Hepatic hyperplasia and cancer in rats: alterations in copper metabolism
Patricia K. Eagon1,2,3,5,
Annette G. Teepe3,
Mary S. Elm1,2,
Stasa D. Tadic1,2,
Marilyn J. Epley1,2,
Bonnie E. Beiler4,
Hisashi Shinozuka4 and
Kalipatnapu N. Rao4
1 VA Medical Center, Pittsburgh, PA 15240,
2 Department of Medicine,
3 Department of Molecular Genetics and Biochemistry and
4 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
 |
Abstract
|
---|
We previously demonstrated that rats exposed to the peroxisome proliferator (PP) diethylhexylphthalate (DEHP) had reduced serum ceruloplasmin (CP) oxidase activity, which suggests tissue copper deposition. Copper is highly toxic in excess, and results in cellular damage and hepatocellular carcinomas (HCC). This study addresses changes in expression of copper-related genes and metal accumulation in hyperplastic liver and tumors induced by PP. Male rats were fed diets containing DEHP or clofibrate (CLF) for 360 days (hyperplasia) and 4-chloro-6-(2,3 xylidino)-2-pyrimidinyl-thio(N-ß-hydroxyethyl) acetamide for 10 months (HCC). During hyperplasia, an immediate and progressive decrease in serum CP activity was observed (P < 0.05), as were reductions in mRNA levels for both CP and Wilson's disease gene (WD gene, a P-type ATPase) (P < 0.05). Tumor-bearing rats had lower serum CP activity (P < 0.05), and CP and WD gene mRNA levels were reduced in tumors (P < 0.05), and in liver surrounding tumors (SL) (P < 0.05). Metallothionein mRNA showed no consistent changes during hyperplasia. Tumors showed a 2.5-fold induction of metallothionein mRNA (P < 0.05), and a 1.2-fold increase in SL. Temporal increases in liver copper content occurred during hyperplasia, with increases of 2-fold (DEHP) and 3.3-fold (CLF) at 60 days (P < 0.05). Copper content was 2.2-fold higher in tumors (P < 0.05) and 1.7-fold higher in SL; iron did not increase and zinc decreased temporally. Thus, copper accumulation and changes in copper-related gene expression may be contributing factors in liver neoplasia in PP-treated rats. Loss of CP results in decreased free radical scavenger capacity and thus may enhance oxidative damage induced by PPs.
Abbreviations: BR931, 4-chloro-6-(2,3 xylidino)-2-pyrimidinyl-thio(N-ß-hydroxyethyl) acetamide; CLF, clofibrate; CP, ceruloplasmin; DEHP, di(2-ethylhexyl)phthalate; HCC, hepatocellular carcinoma; LEC, LongEvans Cinnamon rats; MT, metallothionein; PP, peroxisome proliferator; WD gene, Wilson's disease gene or Atp7b in rat.
 |
Introduction
|
---|
Peroxisome proliferators (PP) are a group of structurally unrelated industrial and household chemicals and medications with a wide human exposure. Among the PPs are therapeutic hypolipidemic agents, such as clofibrate (CLF) and gemfibrozil, and the industrial chemical diethylhexylphthalate (DEHP) commonly used as a component of plastics. Others are used only in animal experimentation, such as WY-14 643 and 4-chloro-6-(2,3 xylidino)-2-pyrimidinyl-thio(N-ß-hydroxyethyl) acetamide (BR931). These PP agents are well-known inducers of hepatic hyperplasia and hepatocellular carcinoma (HCC) in rodents (1,2), but their mechanism(s) of action in this respect have not been well defined. PPs are not mutagenic and do not interact directly with DNA. One postulate, the oxidative stress model, is that these agents induce liver tumors via oxy and peroxy radicals (3,4) produced in peroxisomes, which have disproportionate activities of peroxide-producing enzymes versus peroxide-destroying catalase. However, the validity of this postulate has not been established experimentally and, in fact, the tumorigenicity of the agent does not necessarily correspond to its ability to induce peroxisomes (5,6). Another theory suggests that the most likely cause of tumorigenesis is a sustained stimulation of mitogenesis, possibly sufficient alone to induce tumors (7), but which may be enhanced by an apparent inhibition of apoptosis by these PP agents (8,9). Exposure to PPs also enhances expression of factors that stimulate liver cell proliferation, such as nuclear factor kappa-B (10) and estrogens (11,12). The mitogenesis and the oxidative stress theories are not necessarily exclusive (9).
Our previous work showed that endogenously produced estrogens are likely to be an important factor in the induction of hepatic hyperplasia. Administration of DEHP to male rats resulted in increased hepatic estrogen receptor mRNA, and in an impaired liver metabolism of estradiol, which resulted in increased circulating estradiol levels (11) and which can predispose the liver to hyperplasia (reviewed in 13). Further studies showed that during hyperplasia, which can be considered as a preneoplastic stage, there is an increase in estrogen and androgen receptor activity, whereas progression of hyperplasia to cancer results in a down-regulation of wild-type estrogen receptor (12) with an induction of variant estrogen receptor expression (14). Androgen receptor is maintained and, in fact, is induced in neoplastic liver (12). These findings resulted in our proposal that two types of hepatocytes emerge during the progression from hyperplasia to HCC: those that retain their estrogen receptor positivity and those that become negative for estrogen receptor or express only a variant receptor form.
