Changes in essential fatty acid patterns associated with normal liver regeneration and the progression of hepatocyte nodules in rat hepatocarcinogenesis

S. Abel3,, C.M. Smuts1,, C. de Villiers2, and W.C.A. Gelderblom

Programme on Mycotoxins and Experimental Carcinogenesis,
1 National Research Programme for Nutritional Intervention,
2 Experimental Biology Programme–Primate Unit, Medical Research Council (MRC) ,PO Box 19070, Tygerberg 7505, South Africa


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Changes in lipid metabolism were monitored in rat hepatocyte nodules at certain time points over 9 months. Tissue obtained from partially hepatectomized rats, collected over a period of 7 days, were included as a control for normal hepatocyte cell proliferation. Two important features regarding the lipid profiles of hepatocyte nodules and normal regenerating liver were the increased concentrations of phosphatidylethanolamine (PE), resulting in a decreased phosphatidylcholine/phosphatidylethanolamine (PC/PE) ratio, and cholesterol. These changes coincided with increased membrane fluidity in the nodules and regenerating liver. With respect to the fatty acid (FA) profiles of the nodules, C18:1{omega}9 and C18:2{omega}6 increased in PE and PC whereas C20:4{omega}6 decreased in PC and increased in PE. C22:5{omega}6 and C22:6{omega}3, the end products of the {omega}6 and {omega}3 metabolic pathways, respectively, decreased in PC and remained unchanged in PE. The FA levels in PC reflected an impaired {delta}-6 desaturase enzyme, whereas this effect was masked in PE due to the increased concentration of this phospholipid fraction. In regenerating liver, the FA profiles of PC and PE showed the same pattern as described for the hepatocyte nodules, except for C18:1{omega}9 which decreased in PC and increased non-significantly in PE. The increased C18:1{omega}9 level, a FA with anti-oxidative properties, as well as the decreased levels of the long-chain polyunsaturated fatty acids (C20 and C22 carbon chains), have been associated with the decreased lipid peroxidation level in hepatocyte nodules. The resultant decrease in peroxidative metabolites, known to affect apoptosis, could be important in the progression of the nodules into neoplasia. The present results indicate that the altered lipid parameters associated with hepatocyte nodules closely mimics cellular proliferation in regenerating liver and could be responsible for the enhanced proliferation and/or altered growth pattern in these lesions. The altered FA profiles suggest various pathways in which FA could play a role in transmembrane signalling related to the altered cell proliferative and apoptotic pathways. The persistent changes in the hepatocyte nodules suggest that the lipid metabolism escapes the regulatory mechanisms required for normal cellular homeostasis at different levels.

Abbreviations: Chol, cholesterol; CM, chloroform/methanol; DPH, 1,6-diphenyl-1,3,5-hexatrine; FA, fatty acid(s); FAME, fatty acid methyl esters; LC-PUFA, long chain polyunsaturated fatty acids; P/S, polyunsaturated to saturated fatty acid ratio; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PEMT2, N-methyltransferase-2; PL, phospholipid(s); PUFA, polyunsaturated fatty acid(s).


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The process of carcinogenesis is complex, resulting from alterations in the normal patterns of cellular growth (1). In the resistant hepatocyte model for liver carcinogenesis in the rat, a key event is the appearance of numerous altered or `resistant' cells during initiation which, upon promotion, results in the formation of hepatocyte nodules with a characteristic altered phenotype (2). Although the majority of these hepatocyte nodules disappear or re-differentiate to normal appearing liver, a few `persistent nodules' develop into malignant tumours (3). In the hepatocyte nodules, the balance between cell death and proliferation is disrupted resulting in a net increase in cell proliferation. This phenomenon changes, though, in the persistent nodules where cell death increases to counteract the increased cell proliferation, resulting in the retardation in growth of the persistent nodules. With the onset of cancer, this balance is again disturbed by an increased growth rate observed in the neoplastic tissue (4).

Studies have shown that the occurrence of potentially `neoplastic' hepatocyte lesions is associated with changes in the polyunsaturated fatty acid (PUFA) profile, especially the long-chain PUFA (C20 and C22 carbon chains, LC-PUFA) and the lipid peroxidative status (5,6). The differences in LC-PUFA levels and extent of lipid peroxidation in pre-neoplastic lesions are possibly due to an abnormal essential FA metabolism involving {Delta}-6 desaturase (79). Changes in the FA profiles have a wide range of effects regarding the integrity of cellular membranes. These changes are known to affect the membrane structure and fluidity, the activity of membranal enzymes and the affinity of growth factor receptors. Furthermore fatty acids act as signalling molecules involved in cell proliferation and/or apoptosis (5,10).

