The promotion effect of anorectic drugs on aflatoxin B1-induced hepatic preneoplastic foci

Xu Lin2,3, David A. Levitsky, John M. King1 and T. Colin Campbell

Division of Nutritional Sciences and
1 Department of Pathology, Veterinary Medicine College, Cornell University, Ithaca, NY 14853, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The ability of three extensively used anorectic drugs, namely fenfluramine (FN), fluoxetine (FX) and amphetamine (AM), to alter the development of aflatoxin B1 (AFB1)-induced {gamma}-glutamyl-positive (GGT+) preneoplastic liver foci was investigated in 135 male weanling F344 rats. Following AFB1 administration, 15 rats were killed, while the rest were divided into four groups and fed diets containing either FN, FX, AM or control diet, with half of the animals in each group subsequently being killed at 4 weeks and half at 10 weeks. All three anorectic drugs as expected suppressed initial food intake, growth rate, body weight gain and food efficiency. They also tended to suppress body fat mass and to decrease plasma levels of T3 and T4. FN significantly (P < 0.05) increased GGT+ foci number/cm2 and number/cm3, while FX significantly increased GGT+ foci number/cm2 and the volume fraction of foci. Histopathological staining also revealed that FN- and FX-treated animals had more serious morphological alterations in their liver tissue. In contrast, foci development was, if anything, suppressed by AM feeding. These results indicate that serotoninergic drugs (FN and FX), as opposed to dopaminergic drugs (AM), may have tumor promoter activity, at least for liver tissue.

Abbreviations: AFB1, aflatoxin B1; AM, amphetamine; BAT, brown adipose tissue; DIT, diet-induced thermogenesis; FN, fenfluramine; FX, fluoxetine; GGT, {gamma}-glutamyltranspeptidase; GGT+, {gamma}-glutamyl-positive; H&E, hematoxylin and eosin; T3, triiodothryonine; T4, thyroxine.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An anorectic drug, d-fenfluramine (FN) (Redux), was previously shown in this laboratory to promote hepatic preneoplastic lesions as indicated by significantly increased number and volume fraction of foci in aflatoxin B1 (AFB1)-induced Fisher 344 rats (1). The original objective for that study was to determine whether FN-enhanced energy expenditure could inhibit tumorigenesis, as a means of testing potential mechanisms associated with the anti-tumor properties of feeding a low protein diet (2,3). Indeed, low protein feeding enhances energy expenditure in experimental animals by stimulating both diet-induced thermogenesis (DIT) (2) and increased voluntary physical activity (4). It therefore was of considerable interest that this apparent promotion activity by d-FN occurred in spite of decreased food consumption and elevated DIT.

This earlier report (1) was one of the first to show that one of the most frequently prescribed anorectic agents was associated with carcinogenic potential in an experimental animal model. Because of the possible implications of this finding, the present study was undertaken to determine whether other anorectic drugs might have similar promotion activities.

Three anorectic drugs, namely amphetamine (AM), FN and fluoxetine (FX) were investigated in the AFB1-induced {gamma}-glutamyl-positive (GGT+) foci rat model. AM, which acts via the hypothalamic dopaminergic system, was the first anti-obesity drug introduced in 1936 (5,6) and has been used as a reference drug in pharmacological studies (7). FN (a racemic mixture of d-FN and l-FN) and FX, which act mainly through the serotoninergic system, have been used by more than 100 000 000 patients (8) for various therapeutic applications including weight loss, antidepression and control of attention deficit hyperactivity disorder in children (9,10). In contrast to their widespread clinical applications, investigations of the potential carcinogenic activities of these drugs have been inadequate and controversial. The manufacturers' have reported that d-FN and FX exhibited no carcinogenic and genotoxic activities in rodent bioassays (11,12). However, our earlier report (1), along with that of others, showing that FX could enhance 7,12-dimethylbenz[a]anthracene-induced mammary tumors (13), brings into question this conclusion. To date, no test of carcinogenicity has been performed for FN (14), while AM has been reported to inhibit spontaneous tumors in both mice and rats (15). Certainly, more research is needed to clarify whether chronic use of these drugs possesses tumor-promoting potential.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
A total of 135 Fisher 344 male rats, 80 g body weight, were received at 30 days of age (Charles Rivers, Kingston, ME). All animals were housed individually in hanging stainless steel wire cages with access to food and water ad libitum. The room was maintained at ~24°C with a 12 h light/dark cycle. Routine animal care followed institutional guidelines for good laboratory practice. Animals were monitored daily for their general physical condition and signs of illness. Body weight, food intake and spillage were also recorded.