The PPs induce many other biochemical alterations, such as induction of peroxisomal ß-oxidation enzymes (15,16), inhibition of cholesterogenesis (17,18) and inhibition of the hexose monophosphate pathway (18). The PPs have been shown to have thyromimetic activity by their induction of thyroid hormone-responsive genes, such as malic enzyme (19,20). Induction of malic enzyme partially compensates for the loss of NADPH that results from the inhibition of the hexose monophosphate pathway. Given the increased estrogen levels noted above, we expected that an estrogen-responsive protein in liver, ceruloplasmin (CP), would be induced; to our surprise, however, exposure to DEHP reduced the serum CP oxidase activity in a time-dependent manner (11). Since CP is a copper-containing protein, these findings suggested that PPs may induce changes in copper metabolism and may lead to the accumulation of copper in the liver.
Copper, as a co-factor in several enzymatic reactions, is an essential trace element (21,22). However, copper excess, as occurs in Wilson's disease or as a result of environmental exposure, is toxic to cells. Low serum CP levels are observed in patients with Wilson's disease because of a defective P-type ATPase, the gene product of the Wilson's disease gene (WD gene or ATP7B) (23): this enzyme is involved in insertion of copper into the CP protein prior to secretion and possibly biliary transport of copper as well (24,25). These patients accumulate copper in their livers and other tissues and demonstrate impaired biliary transport of copper, and they may display cirrhosis and fulminant hepatic failure if untreated.
LongEvans Cinnamon (LEC) rats exhibit a homologous genetic defect in the Atp7b gene (26), which also results in an inability to mobilize copper from the liver via CP and biliary routes and in a decreased serum CP oxidase activity. Of note, this strain of rats is highly susceptible to development of fulminant hepatitis and HCC (27). The susceptibility of LEC rats to HCC has been linked to the accumulation of hepatic copper, since treatment with penicillamine or trientine reduces both copper accumulation and HCC incidence (28,29). Further, experimental copper overload, presumably via its generation of oxygen radicals, has been shown to result in liver and cellular damage, such as lipid peroxidation of mitochondrial membranes, DNA damage and DNAprotein cross-linking (30).
The copper excess in Wilson's disease patients, LEC rats and under experimental overloading conditions, appears to overwhelm the ability of cells to remove excess copper via bile and in the blood attached to CP, since these routes involve the defective ATPase protein, and may also overwhelm the cells' ability to bind copper to metallothionein (MT). The deposited copper then produces a spectrum of oxidative injury that includes mitochondrial damage, lipid peroxidation, lysosome fragility and DNA damage (reviewed in 3133).
Because defects in liver copper metabolism appear to be a critical factor in the pathogenesis of Wilson's disease and the hepatocarcinogenesis in LEC rats, and because of our earlier observation of low serum CP activity in DEHP-treated rats (11), we tested whether copper metabolism is altered in this PP model, which involves changes in metabolic and epigenetic factors in carcinogenesis. In the present studies, we demonstrate temporal changes in expression of several parameters related to copper metabolism in both hyperplastic liver and tumors, and suggest a role for copper in the genesis of PP-induced HCC.
 |
Materials and methods
|
---|
Animals and diets
Control and PP-containing diets were prepared by Dyets (Bethlehem, PA) as described elsewhere (11,18) and were administered to male Fischer 344 rats (Hilltop Laboratories, Scottsdale, PA). All animals had free access to food and water, and were housed in special rooms for carcinogen-treated animals. Treatment protocols were approved by the University of Pittsburgh and renewed yearly. Rats, six per group and 11 weeks old, were fed a basal diet that contained 1.2% DEHP or 0.5% clofibrate CLF for 3, 7 and 60 days to induce hepatic hyperplasia. Rats, 10 per group, were fed basal diets that contained 0.16% BR931 for 10 months to induce HCC: all BR931-treated rats developed multiple liver tumors. Control rats were age-matched and were fed a basal diet for the same time periods. Additional details of the animal model have been published (10,11,18). Animals were killed at 9:00 a.m. by exsanguination through the inferior vena cava following anesthesia with ether: body and liver weights were measured. Serum was separated by low speed centrifugation and was frozen at 20°C until analyzed. In all animals, some portions of the liver were processed immediately for assays, while the remaining tissue was frozen in liquid N2 and stored at 70°C for further analyses. In animals fed BR931, tumors were dissected from the livers, and samples of liver surrounding the tumors were also collected. Tumors used for the study were 0.51.5 cm in diameter.