Both LC-PUFA, which form the main substrates for lipid peroxidation, and membrane lipid peroxidation have been found to be lower in hepatocyte nodules than in surrounding `normal' tissue (11,12). The level of lipid peroxidation has also been found to influence tumour growth (11) and, together with changes in the membrane lipid status, is likely to play an important role in the abnormal cellular growth which prevails in pre-neoplastic lesions. The integrity of the cellular membrane is therefore important in the normal functioning of the cell and its responses to external growth stimulatory and/or inhibitory factors.

Studies in experimental animals have indicated that focal hepatocyte proliferations or hepatocyte nodules are the critical, relatively early lesions in the development of liver cancer in rats (2). The current study investigated the lipid profiles associated with the progression of hepatocyte nodules as well as in normal regenerating liver in order to delineate alterations in lipid metabolism with respect to cancer development in the liver of rats.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental animals
Male Fischer rats (n = 144) were fed the AIN 76A diet (13) ad libitum when weaned, and housed under controlled lighting (12 h cycles) and temperature (23–25°C) with free access to water. Upon reaching a body weight of 150 g, they were housed separately in wire-bottomed cages and weighed three times a week.

Resistant Hepatocyte Model
Hepatocyte nodules were induced according to the method described by Solt and Farber (14). Briefly, the rats (body weight approximately 150 g) were injected intraperitoneally (i.p.) with diethylnitrosamine (DEN, 200 mg/kg body weight) to effect cancer initiation. Promotion was effected 3 weeks later by a daily intragastric dose of 2-acetylaminofluorene (2-AAF, 20 mg/kg body weight) on three consecutive days followed by partial hepatectomy on the fourth day. The rats (n = 60) were terminated at intervals of 1 (n = 15), 3 (n = 15), 6 (n = 15) and 9 (n = 15) months after cancer promotion and the hepatocyte nodules and surrounding tissue were collected. Control tissue was collected at similar time intervals from rats (n = 60) that did not receive the initiating and promoting treatments. The tissue samples were immediately frozen on dry ice and stored at –80°C prior to analyses.

Regenerating liver
Rat liver samples (n = 18) were collected at intervals of 1, 2 and 7 days following partial hepatectomy in order to obtain tissue sections representing different stages of the regenerative response (15). Livers of untreated rats (n = 6) were also collected at the time of partial hepatectomy. All the samples were stored at –80°C until analysed.

Lipid analyses
Lipids were extracted from the control, nodule, surrounding and regenerating liver tissue with chloroform/methanol (CM; 2:1; v/v) (16) containing 0.01% butylated hydroxytoluene (BHT) as antioxidant according to the method of Smuts et al. (17). In short, approximately 100–150 mg liver was ground to a fine powder in liquid nitrogen and weighed in glass-stoppered tubes. The tissue was suspended in 0.5 ml saline and the lipids were extracted with 24 ml CM. The CM mixture was filtered (sinterglass filters using Whatman Glass Microfibre filters; Whatman International, Maidstone, UK) and the filtrate was evaporated to dryness in vacuo at 40°C, transferred to glass-stoppered tubes, washed with saline saturated with CM, and stored at 4°C under nitrogen for 2 weeks until analysed. The lipid extracts were fractionated by thin layer chromatography (TLC) and the major phospholipid fractions, PC and PE, were collected for phospholipid and FA analyses (18). Phospholipid levels were determined colorimetrically using a malachite green dye after digestion with perchloric acid (16 N) at 170°C for approximately 1 h (19). For the FA analyses, the phospholipid fractions, PC and PE, were transmethylated with 2 ml methanol/18 M sulphuric acid (95:5; v/v) at 70°C for 2 h. The FA methyl esters (FAME) were extracted in hexane and analysed by gas chromatography on a Varian 3400 Gas Chromatograph equipped with 30 m fused silica Megabore DB-225 columns of 0.53 mm internal diameter (J&W Scientific). The individual FAME were identified by comparison of the retention times to those of a standard mixture of free FA, C14:0 to C24:1, and quantified using an internal standard (C17:0) and expressed as µg FA/100 mg liver weight.

