Failure to demonstrate chemoprevention by the monoterpene perillyl alcohol during early rat hepatocarcinogenesis: a cautionary note

Alexandra Löw-Baselli, Wolfgang W. Huber, Monika Käfer, Krystyna Bukowska, Rolf Schulte-Hermann and Bettina Grasl-Kraupp1

Institut für Tumorbiologie-Krebsforschung, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The monoterpene perillyl alcohol (PA) is being considered as a useful chemopreventive and therapeutic agent against human cancers. However, no data are available on the effects of PA in the first stages of hepatocarcinogenesis. To study such effects, putatively initiated cells and preneoplastic foci in hepatocarcinogenesis were used as a model. Male Wistar rats were treated with a single dose of N-nitrosomorpholine (NNM). Between days 4 and 91 after NNM, subgroups of rats received either PA (1 g/kg body wt/day) or phenobarbital (PB) (50 mg/kg body wt/day) in the diet. Since PA treatment reduced food intake, one control group was fed ad libitum, while a second control was pair fed between days 4 and 91. In order to enhance any treatment effects, all groups, including the controls, were treated with the potent tumor promoter PB after day 91 until the end of the experiment at day 266. Rats were killed at multiple time points and putatively initiated cells and preneoplastic foci were identified by staining positively for placental glutathione S-transferase (G+). The following results were obtained. (i) A few days after NNM treatment single G+ cells emerged; a considerable portion of which developed into foci. (ii) Treatment with PB resulted in an increase in number and size of G+ foci. (iii) PA treatment failed to reduce the number of G+ cells; it somewhat lowered rates of apoptosis in G+ foci and clearly increased their average size. (iv) Eighty-seven days of PA revealed no protective effect on day 266, but, similar to PB treatment, increased the growth of foci. In conclusion, PA exerted no detectable chemopreventive effect in the early stages of rat hepatocarcinogenesis. It rather exerted a PB-like tumor promoting activity. These data argue against a recommendation of PA as a chemopreventive agent for healthy humans.

Abbreviations: ABs, apoptotic bodies; AI(%), apoptotic index; COal, ad libitum fed control group; COpf, pair fed control group; G+, placental glutathione S-transferase-positive; GST-P, placental glutathione S-transferase; H&E, hematoxylin and eosin; LI(%), labeling index; NNM, N-nitrosomorpholine; PA, perillyl alcohol; PB, phenobarbital; TGF-ß, transforming growth factor ß.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The monoterpene perillyl alcohol (PA) is currently being investigated in clinical trials for the treatment of advanced breast cancer (1). In addition, the use of PA for chemoprevention of human carcinogenesis in the near future has been proposed (2). In view of the prospect of application of PA to healthy individuals the present study was designed to investigate the effects of PA on the first stages of carcinogenesis.

PA and other monoterpenes such as d-limonene are naturally occurring components of extracts of lavender oil and orange peel (for formulae see Figure 1Go). Both PA and limonene appear to serve as prodrugs, their therapeutic effects being attributed to their main metabolites, perillic acid and dihydroperillic acid (25). PA was shown to be at least five times more potent than limonene in inhibiting rat mammary carcinogenesis, which might result from differences in pharmacokinetics (4). It appears unlikely that a dose of limonene of ~5 g/kg body wt, as was applied to rats, will be applicable to humans. Therefore, investigations on chemopreventive and therapeutic uses have concentrated on the more potent monoterpene PA.



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Fig. 1. Chemical structure of d-limonene and PA.

 
Cancer prevention may occur by various different mechanisms. These include reduced metabolic toxification and/or enhanced detoxification, which lower the amount of the ultimate initiating carcinogen. Furthermore, in the post-initiation phase reduced growth of initiated/preneoplastic cells may impair the process of tumor promotion. In animal studies limonene was shown to antagonize initiation of mammary carcinogenesis by 7,12-dimethylbenz[a]anthracene, probably via the induction of enzymes that inactivate the carcinogen (68). Additionally, limonene and PA induced complete regression of tumors of the mammary gland and other organs, suggesting interference with the advanced stages of tumor development (4,9,10). However, the effects of PA in early carcinogenesis have as yet not been investigated.