RNA isolation and hybridization
Total RNA was prepared by the guanidium isothiocyanate method of Chomczynski and Sacchi (34) and was quantitated by measuring absorption at 280/260 nm and then separated on a 1% agaroseformaldehydeMOPS gel. The RNA was blotted onto Gene ScreenTM (NEN Life Science Products, Boston, MA) membranes and cross-linked by exposure to UV light. The blots were hybridized at high stringency with different probes (see below) and probe binding was quantitated by densitometer or phosphoimager. All blots were either simultaneously hybridized or rehybridized to a rat glyceraldehyde phosphate dehydrogenase (GAPDH) probe (a gift from Dr Christine Milcarek, University of Pittsburgh) to normalize RNA concentrations. The following probes were labeled with 32P (Amersham Multiprime DNA labeling system, Arlington Heights, IL) and used for hybridizations: CP (partial cDNA corresponding to base pairs 421711) and the WD gene (ATPase 7B or WD800, ref 35), both generous gifts from Dr Jonathan Gitlin (Washington University); MT, a gift from Dr Sidney J.Morris, Jr (University of Pittsburgh) and originating in the laboratory of the late Dr Robert D.Andersen (UCLA) (36).
Other assays
Serum CP was assessed by measuring the oxidation of p-phenylenediamine (37). Liver copper, iron and zinc were measured by atomic absorption spectrophotometry using a PerkinElmer 4000 with graphite furnace; results were expressed as µg/g dry wt of liver.
Statistical analysis
All experimental groups were compared with their age-matched control group, using Student's t-test. Results were expressed as mean ± SEM, and a P-value of <0.05 was considered significant. The number of determinations in each analysis is included in the legend of the particular figure.
Reagents and materials
Amersham Life Sciences and ICN Radiochemicals supplied 32P. DEHP was purchased from Aldrich (Milwaukee, WI). CLF was obtained from Fluka Chemical (Ronkonkoma, New York, NY) and BR931 from LPB Instituto Farmaceutico SpA (Milan, Italy). The sources of other material have been described (11,12,18).
 |
Results
|
---|
Our previous studies documented a decrease in serum CP oxidase activity in male rats exposed to DEHP for 4, 8 and 16 weeks (11). In the current study, both shorter and longer periods of exposure to DEHP, as well as to the additional agents CLF and BR931, were tested, and additional parameters of copper metabolism were examined. These three agents were selected because they are known to be weak, moderate and strong inducers, respectively, of peroxisomes and liver hyperplasia, and upon continuous feeding, result in HCC. Figure 1
demonstrates that animals exposed to DEHP and CLF had a decrease in serum CP oxidase activity. CLF treatment for 3, 7 and 60 days resulted in an immediate and progressive decrease in activity (P < 0.05, compared with control rats). CLF produced a greater and more immediate effect than DEHP, which is in keeping with its greater potency. Tumor-bearing rats that had been treated for 10 months with BR931 also had significantly lower serum CP oxidase activity as compared with their age-matched controls (P < 0.05). It is important to note that CP oxidase activity is measurable by the p-phenylaminediamine assay only if the CP protein contains copper. We also tested whether the addition of PPs to the CP assay system inhibited the oxidase activity: no inhibition was demonstrated, which indicates that the observed reduction of CP oxidase activity is an in vivo effect.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1. Serum CP oxidase activity in rats with hepatic hyperplasia and HCC. Rats were treated for the times indicated with DEHP (open bars) or CLF (hatched bars) to induce hepatic hyperplasia (left) or with BR931 to induce HCC (right, cross-hatched bars). The CP oxidase was measured as indicated in Materials and methods and the values for the treated animals were compared with those of age-matched controls, indicated by the horizontal dotted line at 100%. Each bar represents determinations from six rats with hyperplasia and eight or nine rats from the HCC study. The bars represent means ± SEM, and those marked with asterisks differ from control values, P < 0.05.
|
|
Because of this decrease in serum CP oxidase activity, the steady-state level of hepatic CP mRNA was determined. As shown in Figure 2
, during hyperplasia, the decrease in serum activity was preceded by immediate reductions in hepatic CP mRNA levels at 3 days, followed by a sustained inhibition of CP mRNA expression, especially with CLF, which is <20% of control at 60 days, P < 0.05. Steady-state CP mRNA levels were reduced in tumors to ~30% of the level in age-matched control liver (P < 0.05), and CP mRNA levels in liver surrounding the tumors were also reduced (22% of control liver, P < 0.05). Thus, the reduction in hepatic CP mRNA levels probably accounts for the decreased CP oxidase activity in serum.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2. Liver expression of steady state CP mRNA during hyperplasia and in tumors and surrounding liver. Rats were treated for the times indicated with DEHP (open bars) or CLF (hatched bars) to induce hepatic hyperplasia (left) or with BR931 to induce HCC (right). In rats treated with BR931, tumors (cross-hatched bars) were analyzed separately from non-tumorous surrounding liver (dotted bars). The values for the treated animals were compared with those of age-matched controls, indicated by the horizontal dotted line at 100%. Each bar represents determinations from four rats with hyperplasia and six rats from the HCC study. The bars represent means ± SEM, and those marked with asterisks differ from control values, P < 0.05.