Total cholesterol of the lipid extracts was determined by an enzymatic iodide method using cholesterinoxidase and -esterase (20). The cholesterol/phospholipid molar ratio was calculated using the molar weights of 386.7, 787 and 744 for cholesterol, PC and PE, respectively.

Membrane fluidity/fluorescence polarization
Fluorescence polarization studies were performed on homogenized control, surrounding, nodule and regenerating liver tissue with a fluorescence spectrofluorimeter (MPF 44A, Perkin-Elmer). Samples were diluted to a concentration of 0.2–0.3 mg protein/ml with 10 mM Tris–HCl, pH 7.4, sonicated for 10 s and 5 µl 1,6-diphenyl-1,3,5-hexatrine (DPH; 2 mM in tetrahydrofuran) added (21,22). The suspension (2.5 ml) was incubated in a water bath (37°C) for 30–60 min in the dark. Measurements were done manually with an emission polarizer at 0° (V component) and 90° (L component) with the excitation polarizer first at 0° (vertical component v) and then at 90° (horizontal component h). The excitation and emission slit widths were 14 nm and the excitation and emission wavelengths were 357 and 425 nm, respectively. The temperatures selected for screening ranged from 25 to 41°C.

Protein determination
Powdered liver preparations (10–15 mg) were solubilized in 5% SDS at 37°C and the protein content determined using a modified method of Lowry (23).

Statistical analyses
Statistical analyses were performed using the analyses of variance (ANOVA). The Tukey Studentized Range Method was used to determine differences between the means. Lipid changes as a function of time were analysed with the Parametric Paired Difference t-test. Values were considered significant if P < 0.05.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
Cholesterol content (Table IGo)
The cholesterol content was significantly increased within the control, nodule and surrounding tissue, as a function of time. This increase was significant between months 3 and 6 (P < 0.01) in the control group, between months 6 and 9 (P < 0.05) in the nodules and between months 6 and 9 (P < 0.05) in the surrounding tissue. In the hepatocyte nodules, the cholesterol was significantly increased at months 1 (P < 0.05) and 9 (P < 0.01) compared with the respective controls. Compared with the surrounding tissue, the cholesterol in the nodules was significantly increased at months 6 (P < 0.05) and 9 (P < 0.01). No significant changes were observed between the controls and the surrounding tissue. The cholesterol concentration in the regenerating liver was significantly increased (P < 0.01) at days 2 and 7 when compared with the control.


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Table I. Comparative lipid parameters in the Resistant Hepatocyte model in control, nodule and surrounding tissue and in regenerating liver
 
Phospholipid content (Table IGo)
The PE level in the hepatocyte nodules, increased significantly at 1 (P < 0.01), 3 (P < 0.05), 6 (P < 0.01) and 9 (P < 0.01) months when compared with the respective controls and surrounding tissue (P < 0.01). PE in the surrounding tissue was only significantly increased at months 1 (P < 0.05) and 6 (P < 0.05) compared with the respective controls. In contrast, PC in the nodules increased significantly only at month 1 compared with the control and surrounding tissue. The level of PE in the regenerating liver was significantly increased (P < 0.01) at days 1, 2 and 7 compared with the control, whereas PC was significantly decreased (P < 0.05) at day 2.

Membrane fluidity (Table IIGo)
The measurement of DPH-labelled membranes relates to membrane micro-viscosity which is inversely related to membrane fluidity i.e. an increase in micro-viscosity indicates a decrease in membrane fluidity (19). The membrane fluidity in the nodular tissue, decreased at months 1 (P < 0.01), 3 (P < 0.01) and 6 (P < 0.05) as compared with the respective controls. However, at month 9 the membrane fluidity was significantly higher (P < 0.01) than the respective control. The same pattern was observed when comparing the nodular tissue with the surrounding tissue. No significant differences were observed between the surrounding tissue and the respective controls. In the regenerating liver, the membrane fluidity was significantly increased at days 1, 2 and 7 (P < 0.01) when compared to the control.