The rat liver offers the unique possibility to study the complete sequence of cancer development, including initiation. Within a few days after administration of various genotoxic hepatocarcinogens single hepatocytes emerge that express placental glutathione S-transferase [single placental glutathione S-transferase-positive (G+) cells] (11,12). A certain fraction of these G+ single cells develop into G+ foci, which increase further in number and size on treatment with tumor promoters and which may then evolve into G+ tumors. Therefore, single G+ cells are considered `initiated'; their number as well as the number and size of G+ foci can be used as quantitative indicators of subsequent cancer risk (11).

Growth of G+ single cells and G+ liver foci is determined by the balance between replication and death of cells. One of the main characteristics of G+ liver foci is an elevated rate of DNA replication that is partly counterbalanced by an increased rate of cell death via apoptosis, resulting in little net growth of the lesions (1315). However, treatment with tumor promoters such as phenobarbital (PB) inhibits apoptosis in the foci, thereby accelerating their further growth into neoplasias (14,15). On the other hand, increased apoptotic activity in preneoplastic lesions leads to their preferential elimination and may provide protection against cancer development (16,17).

Based on these findings, we expected that a tumor preventive agent, when active in the post-initiation phase, would exert effects opposite to those of tumor promoters, i.e. it should decrease cell replication and/or induce apoptosis in G+ single cells and G+ foci. Accordingly, we studied the effects of PA on the growth of initiated/preneoplastic cells that had been induced by treatment with N-nitrosomorpholine (NNM). This agent is one of several genotoxic nitrosamines occurring in tobacco smoke and in a variety of foods and alcoholic beverages, which likely contribute to the formation of human cancer (18,19). In contrast with our expectations we found that PA treatment suppressed cell death of G+ cells, resulting in an increased number and size of G+ foci. Since initiated cells may always be present in human organs, the possibility of a tumor promoting activity of PA in early hepatocarcinogenesis raises doubts over the safety and efficacy of this agent for chemopreventive use in healthy humans.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and treatment
Male SPF Wistar rats, 3–5 weeks old, were obtained from the Forschungsinstitut für Versuchstierzucht und Versuchstierhaltung (Himberg, Austria). In these animals the hepatocarcinogen NNM induces considerably more preneoplastic liver lesions than in male SPF Wistar rats from the Zentralinstitut für Versuchstierzucht (Hannover, Germany) (20,21). Rats were randomly assigned to experimental groups and kept under standardized conditions (macrolon cages, 20 ± 3°C ambient temperature, 40–70% relative humidity, inverse 12 h light/dark rhythm with lights off from 10 a.m. to 10 p.m.) (16). They had access to water ad libitum and were fed Altromin 1321N powder diet, which is poor in nitrosamines and other toxic substances (Altromin, Lage, Germany). Body weights were registered once weekly. Three weeks before the first treatment animals were adapted to rhythmic feeding (food supplied from 10 a.m. to 3 p.m.). This procedure was continued until day 91 after initiation with NNM (see below). The feeding rhythm synchronizes DNA synthesis and, presumably, apoptosis in the liver to single peaks per day, which are ~12 h apart (16,17).

All animals were treated with NNM (Serva, Heidelberg, Germany) for initiation, indicated as day 0 in the experimental schedule (Figure 2Go). Immediately before application, NNM was dissolved in phosphate-buffered saline (pH 7.4) and was given as a single dose of 250 mg NNM/10 ml solution/kg body wt by gavage between 8 p.m. and 9 p.m., when the wave of hepatic DNA synthesis was at its peak (16,17).



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Fig. 2. Animals of all experimental groups were treated with a single dose of NNM (250 mg/kg body wt) on day 0 in the time schedule. Small arrows indicate time points of death; four animals were analyzed per group at days 8, 14, 26, 46, 59 and 91 and five per group at day 266.

 
Experimental protocol 1 (Figure 2Go)
On days 4, 5 and 6 after NNM treatment sodium phenobarbital (Fluka AG, Buchs, Switzerland) was dissolved in tap water and administered by gavage as a dose of 50 mg/10 ml/kg body wt at the end of the daily feeding period at 3 p.m. From day 7 onwards, when the animals had regained their normal food intake, PB was admixed with the powder diet (Altromin 1321N) and PB concentrations were adjusted every 14 days to provide a daily dose of 50 mg/kg body wt. PB treatment did not affect food intake or body weights (Figure 3Go), therefore controls (COal) and PB-treated animals were fed ad libitum. At day 91 after NNM the ad libitum fed controls were also switched to a PB diet until the end of the experiment at day 266.