|
|
However, low serum CP could also be explained by a decrease in the ability of hepatocytes to incorporate copper into the CP molecule via the activity of the protein product of the WD gene (or Atp7b). To address this issue, the hepatic steady-state level of mRNA for WD gene was assessed. As shown in Figure 3
, the expression of this gene was also immediately reduced during hyperplasia at 3 days and further reduced at 7 days of exposure. The level of WD gene mRNA was also significantly reduced in both surrounding liver and tumors as compared with the levels in livers from control rats (Figure 3
).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 3. Liver expression of steady state WD mRNA during hyperplasia, and in tumors and surrounding liver. Treatment groups and times are described in the legend of Figure 2 . Each bar represents determinations from two to four rats from the hyperplasia study and five rats with HCC, with six control rats from the HCC study.
|
|
Because the ATPase coded for by Atp7b is critical in mobilizing copper into CP and thus in the secretion of copper from hepatocytes, we examined whether under the observed conditions of reduced expression of both the CP and WD genes, copper would be retained in the livers of the PP-treated animals. As noted in Figure 4
, temporal increases in liver copper content were observed during hyperplasia, with increases of 2-fold (DEHP) and 3.3-fold (CLF) at 60 days of treatment (P < 0.05). In rats treated for 10 months with BR931, the copper content was significantly higher (2.2-fold, P < 0.05) in tumors and 1.7-fold higher in surrounding liver compared with normal liver of age-matched control rats. The specificity of this increase in copper was determined by measuring the liver content of zinc and iron. In contrast to the increase observed in copper content, hepatic zinc levels were reduced by 50% at 60 days in hyperplastic livers and by 40% in liver surrounding tumors (Figure 5
). At the 60 day point with CLF treatment, the copper:zinc ratio was >5-fold higher that in normal liver (P < 0.05, data not shown). Iron content was not significantly increased during hyperplasia and, in fact, the iron level was reduced by 60% in tumors as compared with normal liver (Figure 6
).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 4. Liver content of copper. Rats were treated as described in the legend of Figure 2 ; copper content was analyzed by atomic absorption spectrophotometry. Each bar represents determinations from four rats.
|
|

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5. Liver content of zinc. Rats were treated as described in the legend of Figure 2 ; zinc content was analyzed by atomic absorption spectrophotometry. Each bar represents determinations from four rats with hyperplasia and three rats from the HCC study.
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 6. Liver content of iron. Rats were treated as described in the legend of Figure 2 ; iron content was analyzed by atomic absorption spectrophotometry. Each bar represents determinations from four rats.
|
|
Excess hepatic copper in patients with Wilson's disease and in LEC rats is thought to be bound to MT as a cellular detoxification mechanism; thus, we measured MT mRNA levels during hyperplasia and in animals that had tumors. As noted in Figure 7
, MT mRNA levels showed no consistent change during hyperplasia, although there was somewhat of an increase at 60 days, which corresponds to the increase in liver copper. However, MT mRNA was significantly increased in tumors as compared with the surrounding liver and the livers of age-matched normal control rats.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 7. Liver expression of steady state metallothionein mRNA during hyperplasia, and in tumors and surrounding liver. Treatment groups and times are described in the legend of Figure 2 . Each bar represents determinations from four rats with hyperplasia and five or six rats that had HCC.
|
|
 |
Discussion
|
---|
These studies clearly demonstrate temporal changes in expression of parameters relating to copper metabolism in PP-induced hyperplasia and carcinogenesis: a decrease in serum CP oxidase activity, a decrease in liver mRNA levels for CP and WD gene, and an induction of MT mRNA in tumors but not significantly in hyperplastic liver. The changes in CP and WD mRNA expression are rapid, and appear as early as 3 days after exposure. These changes are followed by a significant increase in liver copper levels after 60 days, which appears to be specific, since a decrease in zinc levels and no significant sustained change in hepatic iron levels were noted.
To our knowledge, alterations in copper metabolism in hepatic hyperplasia and HCC as a result of PP treatment have not been previously documented. It is not obvious why the CP and WD genes are down-regulated. Possibilities include a down-regulation of these genes by PP-activated receptor, or a limitation in the availability of transcription factors. Others have shown that PP administration suppresses liver transferrin gene expression and results in reductions in serum iron, iron binding capacity and plasma transferrin levels (38). This study determined that the suppression of transferrin gene expression was caused by, at least in part, the unavailability of HNF-4. This may not be the case with the CP gene, since analysis of its 5' flanking region does not reveal HNF-4 binding sites (35). Alterations in metal levels have been demonstrated in human cancers and in other rat models. In rats treated with genotoxic carcinogenic agents, preneoplastic foci incorporate less iron than surrounding liver or normal liver (39,40). However, the possibility of changes in copper metabolism in genotoxic models has not been explored. Abnormalities in zinc nutrition have been associated with human cancer; serum zinc levels are generally lower in cancer patients, and almost all tumors have low zinc levels (41).