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Table II. Comparative membrane fluidity parameters of regenerating liver versus control, hepatocyte nodules and surrounding tissue generated in the Resistant Hepatocyte Model
 
A significant increase in the cholesterol/phospholipid molar ratio was observed in the surrounding tissue (P < 0.05) at 3 months. At 9 months the ratio was significantly increased (P < 0.05) in the nodules. The PC/PE ratio in the nodules was significantly decreased at months 1, 3, 6 and 9 (months 1, 6 and 9, P < 0.01; month 3, P < 0.05) compared with the respective controls. The surrounding tissue PC/PE ratio was also significantly decreased from the controls at months 1 and 6 (P < 0.01 and P < 0.05, respectively). In the regenerating liver, the cholesterol/phospholipid molar ratio was significantly (P < 0.01) increased at days 2 and 7 compared with the control and day 1. The PC/PE ratio was significantly (P < 0.01) decreased from the control level at days 1, 2 and 7.

Comparative FA profiles in PC and PE of hepatocyte nodules compared with surrounding and control tissue (Tables III and IVGoGo)
The general trend of the FA profiles in the surrounding tissue tended to mimic that of the control tissue, especially after 3 months. Before 3 months, some values fell in between the control and nodule values.


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Table III. Fatty acid analyses of the phosphatidylcholine (PC) fraction of control liver, nodule and surrounding liver in the Resistant Hepatocyte Model
 

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Table IV. Fatty acid analyses of the phosphatidylethanolamine (PE) fraction of control liver, nodule and surrounding liver in the Resistant Hepatocyte Model
 
Saturated FA: (C16:0, C18:0)
In the nodules, the levels of C16:0 in PC increased significantly at months 1, 6 and 9 (P < 0.01) compared with the controls, whereas C18:0 was significantly decreased at months 6 and 9 (P < 0.01). However, in PE C16:0 was significantly decreased at month 3 (P < 0.05), whereas C18:0 was significantly increased at months 1, 6 and 9 (P < 0.01). No significant changes were observed in the total saturated FA levels of PC, but in PE the levels increased significantly at months 1, 6 and 9 (P < 0.01).

Monounsaturated FA: (C16:1, C18:1)
The values of C16:1 increased significantly in PC and PE in the nodules at months 1, 6 and 9 (P < 0.01), while C18:1 increased significantly (P < 0.01) in PC and PE at months 1, 3, 6 and 9 (P < 0.01). The total monounsaturated FA level was significantly increased in PC at 1, 6 and 9 months (P < 0.01) in the nodules and in PE at 1, 3, 6 and 9 months (P < 0.01).

Polyunsaturated FA
{omega}6 PUFA: (C18:2, C20:4, C22:4, C22:5)
The level of C18:2 in the nodules increased (P < 0.01) at months 1, 3, 6 and 9 in PC and PE. In PC, C20:4 was significantly decreased only at month 3 (P < 0.05), but tended to be slightly (not significant) lower at months 1, 6 and 9. However, in PE C20:4 was significantly increased at 1, 6 and 9 months (P < 0.01) with a non-significant increase at 3 months. The level of C22:4 in PC was significantly decreased at months 3 (P < 0.05) and 6 (P < 0.01), but in PE the level was increased (P < 0.01) at 1, 3, 6 and 9 months. In PC, C22:5 was significantly decreased at 1 (P < 0.05), 3, 6 and 9 months (P < 0.01). No significant changes were observed in PE. The total {omega}6 levels in PE were increased significantly at 1, 3, 6 (P < 0.01) and 9 (P < 0.05) months, while in PC it was significantly lower only at 3 months.

{omega}3 PUFA: (C22:5, C22:6)
In PC, C22:5 was significantly decreased in the nodules at 3 and 6 months (P < 0.01) but increased significantly in PE at 1, 6 (P < 0.01) and 9 months (P < 0.05). C22:6 was significantly decreased in PC at 1, 3, 6 and 9 months (P < 0.01) compared with the controls, while no changes were observed in PE. The total {omega}3 level in PC was significantly decreased at 1, 3, 6 (P < 0.01) and 9 months (P < 0.05). In PE, no significant changes were observed although the levels tended to increase (not significant) at months 1, 6 and 9.

Due to the decrease in {omega}3 FA, the {omega}6/{omega}3 ratio was significantly increased in PC at 1, 3, 6 and 9 months (P < 0.01), but only at months 1 and 6 (P < 0.01) in PE. The PUFA level increased in PE at 1, 6 and 9 (P < 0.01) months, and decreased significantly (P < 0.01) at 3 months in PC. No significant changes in the P/S ratio were observed in the nodular tissue compared with the respective controls.