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Fig. 3. Effect of initiation with NNM and subsequent treatment with PB or PA on body weight. Means ± SD are given; statistics by Kruskal–Wallis test: COal versus PB, not significant; COpf versus PA, not significant.

 
Experimental protocol 2 (Figure 2Go)
From day 4 after NNM treatment onwards one group of animals received PA (Aldrich, Steinheim, Germany) in the diet. Within 24 h a 2% PA diet was reported to lose ~30–50% of its terpene content by evaporation at room temperature (4). Therefore, PA was admixed with the powder diet (Altromin 1321N) every 7–10 days, which was stored in sealed plastic bags at –20°C. Animals were provided daily with a new aliquot of the stored food, a great proportion of which was consumed immediately at the beginning of the 5 h feeding period. Since PA reduced food intake, the pair fed control group (COpf) on basal diet received the average amount of food that had been consumed by the PA rats the day before. The concentration of PA in the diet was gradually increased; at day 20 after NNM treatment the final concentration of 1.8% PA was reached, providing a dose of 1 g/kg body wt/day (PA intake per kg body wt: on day 4, 0.07 g; on day 8, 0.17 g; on day 11, 0.56 g; on day 13, 0.67 g; on day 20, 1.06 g). This concentration was the maximum tolerated one and caused no additional body weight loss when compared with COpf. On day 91 after initiation PA treatment was discontinued. Both groups were fed basal diet ad libitum for 4 days, followed by promotion with PB (50 mg/kg body wt/day) for a further 171 days. The experiment was terminated at day 266.

All experiments were performed according to the Austrian law on animal care and protection.

Biochemical determinations
Liver weight was registered and samples of liver tissue were deep frozen at –70°C until use. Determination of hepatic protein and DNA content was performed as described previously (22).

Histology and morphology
Specimens of liver tissue were fixed in Carnoy's solution and processed for histology as described (17). Two serial sections, 2 µm thick, were used for hematoxylin and eosin (H&E) staining and immunohistochemical detection of placental glutathione S-transferase (GST-P).

Immunostaining for GST-P
One serial section was stained for GST-P by the unlabeled antibody peroxidase–antiperoxidase technique as previously described (23). The applied dilutions of antisera were as follows: anti-GST-P, 1:320 (Biotrin International, Dublin, Eire); anti-rabbit IgG, 1:50 (Nordic, Tilburg, The Netherlands); horseradish peroxidase–antiperoxidase complex, 1:50 (Dako, Glostrup, Denmark). The specificity of immunohistochemistry was confirmed by omitting the primary antibody.

Autoradiography
Autoradiography was performed on the GST-P stained serial section using Ilford K5 photo emulsion (Ilford, UK) as described previously (17).

Determination of DNA synthesis and apoptosis per day
After synchronization of cell replication and apoptotic activity by feeding rhythm (see above) [3H]thymidine (6.7 Ci/mmol; NEN, Frankfurt, Germany) was injected into the peritoneal cavity as a single dose of 0.2 mCi/kg body wt at the daily peak of DNA synthesis between 8 p.m. and 9 p.m. Thirty-six hours later, at the daily maximum of apoptotic activity, animals were killed by decapitation under CO2 narcosis. For a more detailed description see Grasl-Kraupp et al. (16,17).

DNA synthesis was determined by counting the number of [3H]thymidine-labeled nuclei per 100 nucleated hepatocytes (labeling index, LI); LIs were determined for at least 2000 nuclei of unaltered cells in each liver and in all nucleated cells within individual G+ foci. Residues of hepatocytes undergoing cell death by apoptosis were detected according to previous morphological descriptions (24). Only apoptotic bodies (ABs) containing chromatin fragments were used for the present evaluation. For the determination of ABs in preneoplastic lesions, G+ foci were first identified in the GST-P stained serial section and were then evaluated for ABs in the H&E stained serial section using two microscopes linked by a bridge for overprojection (Zeiss, Germany). The apoptotic index (AI) indicates the number of ABs per 100 intact hepatocytes. AIs were assayed by scoring at least 4000 normal hepatocytes per animal and all cells within individual G+ foci. Since interindividual variations were small, LIs and AIs of all foci obtained from different livers of the same experimental group and time point were pooled.