AFB1 administration
After a 5 day acclimation period, all animals were treated with AFB1, dissolved in tricaprylin, in 12 intragastric doses (5 days dosing, 2 days rest, 5 days dosing, 2 days rest and 2 days dosing) over a 16 day period to achieve a total dose of 4.5 mg AFB1/kg body wt. An additional 8 days were allowed for body clearance of AFB1 and its metabolites before initiation of drug treatment. No animal was lost or suffered severe weight loss during the AFB1 dosing period. All animals remained in a healthy condition throughout the entire study.

Diet and drug treatment
Animals were fed with AIN-76A purified diet (no. 111024; Dyets, Bethleham, PA) for the entire experimental period. The diet contained (by weight) 22% protein as casein, 63% carbohydrate (as 3:1 sucrose:corn starch) and 5% fat as corn oil. Other ingredients included 5% cellulose, 3.5% vitamin mix and 1% mineral mix.

After the 8 day AFB1 clearance period, all animals were randomly assigned into five groups (Figure 1Go) blocked to achieve a similar mean body weight (due to the large variation in body weight, 144–203 g). In order to obtain a baseline for various parameters, 15 rats were killed on the day when the drug treatment began while the remaining four groups of animals (30 rats each) were fed FN-, FX- or AM-containing diets or control diet, respectively. After 4 weeks of drug treatment, half of the rats in each group were killed and the rest continued to receive their drug treatments for another 6 weeks. Finally, all animals were killed at the end of the 10 weeks.



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Fig. 1. Study protocol in the AFB1-induced model.

 
FN hydrochloride and d-AM sulfate were obtained from Sigma Chemical Co. (St Louis, MO), while FX hydrochloride (Prozac) was purchased from Eli Lilly (Indianapolis, IN), as 20 mg capsules. The initial dietary concentrations of FN, FX and AM were 0.3, 0.1 and 0.0375 g/kg, respectively. The dose of FN in this study was twice as much as used in one previous study (1), because this racemic FN preparation was half as anorectic as d-FN (7). The doses of FX and AM were selected to be clinically relevant to that for FN (5). After 12 days treatment, however, FX and AM at these doses did not effectively suppress body weight and food intake compared with FN (Figures 2 and 3GoGo), therefore, the doses of FX and AM were increased to 0.2 and 0.075 g/kg diet, respectively, for the remainder of the study.



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Fig. 2. Mean body of rats untreated (CN) or treated with FN, FX or AM for 10 weeks post-AFB1 (error bars represent SEM).

 


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Fig. 3. Mean daily food intake of rats untreated (CN) or treated with FN, FX or AM for 10 week post-AFB1 (error bars represent SEM).

 
During the preparation of the drug-containing diets, all drugs were first mixed with very small amounts of diet, using blenders, and then more diet was gradually added to achieve the final concentrations. All drug-containing diets were prepared twice per month and were stored at 4°C until use.

Termination procedure and brown adipose tissue (BAT) measurement
Animals were randomly selected and killed at 0, 4 and 10 weeks of drug treatment under non-fasting conditions. Animals were first anesthetized with CO2 and blood was collected in heparinized monovettes (Sarstedt, Newton, NC) through cardiac puncture. Plasma was obtained by centrifugation and was kept in a freezer at –70°C for hormone assays.

During killing, intrascapular BAT, which reflects increased energy expenditure when elevated (16), was excised and weighed for each carcass, after adhering muscle and white adipose tissue were carefully removed.

Determination of GGT+ foci and other lesions
At the termination of the study, livers were weighed and four slices from the left median lobe of each liver were taken. One slice was fixed in 10% neutral formalin and stained with hematoxylin and eosin (H&E) and reticulin for the evaluation of morphological abnormalities and lesions. The remaining three slices were placed flat on dry ice and these frozen slices were then stored at –70°C until sectioned.