FA profiles of PC and PE in regenerating liver (Table VGo)
Saturated FA: (C16:0, C18:0)
The levels of both C16:0 (1, 2 and 7 days: P < 0.05) and C18:0 (1 and 2 days: P < 0.05; 7 days: P < 0.01) decreased significantly in PC. No changes were observed with regards to C16:0 and C18:0 in PE, although a non-significant increase was noticed at days 1 and 2. As a result of these changes, the total saturate level in PC was significantly decreased at 1, 2 (P < 0.05) and 7 days (P < 0.01). In PE, no significant changes were observed, but there was a slight increase (not significant) in the level at days 1 and 2.


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Table V. Fatty acid analyses of the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) fractions of regenerating liver
 
Monounsaturated FA: (C16:1, C18:1)
No significant changes occurred with regards to C16:1 in PC and PE, although it decreased initially (not significant) in PC. The level of C18:1 was significantly decreased at 1 day (P < 0.05) in PC, but increased thereafter at 2 and 7 days towards the control level. In PE, the level of C18:1 was significantly increased at 7 days after partial hepatectomy (P < 0.05). The total monounsaturate level was significantly decreased in PC at day 1 (P < 0.05) and thereafter the levels at days 2 and 7 tended to increase towards the control level. In PE, the total monounsaturate level was significantly increased at day 7 (P < 0.05).

Polyunsaturated FA
{omega}6 PUFA: (C18:2, C20:3, C20:4, C22:4, C22:5)
At day 1 C18:2 increased in PC (P < 0.05) and PE (P < 0.01), after which the level tended to revert back to that in the controls. In PE, C20:3 was significantly increased 7 days (P < 0.01) after partial hepatectomy (data not shown). The C20:4 level significantly decreased at 1, 2 (P < 0.01) and 7 days (P < 0.05) after partial hepatectomy in PC, but increased over time towards control levels. No significant changes were observed in PE, although there was a slight increase at day 1, but then tended to decrease back to the control level. C22:4 was significantly decreased in PC at day 1 only (P < 0.05), but tended to increase thereafter. There was a significant increase in the level of C22:4 in PE at days 2 and 7 (P < 0.05). The level of C22:5 decreased in PC at days 1 and 2 (P < 0.01) and in PE at day 1 only (P < 0.05). In both PC and PE, C22:5 was decreased initially at day 1 but tended to increase to the control levels at day 7. The total {omega}6 FA level was significantly decreased at days 1, 2 (P < 0.01) and 7 (P < 0.05) in PC. No significant changes were observed in PE, although the level tended to be higher than control levels.

{omega}3 PUFA: (C22:5, C22:6)
The level of C22:5 was significantly decreased at day 1 (P < 0.05) only in PC, but increased significantly in PE at days 1 (P < 0.05), 2 (P < 0.01) and 7 (P < 0.05). In PC, C22:6 was significantly decreased at days 1, 2 and 7 (P < 0.01). No changes were observed in PE. The total {omega}3 FA level was significantly decreased in PC at 1, 2 and 7 days (P < 0.01) compared with the control. Once again, no changes were observed in PE.

The {omega}6/{omega}3 ratios were significantly increased in PC (P < 0.05) and PE (P < 0.01) 1 day after partial hepatectomy compared with the control levels. The PUFA level was significantly decreased at 1, 2 (P < 0.01) and 7 days (P < 0.05) in PC, but no significant changes were observed in PE, although the level tended to be higher (not significant) over the experimental period. The P/S ratio in PC and PE was significantly increased from the respective controls at 7 days (P < 0.01) after partial hepatectomy.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The structure of plasma membranes in regenerating liver concerning the patterns of membrane proteins have shown no changes compared with that of control liver (5,10). However, changes in the plasma membrane enzyme activity and receptor expression in regenerating liver have been described (24,25). In hepatoma cells, alterations in membranal protein profiles have been reported resulting in changes in the activity of certain membrane enzymes, such as a decrease in 5-nucleotidase, an increase in {gamma}-glutamyltranspeptidase, as well as changes in the affinity of receptors (10,26,27). Changes in membrane protein turnover have also been shown to occur in HTC cells (Morris hepatoma 7288C) (28). Alterations in membrane fluidity can also influence the activity of certain enzymes and the affinity of receptors to their ligands (5,10). Membrane fluidity has been shown to increase in the nuclear membrane following partial hepatectomy in rats (29). This increase is linked to nuclear membrane neutral-sphingomyelinase activity and the content of sphingomyelin. Alterations in lipid content have been closely linked with changes in membrane fluidity which could play an important role in the control of signal transduction pathways and cellular regeneration in the altered growth pattern and progression of hepatocyte nodules to cancer development (5,10).