Changes in copper metabolism may result in a number of potentially toxic changes in the liver cells. The reduced expression of CP represents a loss of oxidase activity and free radical scavenger capacity in the treated rats, which in turn may contribute to both liver damage and the carcinogenic process. The accumulation of copper in the liver is also likely to contribute to the tumorigenic process, in that copper is well-known to promote free radical formation. Thus, copper accumulation in the liver may act in synergy with the peroxides generated by the PP treatment. The role of copper in hepatocarcinogenesis is dramatically illustrated by the very high incidence of HCC in LEC rats, and was underscored by experiments in which the copper chelators penicillamine (28) and trientine (29) were shown to significantly reduce the incidence of HCC. Thus, the information generated in our current study suggests that alterations in copper metabolism as a factor in hepatocarcinogenesis may not be confined to the LEC rat model, and may be an important mechanism that contributes to liver neoplasia in the non-genotoxic PP model as well.
Another effect of copper hepatocellular toxicity may be an inhibitory effect on hepatocyte growth and regenerative ability. Copper overload in LEC rat liver, which is ~40-fold higher than normal liver (42), induces mRNA levels for cell growth inhibitors, such as p53 and p21waf 1/cip 1, which may result in diminished growth potential of hepatocytes, and suggests that differences in sensitivity to growth stimuli between initiated and normal hepatocytes may eventually result in the development of HCC in the LEC rat liver (43). Our preliminary evidence shows that p21waf 1/cip 1 mRNA is also induced in the PP-treated rats (44). Further, our previous work has demonstrated an increase in serum estrogens, with a significantly reduced hepatic estrogen-metabolism capacity in animals exposed to PPs, which suggests that intrahepatic estrogens would also be elevated (11,12). Recent evidence shows that increased estrogen levels in the presence of copper further enhances the reactivity of estrogens in producing DNA adducts; Li et al. (45) demonstrated that the oxidation of estrogen catechols by Cu2+ leads to a copper-dependent mechanism of hydroxyl radical production via a hydrogen peroxide intermediate, thus suggesting a mechanism for estrogen-associated site-specific DNA damage and mutagenesis.
MT mRNA is not induced significantly or consistently in hyperplastic liver of PP-treated animals, in spite of the increase in copper. However, copper is a much weaker inducer of MT than zinc, and the zinc levels in these animals are significantly reduced. Even if the available MT is sufficient to chelate the excess copper, there is evidence that MT in the presence of copper and the absence of zinc can act as a pro-oxidant (46). Also, under conditions in which the copper:zinc ratio is high, the half-life of MT is shortened (47). Further, MTcopper complexes tend to accumulate in lysosomes, and may therefore contribute to lysosomal fragility in situations involving copper excess (48,49). Other cellular defenses for protection against excess metals or their sequelae are known to be disrupted by PP treatment, in particular, glutathione (50,51) and superoxide dismutase (51). It should be noted, however, that the tumors appear to induce MT mRNA at higher levels than surrounding liver; this situation has also been observed in LEC rats (42,52).
A consideration of the documented cellular injury induced by PPs and by copper shows a number of similarities. First, both PP treatment and copper overload result in mitochondrial damage. Copper overload alters mitochondrial respiration and cytochrome C oxidase activity (30), and ultrastructural changes in mitochondria have been demonstrated in LEC rats (53). Further, Wilson's disease is associated with what is termed premature oxidative aging of mitochondrial DNA, i.e. frequent, diverse and early deletions of mitochondrial DNA (54). The PP agents also alter mitochondrial function and gene expression. In a study by Cai et al. (20), three different PPs increased mRNA levels for several critical mitochondrial genes, including cytochrome c oxidase subunit I and NADH dehydrogenase subunit I. Our studies suggest that the observed copper accumulation in PP-treated liver may be a major focus for the process of PP-induced hepatocarcinogenesis. It is important to note that the copper overload is most pronounced during hyperplasia, the period in which the cellular changes that lead to neoplasia occur. Thus, it is not surprising that the copper overload is less apparent in tumors and in liver surrounding tumors than in hyperplastic liver, since neoplastic liver is very different metabolically from hyperplastic liver and represents the endpoint rather than the process.
The combined effect of copper excess and PPs on cell turnover may also play a role in carcinogenesis. Copper accumulation can suppress growth of normal hepatocytes, as noted above, but may not suppress the growth of neoplastic cells, since the latter cells can induce MT effectively, as has been shown previously (42,52) and in this study (Figure 7
). Copper also inhibits apoptosis of neoplastic cells (8). Further, PPs have been shown to inhibit apoptosis of normal hepatocytes in culture (9). These effects, in combination, may provide a relative growth advantage to neoplastic cells while inhibiting the growth of normal cells and the ability to achieve cell turnover by apoptosis. Although the hepatic copper levels in the PP model are less than that in LEC rats, the possible synergism between copper accumulation and PPs in creating cellular damage may enhance tumorigenesis. In addition, the livers of PP-treated rats demonstrate significant depletion of the cytoprotective element zinc, whereas the LEC rat livers show an increase in zinc (42).
