procedure between control and treated animals or alternatively may differentially affect the propidium iodide staining. There are no controls to determine whether this is so; and both of these issues could introduce artifactual error into the determination of the proportion of labeled hepatocytes by flow cytometry. Miller et al. were unable to replicate the results of Styles on rat hepatocyte ploidy (Miller et al., 1996; Styles et al., 1987), which shows difficulty in reproducing results obtained by the flow cytometry methodology. The reason for the difference in results remains obscure, but we have excluded the choice of PPAR ligand, dose, sex, and strain differences as possible variables. Further, our results are consistent with other reports (Ledda-Columbano et al., 2003), and so we conclude that the induction of hepatocyte DNA synthesis is delayed until 3 days after administration of PPAR ligand, and we propose that the results obtained by Styles are an artifact arising from the cell isolation and flow cytometric analysis of liver cells.
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The delayed induction of hepatocyte DNA synthesis in the mouse is distinct from the rapid induction of DNA synthesis in the rat. Our results in the rat are consistent with our previous demonstration that PPAR
ligands cause rapid (ca. 24 h) induction of DNA synthesis in rat hepatocytes in vitro (Plant et al., 1998a,b) and the finding that the PPAR ligands, nafenopin and Wyeth-14,643, cause induction of hepatic DNA synthesis at 24 h after dosing in Wistar and Fisher344 rats, respectively (Menegazzi et al., 1997; Miller et al., 1996). However, it is essential to use the same methodology in the same laboratory to obtain a reliable comparison of mouse and rat. This delayed response of the mouse to induction of hepatocyte DNA synthesis is reminiscent of the response to partial hepatectomy, where mouse hepatocyte DNA synthesis commences 12–16 h later than in the rat; after partial hepatectomy, it has been shown that the faster response of the rat hepatocyte is cell autonomous (Weglarz and Sandgren, 2000). However, the delayed induction of DNA synthesis in mouse does not reflect an intrinsic lack of capability in the mouse hepatocyte, as TCPOBOP triggers a fast DNA synthesis response, signaling via the CAR receptor. Thus, the kinetics of induction of hepatocyte DNA synthesis are specific to the signaling pathway initiating DNA synthesis. The delayed induction of DNA synthesis response in mouse by PPAR ligands is unlikely to be due to a species difference in the amount of the PPAR since the receptor is present at high levels in mouse, compared to other rodents (Bell et al., 1998; Choudhury et al., 2000, 2004).
The distinct species-specific kinetics of induction of hepatocyte DNA synthesis is associated with altered zonation of induced DNA synthesis, where the rat shows preferential induction of DNA synthesis in the periportal region and the mouse shows panlobular induction of DNA synthesis. The zonation of induction of enzymes by various xenobiotics has been extensively demonstrated (Bars et al., 1992; Oinonen et al., 1994), but the zonal induction of DNA synthesis is much less well characterized, although reliable methods have been established (Barrass et al., 1993). Our results confirm and extend the findings of Barrass et al. by showing that a distinct PPAR ligand, ciprofibrate, also induces zonal induction of hepatocyte DNA synthesis in rat and by showing that DNA synthesis does not show zonal distribution in several mouse strains. The periportal distribution of induced hepatocyte DNA synthesis in rat is distinct from the preferentially centrilobular induction of cytochrome P450 and peroxisomal enzymes induced by PPAR ligands (Bars et al., 1993; Bell et al., 1991), yet the induction of both DNA synthesis and enzymes are PPAR dependent. This suggests that PPAR-independent mechanism must be responsible for the distinct zonation of DNA synthesis and peroxisomal enzymes. Characterization of the zonal distribution of the PPAR-associated coactivators, or microRNAs that are known to be required for induction of DNA synthesis (Matsumoto et al., 2007; Shah et al., 2007), are obvious candidates for mediating these PPAR-independent effects. While it is known that Apc is the "zonation keeper" in mouse liver (Benhamouche et al., 2006), it is not clear that exactly the same pathway applies in rat liver. Given the complex web of interactions between Apc and β-catenin, and that β-catenin signaling regulates liver growth pathways (Tan et al., 2006), it can be hypothesized that species differences in regulation of the β-catenin signaling pathway mediate PPAR ligand–induced hyperplasia and lobular localization. Given the availability of liver-specific knockouts of β-catenin (Tan et al., 2006), this hypothesis is partly experimentally tractable.
The definition of the rapid induction of hepatocyte DNA synthesis in rat by PPAR ligands opens up opportunities for investigating the mechanism of induction of the growth response, by relating the early gene induction events to subsequent hyperplasia. There is evidence that the liver growth program induced by PPAR ligands is quite distinct from that seen during regeneration or after treatment with growth factors (e.g. Menegazzi et al., 1997; Plant et al., 1998a,b): the PPAR ligand–induced growth defined in this paper offers a tractable system for investigating how augmentative liver growth is regulated.
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Sanofi-Synthelabo to B.J., and Saudi Arabian government to A.A.K. and the Libyan government to A.A.
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ACKNOWLEDGMENTS |
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The authors wish to thank Declan Brady for expert technical assistance, Prof. J.M. Behnke for provision of facilities for histological analysis, and Dr Sandy Brown for help with ALT assays and analysis. We wish to thank Frank Gonzalez (National Institutes of Health, Bethesda, MD) and Jeff Peters (Pennsylvania State, PA) for the kind gift of 129S4/SvJae and PPAR
-null mice, and Professor Brian G. Lake for critical review of the manuscript. |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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