Quantitative evaluation of G+ single cells and G+ foci
Numbers of G+ single cells and G+ foci were registered. Size (area of cross-section) of G+ foci and areas of evaluated liver tissue sections were determined by means of a semi-automatic image analyzer (VIDS IV; Ai-Tektron GmbH, Meerbusch, Germany). Wherever indicated we also determined the number of cells per cross-section of individual foci. The size of the G+ foci served to calculate the number and volume of the lesions per liver according to Saltykow, a stereological procedure assuming a spherical shape for preneoplasias (25). Since this condition is not met by very small G+ lesions, only foci of >4 G+ cells/cross-section were subjected to analysis (21,26).

Statistics
If not stated otherwise, means ± SD or SEM are given. For LIs and AIs incidences and 95% confidence limits were determined. The significance of differences of means was calculated using the non-parametric Wilcoxon's test or Kruskal–Wallis test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Design of the study
The experiment was designed to analyze the effects of PA on the first stages of hepatocarcinogenesis. For this purpose, PA was applied to rats beginning at day 4 after initiation with NNM; at that point of time the first putatively initiated liver cells and mini-foci emerged (20,21). Since PA reduces food intake, controls on basal diet were split into an ad libitum (COal) and a pair fed group (COpf). Treatment with the tumor promoter PB served as a positive control. In order to enhance any treatment-related effects all groups received PB from day 91 or 95 until the end of the experiment at day 266 (for the experimental protocol see Figure 2Go).

Survival, food intake and body weights
Two rats died within the first 3 days after NNM application. No further loss of animals occurred until the end of the experiment. Immediatley after treatment the daily food consumption decreased from 15.9 ± 1.0 to 3.6 ± 0.5 g at day 3 and gradually recovered to 15.9 ± 1.1 g at day 9. As a consequence, all animals lost ~30% of their body weight within the first week and regained their initial weight a few days later (Figure 3Go). No significant differences in food intake and body weights were observed between rats of the PB and the COal groups. However, PA reduced the daily food consumption considerably to 11.0 ± 0.8 g. Despite identical food intake of PA and COpf animals, PA exerted a food-independent negative effect on body weight throughout the treatment period, as already described for similar doses of PA (Figure 3Go; 4,27).

Liver weight, hepatic protein and DNA content (Figure 4Go)
When compared to COal, COpf animals showed lower absolute liver weights with reduced protein and DNA contents in the total organ. Thus, continuous restriction of the daily food intake caused some hypotrophy and hypoplasia of the liver, as has been observed before (16,28).



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Fig. 4. Effect of PB and PA treatment on liver weights (a and b), liver protein (c and d) and liver DNA content (e and f). Means ± SD are given; % above the columns indicates relative changes compared to controls; statistics by Wilcoxon's test: *P < 0.05 for COal versus PB and COpf versus PA.

 
PB treatment led to pronounced increases in absolute and relative liver weights (Figure 4a and bGo); this was due to significant elevations of hepatic protein and DNA content, indicating PB-induced hypertrophy and hyperplasia of the organ (Figure 4c–fGo). When compared with COpf, PA-treated livers showed increases in absolute liver weights and, due to lower body weights, even more pronounced increases in relative liver weights (Figure 4bGo); hepatic protein content was raised in parallel (Figure 4c and dGo), while the DNA content was enhanced much less (detectable only if related to 100 g body wt; Figure 4e and fGo). Thus, PA elevated the mass of the liver predominantly by hypertrophy.