Important indicators of membrane fluidity are the cholesterol/phospholipid molar ratio (Chol/PL), the PC/PE ratio and the degree of membrane unsaturation (P/S ratio) (5,10). In the present study, fluorescence polarization indicated that the nodule membranes were more rigid, i.e. less fluid, than the respective controls at 1, 3 and 6 months, in contrast to the increased fluidity in regenerating liver. However, at 9 months the nodule membrane fluidity increased above the control, mimicking regenerating liver. The PC/PE ratio in the nodules and the regenerating liver decreased early on due to the increased PE level. This increase in the PE concentration has also been observed in Morris hepatoma 7777 cells (5,10). The Chol/PL molar ratio in the nodules increased only at 9 months due to a significant increase in the cholesterol level. This mimics regenerating liver where the cholesterol level also increased after 2 and 7 days. Cholesterol appears to play an important structural role in maintaining the fluidity of membranes making the outer part of the membrane less fluid, but causing the inner part of the lipid bilayer to be slightly more fluid by organizing the tails of the FA acyl chains (30). The non-polar membrane components, such as the FA acyl chains and cholesterol, seem to have a greater regulatory effect on the activity of membrane bound proteins than the polar phospholipid head groups (31). In this way cholesterol can organize the movement of membrane bound proteins in regenerating liver and also in hepatocyte nodules. Finally, the unsaturation index (P/S ratio) in the hepatocyte nodules did not change, suggesting that this index is tightly controlled in the nodules and does not appear to have a significant effect on membrane fluidity in the hepatocyte nodule. The increased PUFA level in PE, due to the increased PE concentration, did not correlate with the resultant unchanged membrane fluidity, further indicating that unsaturation did not affect fluidity directly. A recent study showed that the FA unsaturation level in a membrane did not have such a large influence on membrane fluidity when compared with the effect of cholesterol (32). This increase in PE appears to be an early event in the nodular lipid profile compared with changes in the cholesterol and membrane fluidity which appear to be late events. Together, the increases in membrane cholesterol and PE are likely to be the major factors determining fluidity changes in the hepatocyte nodules. In regenerating liver, an increase in these two parameters was also associated with an increase in fluidity.

When comparing the lipid profiles of nodular liver with regenerating liver, some similarity in the pattern of lipid changes exists. In the regenerating liver, cholesterol increased significantly at days 2 and 7 while PE increased from day 1. The latter changes coincided with an increase in the fluidity as well as maximal liver regeneration, which occurs 2–3 days after partial hepatectomy (15). As seen in the hepatocyte nodules, the increased PE concentration in the regenerating liver is an early event with the changes in cholesterol occurring later. With regards to the fatty acids, C18:1{omega}9 decreased significantly in PC only at day 1, but was increased in PE at day 7, presumably due to the increase in the concentration of this phospholipid. It is not known whether the initial decrease in C18:1{omega}9, known to have antioxidant properties (6,7), could be related to the decreased rate in lipid peroxidation noticed in regenerating liver prior to DNA synthesis (7). After an initial increase of C18:2{omega}6 in PC after day 1, the level was significantly reduced after 7 days, presumably due to an increased conversion of this FA to C20 and C22 fatty acids as a result of liver regeneration. In comparison with the nodules, it would therefore appear that the {Delta}-6 desaturase enzyme is not impaired during normal regeneration following partial hepatectomy. The LC-PUFA, C20:4{omega}6, C22:5{omega}6 and C22:6{omega}3, decreased significantly in the regenerating liver PC presumably due to a higher metabolism of these FA during regeneration (7). It has been observed that proliferating cells have a higher utilization of the {Delta}-6 desaturated FA (11). The resultant decrease in these LC-PUFA supports the low lipid peroxidative status present in regenerating liver (33). In the nodules, the higher rate of cell proliferation, as well as the impaired {Delta}-6 desaturase enzyme, resulted in a similar decrease of LC-PUFA in PC which also appears to be an early event in the genesis of the nodules.