In summary, this study shows a clear effect of PPs on liver copper homeostasis. It is intriguing to note the down-regulation of two genes related to copper metabolism, CP and WD genes, which is immediate and sustained. The mechanism by which these two genes are down-regulated is not known at this time. The consequence of this down-regulation appears to account for the observed accumulation of copper in the liver, as well as loss of the free radical scavenger activity of CP. The accumulated copper, particularly in combination with the effects of PPs, has a profound potential for cell damage. Thus, the disruption of copper metabolism in this model is probably a contributing factor in the hepatocarcinogenesis of these agents, especially in light of the evidence that implicates copper deposition in the hepatocarcinogenesis in LEC rats and, possibly, in patients with Wilson's disease.
 |
Acknowledgments
|
---|
We are grateful to Dr Jonathan D.Gitlin, Washington University, for advice and his generous gift of the CP and WD gene probes, and to Drs Benito Lombardi and George Michalopoulos for their invaluable advice and comments on the manuscript. This work was supported in part by the Department of Veterans Affairs (PKE), NIH award CA53453 (HS), Veterans Research Foundation of Pittsburgh (SDT), and Pathology Education and Research Foundation, University of Pittsburgh School of Medicine (KNR). PKE is the recipient of a VA Associate Research Career Scientist Award.
 |
Notes
|
---|
5 To whom correspondence should be addressed at Department of Medicine, 566 Scaife Hall, University of Pittsburgh, Pittsburgh, PA 15261, USA Email: pkeagon2+{at}pitt.edu 
 |
References
|
---|
-
Perera,F.P. (1991) Perspectives on the risk assessment for nongenotoxic carcinogens and tumor promoters. Environ. Health Perspect., 94, 231235.[ISI][Medline]
-
Marsman,D.S., Goldsworthy,T.L. and Popp,J.A. (1992) Contrasting hepatocyte and peroxisome proliferation, lipofuscin accumulation and cell turnover for the hepatocarcinogen Wy-14 643 and clofibric acid. Carcinogenesis, 13, 10111017.[Abstract]
-
Reddy,J.K. and Lalwani,N.D. (1983) Carcinogenesis by hepatic peroxisome proliferators: evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. CRC Crit. Rev. Toxicol., 12, 158.[ISI]
-
Reddy,J.K. and Rao,M.S. (1977) Transplantable pancreatic carcinoma of the rat. Science, 198, 7880.[ISI][Medline]
-
Cook,J.C., Hurtt,M.E., Frame,S.R. and Biegel,L.B. (1994) Mechanism of extrahepatic tumor induction by peroxisome proliferators in Crl:CD ®BR (CD) rats. Toxicologist, 14, Abstract no. 1163.
-
Orner,G.A., Matthews,C., Hendricks,J.D., Carpenter,H.M., Bailey,G.S. and Williams,D.E. (1995) Dehydroepiandrosterone is a complete hepatocarcinogen and potent tumor promoter in the absence of peroxisome proliferation in rainbow trout. Carcinogenesis, 16, 28932898.[Abstract]
-
Ledda-Columbano,G.M. and Columbano,A. (1994) Compensatory cell proliferation, mitogen-induced liver growth and hepatocarcinogenesis in the rat. In Cockburn,A. and Smith,L. (eds) Nongenotoxic Carcinogenesis. SpringerVerlag, New York, pp. 121139.
-
Roberts,R.A. (1996) Non-genotoxic hepatocarcinogenesis: suppression of apoptosis by peroxisome proliferators. Ann. NY Acad. Sci., 804, 588611.[ISI][Medline]
-
Roberts,R.A., James,N.H., Woodyatt,N.J., Macdonald,N. and Tugwood,J.D. (1998) Evidence for the suppression of apoptosis by the peroxisome proliferator activated receptor alpha (PPAR alpha). Carcinogenesis, 19, 4348.[Abstract]
-
Rusyn,I. Tsukamoto,H. and Thurman,R.G. (1998) WY-14 643 rapidly activates nuclear factor kappaB in Kupffer cells before hepatocytes. Carcinogenesis, 19, 12171222.[Abstract]
-
Eagon,P.K., Chandar,N., Epley,M.J., Elm,M.S., Brady,E.P. and Rao,K.N. (1994) Di(2-ethylhexyl)-phthalate-induced changes in liver estrogen metabolism and hyperplasia. Int. J. Cancer, 58, 736743.[ISI][Medline]
-
Eagon,P.K., Elm,M.S., Epley,M.J., Shinozuka,H. and Rao,K.N. (1996) Sex steroid metabolism and receptor status in hepatic hyperplasia and cancer in rats. Gastroenterology, 110, 11991207.[ISI][Medline]
-
Porter,L.E., Van Thiel,D.H. and Eagon,P.K. (1987) Estrogens and progestins as tumor inducers. Semin. Liver Dis., 7, 2431.[ISI][Medline]
-
Eagon,P.K., Elm,M.S., Dugani,A., Shinozuka,H., Rao,K.N. and Villa,E. (1996) Loss of estrogen receptor expression and detection of a variant estrogen receptor transcript in a rat model of nongenotoxic hepatocarcinogenesis. Hepatology, 24, 340A (abstract no. 856).