Induction of G+ single cells and G+ foci by NNM
In the COal group, G+ single cells and small G+ foci were seen 8 days after initiation (Figure 5aGo). G+ single cells increased dramatically and reached a maximum of 750/cm2 evaluated tissue section on day 26. At the same time a maximum number of lesions consisting of 2–64 G+ cells/cross-section was obtained. Between days 26 and 91 numbers of G+ single cells and small foci (<=8 cells/cross-section) declined. The decline in single cells and small foci was not compensated for by an increase in larger foci (>8 cells/cross-section). As a consequence, the total number of G+ lesions, seen per cm2 section area, decreased (Figure 5bGo).



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Fig. 5. Kinetics of appearance of G+ single cells and G+ foci. Experimental data are expressed per cm2 evaluated tissue section between days 8 and 91 post-NNM treatment. (a) G+ lesions grouped into four different size classes according to the number of component cells per cross-section; (b) the sum of all lesions. Means are given, SDs are not shown. In (a) SDs in the different size classes of foci were in the range 53–90% of the mean at day 8, 95–116% at day 14, 51–172% at day 26, 67–88% at day 46, 31–200% at day 59 and 45–122% at day 91. In (b) SDs, when expressed as a percentage of the mean, were 59% at day 8, 89% at day 14, 64% at day 26, 74% at day 46, 38% at day 59 and 46% at day 91.

 
Effects of pair feeding on G+ single cells and G+ foci
G+ single cells per unit area were more frequent in COpf than in COal livers (Figure 6aGo). This may be due to two effects of lowered food intake: firstly, a reduced volume of the cells due to hypotrophy resulting in a higher cell density; secondly, a delay in the time course of early hepatocarcinogenesis, e.g. retarding both the decline in G+ single cells and G+ foci and the increase in the average size of foci between days 26 and 91. As a result, many but small G+ foci were present and the total area of G+ foci remained unaffected. A similar effect of reduced food intake on early hepatocarcinogenesis was observed in another independent study (20).



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Fig. 6. Effect of PB or PA treatment on G+ single cells and G+ foci. The frequency of G+ single cells (a), the number (c) and area in mm2 (d) of G+ foci (>1 cell/cross-section) were determined per cm2 evaluated tissue section. Mean number of G+ cells per focus cross-section (f.c.s.) is given in (b). Numbers (e) and volumes (f) of G+ foci were calcluated per liver. For symbols see Figure 4Go; means ± SEM are given; statistics by Wilcoxon's test; *P < 0.05 for COal versus PB, no significance for calculated COpf versus PA.

 
Effects of PB and PA on G+ single cells and G+ foci
When animals received either PB or PA the number of G+ single cells tended to be increased, but not to a statistically significant extent (Figure 6aGo). PB treatment also had no clear effect on foci numbers (Figure 6cGo), but raised the average cell number per focus cross-section and the total area of G+ foci until day 91 (Figure 6b and dGo). Thus, PB promoted early hepatocarcinogenesis mainly by increasing focus size; this effect required ~3 months to become obvious. PA-treated livers showed both slightly elevated numbers and areas of G+ foci at all time points evaluated (Figure 6b–dGo).

Stereological estimates of G+ foci
Under the present experimental conditions we observed both hypo- and hypertrophy of the liver, resulting in different cell densities per unit evaluated tissue section. To counter this problem, a stereological method was applied to calculate the number and volume of G+ foci per liver (Figure 6e and fGo). Following this approach, the differences between COal and COpf were no longer evident and PB treatment significantly elevated the number and volume of foci in the total organ until day 91 (Figure 6e and fGo). PA exhibited similar but less pronounced effects that did not reach the level of significance.

DNA synthesis and apoptosis (Figure 7Go)
The percentages of replicating hepatocytes (LI%) and of apoptoses (AI%) were determined in unaltered liver and in G+ foci (>1 cell/cross-section). In both control groups LI(%) and AI(%) were ~3- to 5-fold higher in G+ foci than in the surrounding normal liver tissue (Figure 7Go). This indicates elevated cell turnover in early preneoplasia, as known for more advanced stages of hepatocarcinogenesis (14,17). Administration of PB slightly increased DNA synthesis in normal liver as well as in G+ foci. Furthermore, PB treatment tended to decrease apoptotic activity in G+ foci (Figure 7aGo). This effect has been described in several previous studies; in the present work it was not statistically significant, possibly due to the very early time points of investigation and the general small size of the G+ lesions (14).