The lipid pattern associated with regeneration could be instrumental in the signal for cellular growth in regenerating liver under controlled conditions, while it prevails in hepatocyte nodules resulting in a steady increase in their size. However, in the majority of these nodules the lipid-associated stimulatory signals revert back to that prevailing in normal tissue, as observed in regenerating liver following partial hepatectomy, contributing to the remodelling process in the majority of the nodules. In the present study this was shown in the FA profiles of PC and PE of the 3 month nodules versus surrounding tissue with differences not being as prominent as that obtained at 6 and 9 months, presumably due to the large amount of nodules that are still remodelling at this stage. In a small subset of nodules, however, these changes `persist' supporting the increased rate of cell proliferation and facilitating their ultimate development into cancer. It has been reported that after 6 months post-initiation, certain hepatocyte nodules, termed `persistent' nodules, lose their ability to control cell proliferation (4) i.e. the normal `regulatory' processes of the cell cycle are impaired. After 9 months, the remaining subset of the original pre-neoplastic nodules develop into cancer (4). Therefore, the normal control processes that regulate cell proliferation, seen in regenerating liver, is not present in a small subset of these nodules. Up to a certain stage, 1–6 months, the hepatocyte nodules are still in a transitional phase i.e. at an early stage most of the nodules revert back/regress to normal hepatocytes and at a later stage, i.e. from 6 to 9 months, the persistent nodules lose their `pre-neoplastic' features/phase and advance to the neoplastic stage.

Critical events in the nodules are the persistent alterations in lipid parameters involving changes in FA metabolism ({Delta}-6 desaturase), increased PE and cholesterol levels and changes in membrane fluidity (Figure 1Go). Except for some changes in FA metabolism related to the impaired {Delta}-6 desaturase enzyme, the other changes closely mimic that of regenerating liver but with the difference that in the latter, these changes revert back to normal liver. This implies that the normal regulatory mechanisms, related to lipid metabolism to ensure normal liver homeostasis, are disrupted in hepatocyte nodules. It is not known at present whether the increased level of PE is related to an increased synthesis or to a decrease in the conversion to PC involving phosphatidylethanolamine N-methyltransferase-2 (PEMT2). Recent studies imply the expression of this enzyme in the regulation of hepatocyte growth (34). PEMT2 expression is transiently inactivated after partial hepatectomy (35) and permanently disappears in hepatocellular carcinoma induced by the resistant hepatocyte model (36), while the transfection of the enzyme to a hepatoma cell line inhibits the cell growth rate (37). The higher PE concentration, a phospholipid normally situated on the inside of cellular membranes (38), together with the resultant increases in C20:4{omega}6 appears to be an integral part of the growth stimulus in hepatocyte nodules and could play a role in sustaining cellular proliferation in these lesions (Figure 1Go).



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Fig. 1. Critical events associated with the altered growth pattern of hepatocyte nodules. The growth of pre-neoplastic nodules can be influenced by certain critical events with regards to lipid metabolism as summarized in the figure. This involves an impaired {Delta}-6 desaturase, an increase in PE, cholesterol concentration and membrane fluidity (this study dotted block). The early events involving the {Delta}-6 desaturase and increased PE level establish an environment critical for the continued proliferation of the nodules. This results in the later events such as the increased cholesterol and membrane fluidity affecting the functionality of the cellular membrane involving membrane enzymes and receptor affinity. The increased PE level is an important event leading to an increased membrane C20:4{omega}6 availability affecting various systems such as prostaglandin synthesis and PKC, ceramide and MAP kinase activity. These factors in turn play a role in the regulation of cellular proliferation and apoptosis. The impaired {Delta}-6 desaturase enzyme, as shown by increases in C18:1{omega}9 and C18:2{omega}6 and decreases in the LC-PUFA, can result in a decreased lipid peroxidation status leading to an imbalance in the cell proliferation/apoptosis equilibrium in nodules, thereby favouring cell proliferation.