-
Reddy,J.K. (1994) Peroxisomal lipid metabolism. Annu. Rev. Nutr., 14, 34370.[ISI][Medline]
-
Zakim,D., Paradini,R.S. and Herman,R.H. (1970) Effect of clofibrate (ethyl-chlorophenoxy-isobutyrate) feeding on glycolytic and lipogenic enzymes and hepatic glycogen synthesis in the rat. Biochem. Pharmacol., 19, 305310.[ISI][Medline]
-
West,D.W. and Shand,J.H. (1994) The effects of clofibrate and bezafibrate on cholesterol metabolism in the liver of the male rat. Lipids, 29, 747752.[ISI][Medline]
-
Rao,K.N., Elm,M.S., Kelly,R.H., Chandar,N., Brady,E.P., Rao,B., Shinozuka,H. and Eagon,P.K. (1997) Hepatic hyperplasia and cancer in rats: metabolic alterations associated with cell growth. Gastroenterology, 113, 238248.[ISI][Medline]
-
Hertz,R., Aurbach,R., Hashimoto,T. and Bar-Tana,J. (1991) Thyromimetic effects of peroxisomal proliferators in rat liver. Biochem. J., 274, 745751.[ISI][Medline]
-
Cai,Y., Nelson,B.D., Li,R., Luciakova,K. and DePierre,J.W. (1996) Thyromimetic action of the peroxisome proliferators clofibrate, perfluorooctanoic acid and acetylsalicylic acid includes changes in mRNA levels for certain genes involved in mitochondrial biogenesis. Arch. Biochem. Biophys., 325, 107112.[ISI][Medline]
-
Linder,M. and Hazegh-Azam,M. (1996) Copper biochemistry and molecular biology. Am. J. Clin. Nutr., 63, 797S811S.[Abstract]
-
Kodama,H. (1996) Genetic disorders of copper metabolism. In Chang,L.W. (ed.) Toxicology of Metals. Lewis Publishers, New York, pp. 371386.
-
Yamaguchi,Y., Heiny,M.E. and Gitlin,J.D. (1993) Isolation and characterization of a human liver cDNA as a candidate gene for Wilson's disease. Biochem. Biophys Res. Commun., 197, 271277.[ISI][Medline]
-
Sato,M. and Gitlin,J.D. (1991) Mechanisms of copper incorporation during the biosynthesis of human ceruloplasmin. J. Biol. Chem., 266, 51285134.[Abstract/Free Full Text]
-
Harris,Z.L. and Gitlin,J.D. (1996) Genetic and molecular basis for copper toxicity. Am. J. Clin. Nutr., 63, 836S841S.[Abstract]
-
Yamaguchi,Y., Heiny,M.E., Shimizu,N., Aoki,T. and Gitlin,J.D. (1994) Expression of the Wilson disease gene is deficient in the LongEvans Cinnamon rat. Biochem. J., 301, 14.[ISI][Medline]
-
Li,Y., Togashi,Y., Sato,S. et al. (1991) Abnormal copper accumulation in non-cancerous and cancerous liver tissues of LEC rats developing hereditary hepatitis and spontaneous hepatoma. Jpn J. Cancer Res., 82, 490492.[ISI][Medline]
-
Jong-Hon,K., Togashi,Y., Kasai,H., Hosokawa,M. and Takeichi,N. (1993) Prevention of spontaneous hepatocellular carcinoma in LongEvans Cinnamon rats with hereditary hepatitis by administration of D-penicillamine. Hepatology, 18, 614620.[ISI][Medline]
-
Sone,H., Maeda,M., Wakabayashi,K., Takeichi,N., Mori,.M., Sugimura,T. and Nagao,M. (1996) Inhibition of hereditary hepatitis and liver tumor development in LongEvans Cinnamon rats by the copper-chelating agent trientine dihydrochloride. Hepatology, 23, 764770.[ISI][Medline]
-
Sokol,R.J., Devereaux,M., Mierau,G.W., Hambidge,K.M. and Shikes,R.H. (1990) Oxidant injury to hepatic mitochondrial lipids in rats with dietary copper overload: modification by vitamin E deficiency. Gastroenterology, 99, 10611071.[ISI][Medline]
-
Arora,A.S. and Gores,G.J. (1996) The role of metals in ischemia/reperfusion injury in the liver. Semin. Liver Dis., 16, 3139.[ISI][Medline]
-
Sokol,R.J. (1996) Antioxidant defenses in metal-induced liver damage. Semin. Liver Dis., 16, 3946.[ISI][Medline]
-
Schilsky,M.L. (1996) Wilson's Disease: genetic basis of copper toxicity and natural history. Semin. Liver Dis., 16, 8395.[ISI][Medline]
-
Chomczynski,P. and Sacchi,N. (1987) Single-step method of RNA isolation by guanidinium thiocyanatephenolchloroform extraction. Anal. Biochem., 162, 156159.[ISI][Medline]
-
Bingle,C.D., Fleming,R.E. and Gitlin,J.D. (1993) Interaction of CCAAT/enhancer-binding protein alpha and beta with the rat caeruloplasmin gene promoter. Biochem. J., 294, 473479.[ISI][Medline]
-
Andersen,R.D., Birren,B.W., Taplitz,S.J. and Herschman,H.R. (1986) Rat metallothionein-1 structural gene and three pseudogenes, one of which contains 5'-regulatory sequences. Mol. Cell. Biol., 6, 302314.[ISI][Medline]
-
Sunderman,F.W. and Nomoto,S. (1970) Measurement of human serum ceruloplasmin by its p-phenylaminediamine oxidase activity. Clin. Chem., 16, 903910.[Abstract/Free Full Text]
-
Hertz,R., Seckbach,M., Zakin,M.M. and Bar-Tana,J. (1996) Transcriptional suppression of the transferrin gene by hypolipidemic peroxisome proliferators. J. Biol. Chem., 271, 218224.[Abstract/Free Full Text]
-
Williams,G.M. and Yamamoto,R.S. (1972) Absence of stainable iron from preneoplastic and neoplastic lesions in rat liver in 8-hydroxyquinoline-induced siderosis. J. Natl Cancer Inst., 49, 685692.[ISI][Medline]
-
Cater,K.C., Gandolfi,A.J. and Sipes,I.G. (1985) Characterization of dimethylnitrosamine-induced focal and nodular lesions in the liver of newborn mice. Toxicol. Pathol., 13, 39.[Medline]
-
Poirier,L.A. (1996) An introduction to selected concepts in metal carcinogenesis. In Chang,L.W. (ed.) Toxicology of Metals. Lewis Publisher, New York, pp. 287298.