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Fig. 7. Effect of PB or PA on LI (%) and AI (%) in normal liver and in G+ foci (>1 G+ cell/cross-section). Four animals were evaluated per treatment group and time point. No significant difference was found between foci of different size classes (not shown) and data were therefore combined. In each of the animals LI was determined in 2000 unaltered nucleated hepatocytes and on average in 889 nuclei of G+ cells, whereas a mean of 4000 unaltered hepatocytes and of 1221 G+ cells was screened for AI. Incidences are given; vertical lines indicate 95% confidence limits; no significant differences were found.

 
PA treatment lowered cell replication insignificantly in normal liver and in G+ foci (Figure 7Go). Similar to PB, PA appeared to slightly suppress the apoptotic activity of foci cells at both time points examined.

Effects of prolonged treatment with PB on G+ foci
After the final PB treatment of all groups (see Figure 2Go) no significant difference was evident between COal and COpf (Figure 8Go). PB treatment from day 4 after NNM onwards led to a significantly higher cell number per focus cross-section than in COal at day 266 (Figure 8cGo; P < 0.05). The area of G+ foci also tended to be higher.



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Fig. 8. Effect of tumor promotion with PB after day 91 on G+ foci in groups COal (COal-PB), COpf (COpf-PB), PB (PB-PB) and PA (PA-PB). For details of the experimental protocol see Figure 1Go. The number (a) and area in mm2 (b) of G+ foci per cm2 evaluated tissue section and the average number of G+ cells per focus cross-section (c) are given. Only foci consisting of >4 G+ cells were determined. Means ± SEM are given; statistics by Wilcoxon's test; COal-PB versus PB-PB and COpf-PB versus PA-PB; *P < 0.05.

 
PA treatment between days 4 and 91 exhibited effects on foci at day 266, which were similar to those of PB, although somewhat less pronounced (Figure 8Go). Specifically, the total number of foci appeared to be reduced, but the number of G+ cells per focus cross-section was significantly higher than in COpf. Thus, under the present experimental conditions a chemopreventive effect of PA was not detected. On the contrary, PA seemed to exhibit a weak tumor promoting effect, qualitatively similar to that of PB.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study PA did not exert chemoprevention in the first stages of rat hepatocarcinogenesis but revealed effects similar to those of the tumor promoter PB; PA showed slight promotion rather than inhibition of preneoplastic growth. The implications of these findings include the following.

Agents with tumor promoting activity in the liver generally cause several of the following effects: enzyme induction, enlargement of the liver by hypertophy and/or hyperplasia, an increase in DNA synthesis and/or decrease in apoptotic activity, which is more pronounced in preneoplastic than in unaltered cells, and preferential growth stimulation of (pre)neoplasias (15,28). Under the present experimental conditions PA exhibited several of these characteristics: it increased relative liver weight via hypertrophy, slightly lowered the apoptotic activity of unaltered and G+ cells and led to enlargement of preneoplastic G+ foci. The induction of various enzymes by monoterpenes has been described elsewhere (8). We did not find an elevated hepatic DNA content and a stimulatory effect of PA on proliferation of unaltered and G+ cells. In any case, many of the PA-induced alterations in the liver, including enlargement of preneoplastic foci, were comparable with the effects of the potent tumor promoter PB. Other monoterpenes, such as d-limonene, were also found to promote growth of G+ foci (29). Thus there are several lines of evidence that monoterpenes do not antagonize but even appear to enhance early rat hepatocarcinogenesis.

In a previous and in the present study initiation with NNM induced a dramatic increase in the number of G+ single cells, which dropped dramatically thereafter (20,21). We showed recently that a considerable proportion of these G+ cells disappear, most probably by apoptosis (21). Since the present data suggest that PA tends to suppress such apoptosis of G+ cells, diminished apoptotic activity could have contributed to the elevated number of G+ single cells in PA-treated livers. By the same mechanism PA may also have enhanced growth of G+ foci. This is analogous to the long-known effects of PB, which promotes tumor development mainly via prolongation of the lifespan of preneoplastic cells (14,15). The effects of PA on early liver preneoplasia contrast with induction of apoptosis in various fully developed malignancies after treatment with monoterpenes, indicating that this class of compounds may act differently in the early and late stages of carcinogenesis (30,31).