 
The dual role of C20:4{omega}6, (i) structural as part of membrane phospholipids and (ii) functional as a precursor to the E2-series eicosanoids and signal transduction pathways, are of particular interest with respect to its role in maintaining normal cellular homeostasis in the liver (39). The proteins, phospholipase A2 (PLA2), phospholipase C (PLC) and protein kinase C (PKC) play an important role in cell proliferation and have shown a tendency to be modulated by FA. Recent studies indicated that membranal FA, specifically C20:4{omega}6, play an important role as second messengers in signal transduction pathways via the activation of protein kinase C (PKC), mitogen activated protein kinase (MAP kinase) and the generation of ceramide (40). C20:4{omega}6 can also be involved in apoptosis via the release of ceramide by activating sphingomyelinase, which acts as a second messenger activating the apoptotic process (41). Increased levels of the E2-series prostaglandins from C20:4{omega}6 can also be involved in activating the apoptotic process (42). A recent study investigating the role of C20:4{omega}6 in phenobarbital induced rat liver foci indicated the involvement of this FA in the tumour promoting mechanisms of phenobarbital (43). It would appear that C20:4{omega}6 plays a key role in controlling events which support the altered growth kinetics in hepatocyte nodules (Figure 1Go).

Apart from the role of FA in regulating prostaglandin production and signal transduction pathways, they are also key substrates for lipid peroxidation. LC-PUFA can play a role in the control of cell proliferation by inhibiting cell growth and stimulating and/or enhancing apoptosis by the increase of cellular lipid peroxidation and the subsequent breakdown products such as malondialdehyde (44,45). A key event with respect to the changes in FA metabolism in hepatocyte nodules appears to be a change in the {Delta}-6 desaturase enzyme and the implications this has on the LC-PUFA levels (6,30). The impairment of this enzyme has been observed in BL6 melanoma and Morris hepatoma 9618A cell lines and in various types of liver cancer with different origins (79). In the present study, impaired activity of this enzyme was observed in the nodule tissue indicated by the increased levels of the FA substrates C16:1{omega}7, C18:1{omega}9 and C18:2{omega}6 in PC, while the LC-PUFA products, C20:4{omega}6, C22:5{omega}6 and C22:6{omega}3, were decreased. The decreased levels of C22:5{omega}6 and C22:6{omega}3 in PC can also be related to the impaired activity of {Delta}-6 desaturase, as this enzyme has also been shown to be involved in the conversion of C22:4{omega}6 and C22:5{omega}3 to C22:5{omega}6 and C22:6{omega}3, respectively (46). The increased C18:1{omega}9 level, a FA with anti-oxidative properties, as well as the decrease in the LC-PUFA have been associated with a decreased lipid peroxidation status in malignant lesions (6,7). A loss of lipid peroxidation has also been observed in pre-neoplastic hepatocellular lesions in rats and a decrease in the cytostatic 4-hydroalkenals, other aldehydes and peroxides could be related to the increased growth patterns observed in these lesions (12). The subsequent decrease in peroxidative metabolites, which are known to induce apoptosis (47), is likely to negatively affect the apoptotic process in the nodular environment (Figure 1Go). In normal regenerating liver, the level of C18:1{omega}9 decreased, C18:2{omega}6 increased and the LC-PUFA in PC decreased after day 1, implying a possible controlled involvement of the {Delta}-6 desaturase enzyme to reduce the lipid peroxidation level. It has been shown that the rate of lipid peroxidation is reduced in regenerating liver following partial hepatectomy which fits into the general hypothesis that increased cell proliferation is associated with a decreased rate of lipid peroxidation (7,48,49). This would suggest that the decrease in lipid peroxidation is another important event in sustaining hepatocyte regeneration both under normal and abnormal conditions (Figure 1Go).

The present study indicated that the persistent alterations in lipid metabolism in the hepatocyte nodules (dotted block in Figure 1Go) are likely to play an important role in the development of the malignant phenotype. The dynamic state of the lipid bilayer of cellular membranes would allow the manipulation of the lipid content of the membrane by dietary means, thereby modulating the activity of membrane proteins, the availability of FA for signal transduction pathways and prostaglandin synthesis and thereby determining cell survival (31). The altered cholesterol, phospholipid and FA profiles in the hepatocyte nodules could provide unique targets for developing strategies in chemoprevention with the inclusion of dietary manipulation in order to counteract the increased cellular proliferation and therefore the progression and subsequent development of these lesions into neoplasia.


    Notes
 
3 To whom correspondence should be addressed Email: sabel{at}mrc.ac.za Back


    Acknowledgments
 
The authors wish to thank Mr G.P.Engelbrecht for his assistance with the gas chromatography analyses, Ms Johanna van Wyk for her laboratory expertise and Ms S.Swanevelder for the statistical analyses. This project was partly funded by the Cancer Association of South Africa.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 15, 2000; revised November 15, 2000; accepted January 29, 2001.