-
Sugawara,N., Sugawara,C., Katakura,M., Takahashi,H. and Mori,M. (1991) Copper metabolism in the LEC rat: involvement of induction of metallothionein and disposition of zinc and iron. Experientia, 47, 10601063.[ISI][Medline]
-
Obata,H., Sawada,N., Isomura,H. and Mori,M. (1996) Abnormal accumulation of copper in LEC rat liver induces expression of p53 and nuclear matrix-bound p21waf 1/cip 1. Carcinogenesis, 17, 21572161.[Abstract]
-
Rao,K.N., Chandar,N., Elm,M.S., Shinozuka,H. and Eagon,P.K. (1998) Role of copper and zinc in hepatic hyperplasia and hepatocellular carcinoma in rats by peroxisome proliferators. Proc. Am. Assoc. Cancer Res., abstract no. 2284.
-
Li,Y., Trush,M.A. and Yager,J.D. (1994) DNA damage caused by reactive oxygen species originating from a copper-dependent oxidation of the 2-hydroxy catechol of estradiol. Carcinogenesis, 15, 14211427.[Abstract]
-
Suzuki,K.T., Rui,M., Ueda,J.-I. and Ozawa,T. (1996) Production of hydroxyl radicals by copper-containing metallothionein; roles as prooxidant. Toxicol. Appl. Pharmacol., 141, 231237.[ISI][Medline]
-
Bremner,I., Hoekstra,W.G., Davies,N.T. and Young,B.W. (1978) Effect of zinc status of rats on the synthesis and degradation of copper-induced metallothioneins. Biochem. J., 174, 883892.[ISI][Medline]
-
Myers,B.M., Prendergast,F.G., Holman,R., Kuntz,S.M. and LaRusso,N.F. (1993) Alterations in hepatocyte lysosomes in experimental hepatic copper overload in rats. Gastroenterology, 105, 18141823.[ISI][Medline]
-
Gross,J.B.Jr, Myers,B.M., Kost,L.J., Kuntz,S.M. and LaRusso,N.F. (1989) Biliary copper excretion by hepatocyte lysosomes in the rat. Major excretory pathway in experimental copper overload. J. Clin. Invest., 83, 3039.[ISI][Medline]
-
Lake,B.G., Gray,J.T.B., Korosi,S.A. and Walters,D.G. (1989). Nafenopin, a peroxisome proliferator, depletes hepatic vitamin E content and elevates plasma oxidised glutathione levels in rats. Toxicol. Lett. 45, 221229.[ISI][Medline]
-
Ciriolo,M.R., Mavelli,I., Rotilio,G., Borzatta,V., Cristofari,N. and Stanzani,L. (1982) Decrease of superoxide dismutase and glutathione peroxidase in liver of rats treated with hypolipidemic drugs. FEBS Lett., 144, 264248.[ISI][Medline]
-
Sawaki,M., Enomoto,K., Hattori,A., Tsuzuki,N., Sugawara,N. and Mori,M. (1994) Role of copper accumulation and metallothionein induction in spontaneous liver cancer development in LEC rats. Carcinogenesis, 15, 18331837.[Abstract]
-
Sternlieb,I., Quintana,N., Volenberg,I. and Schilsky,M.L. (1995) An array of mitochondrial alterations in the hepatocytes of LongEvans Cinnamon rats. Hepatology, 22, 17821787[ISI][Medline]
-
Mansouri,A., Gaou,I., Fromenty,B., Berson,A., Letteron,P., DeGott,C., Erlinger,S. and Pessayre,D. (1997) Premature oxidative aging of hepatic mitochondrial DNA in Wilson's disease. Gastroenterology, 113, 509605.
Received October 23, 1998;
revised January 19, 1999;
accepted February 5, 1999.