Monoterpenes attenuate the development of cancer in liver, mammary gland, colon and pancreas (27,30,32,33). Most long-term animal studies, however, did not discriminate between cancer preventive effects during tumor promotion and cancer therapeutic effects in the case of frank malignancy. After initiation with N-ethyl-N-hydroxyethylnitrosamine, d-limonene significantly decreased liver tumor incidence in F344 rats but failed to do so in NCI-Black-Reiter (NBR) rats (34). However, NBR rats generally carry lower numbers of liver tumors than F344 rats, indicating that in this strain the frequency of preneoplasias and/or the formation of neoplasias is reduced. If PA acts mainly on advanced neoplasia, NBR rats probably did not provide adequate numbers of targets for PA to exert its tumor therapeutic effects. In accord with this assumption, limonene significantly and dose-dependently increased numbers and areas of G+ liver foci in F344 rats (29), while in the same strain a dramatic reduction in the size of hepatocellular carcinoma was induced by PA (30).

There is no doubt about the potential benefit of monoterpenes as cancer therapeutic agents, since they appear to be capable of inducing regression of tumors via induction of apoptosis, as has been shown for tumors of mammary gland, liver, colon and pancreas (4,9,10,27,35). d-Limonene induces complete regression even of secondary mammary tumors, indicating efficacy against very advanced stages of carcinogenesis (9). Consequently, monoterpenes are currently being investigated as potential therapeutic agents in clinical studies in advanced cancer patients (1,2,5).

Recent research provided possible explanations for the divergent effects of monoterpenes on the early and late stages of carcinogenesis. Firstly, d-limonene was found to inhibit gap junctional intercellular communication in mouse primary keratinocytes and in cell lines derived thereof (36). Such inhibition of intercellular communication, which may contribute to an altered growth behavior of the target cells, is shared by many different tumor promoting compounds. The inherently altered pattern of gap junctional proteins in G+ foci may further support enhanced growth during monoterpene treatment (37,38).

Secondly, monoterpenes inhibit the isoprenylation of small G-proteins of 21–26 kDa, such as rasp21, which is frequently activated in the late stages of rodent and human cancer (3942). Isoprenylation appears to be essential for this oncogene product to associate with the plasma membrane and to exert transforming activity (43). Accordingly, PA inhibited the growth rate of one hamster and one human pancreatic cell line harboring a K-ras oncogene in a dose-dependent manner (10). However, mutations of ras are rarely seen in the early stages of rat hepatocarcinogenesis, which may explain the lack of growth inhibition of the foci by monoterpenes (44).

Thirdly, PA may increase the sensitivity of tumor cells to the mitoinhibitory and apoptosis-inducing effects of transforming growth factor ß (TGF-ß) by increasing both its uptake by TGF-ß receptors I–III and its activation by the mannose 6-phosphate/insulin-like growth factor II receptor (30). Accordingly, only mammary tumors that exhibit elevated levels of the mannose 6-phosphate/insulin-like growth factor II receptor regress in response to limonene treatment (45). However, we could not find an altered level of TGF-ß mRNA or protein and of TGF-ß receptors I–III in liver tumors (46). It therefore appears likely that the concentration of this cytokine and of its receptors is unchanged in liver foci as well; this may provide a further explanation as to why PA did not antagonize growth of the foci.

In conclusion, PA did not antagonize but slightly enhanced liver carcinogenesis in the early stages. Initiated cells are probably present in many organs and may give rise to tumors. Even if the efficacy of PA for the treatment of human cancers can be proven, caution is recommended in applying this agent to healthy humans as a chemopreventive drug.


    Notes
 
1 To whom correspondence should be addressed. Email: bettina.grasl-kraupp{at}univie.ac.at Back


    Acknowledgments
 
The excellent technical assistance of H.Koudelka and R.Tiefenbacher is gratefully acknowledged. For financial support of this study, which was performed within the training program of the Hochschullehrgang für Toxikologie at the University of Vienna, we thank Hafslund Nycomed Austria. This study was also supported by the Herzfeldersche Familienstiftung.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 30, 1999; revised July 4, 2000; accepted July 7, 2000.