During sectioning, two of the liver slices were first embedded in OCT compound (American Scientific Products, Rochester, NY) and multiple consecutive cryostat sections (6 µm) were obtained for two of the OCT-embedded liver slices. Once being air dried, all sections were kept in ice-cold acetone for later GGT+ focus staining.

To determine {gamma}-glutamyltranspeptidase (GGT) activity, sections were stained according to the method of Rutenburg et al. (17). Four sections per rat liver were incubated for 60 min in a solution containing the substrate of the GGT, i.e. {gamma}-glutamyl-4-methoxy-2-napthylamide and mounted in glycerine jelly.

To prevent investigator bias, a total of 540 GGT+ foci slides (60 slides/group) were coded by another investigator and randomly selected prior to scoring under a Nikon Diaphot-TMD optical microscope. Focus numbers were quantified directly and focal diameters were measured in two dimensions with an ocular micrometer with a fixed magnification. Surface areas of liver were determined by projecting an image of the section with a photographic enlarger at a constant magnification. Images were then compared with a standard 1 cm2 image projected in the same fashion. GGT+ foci were calculated by stereological formulae from Campbell et al. (18).

After being stained by the H&E and reticulin methods, 30 liver sections per group were evaluated by a pathologist who was unaware of the treatment groups. For scoring the liver lesions, each section in both of the stained slices was graded for morphological alterations by an arbitrary scale ranging from 0 to 3, with 0 being that for normal tissue.

Hormone assay
The non-fasting concentrations of plasma triiodothryonine (T3) and thyroxine (T4) were determined in duplicate by radioimmunoassay (Diagnostic Products, Los Angeles, CA) in the Endocrinology Laboratory of the College of Veterinary Medicine, Cornell University.

Body composition study
After removing gastrointestinal contents, carcasses were weighed and homogenized. Duplicate samples were analyzed for protein, fat, ash and water content by the Department of Animal Science, Cornell University. Carcass protein was measured by the micro-Kjeldahl method (19) and was determined by multiplying the nitrogen content by 6.25. Fat content was analyzed by a Soxhlet extraction method (20,21), in which the moisture-free samples were extracted with ethyl ether in a Soxhlet extraction apparatus. Ash content was the residue after burning the samples in a muffle furnace (22). Water content was calculated as the weight difference of the sample before and after lyophilization (23), excluding the portion of water added during sample preparation (24).

Statistical analysis
Statistical differences in mean body weight, daily food intake, food efficiency, plasma hormone levels (T3 and T4), BAT weight, liver weight, body composition and foci responses among drug-treated groups were tested by one-way analysis of variance. Tukey and Fisher pairwise comparisons were also used to make multiple comparisons among groups. Unless otherwise specified, results were considered to be statistically significant at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Body weight and food intake
Body weights for rats treated with AFB1 and fed the 22% casein diet alone (controls) or with FN, FX and AM for the 10 week period are shown in Figure 2Go. Initial body weights, which are referred to as time 0 in Figure 2Go, were approximately the same in all groups. The rates of growth for the three drug-treated groups were significantly depressed within the first week of drug treatment, especially for the FN-treated (0.3 g/kg diet) group. After 12 days of drug feeding, the doses of FX and AM were doubled from 0.1 to 0.2 and 0.0375 to 0.075 g/kg, respectively, in order to bring down the mean body weight to a level similar to that of the FN group. However, doubling the AM dose did not further suppress body weight. The decline in the growth rate with FX was persistent and the mean body weight became the lowest among the three drug-treated groups by the end of the study. The control group was always heavier than the other groups over the entire experimental period.

As indicated in Figure 3Go, all three drugs exhibited an anorectic effect as early as during the first week of drug treatment. The effect of FN was the most potent and was significantly different from that of FX and AM. Doubling the dose of FX and AM amplified the anorectic effect to a magnitude similar to that of FN. The controls had the highest food intake at all times.

Drug intakes of FN, FX and AM at different times are shown in Table IGo. The doses used in this study may be considered as medium to high, when compared with other studies (11,15).


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Table I. Drug intake
 
Food efficiency during the study is shown in Table IIGo. At the beginning of the experiment, food efficiency of the drug-treated groups dropped sharply, particularly in the FN group, and then returned to normal within a few days. The increase in dose at 12 days for FX and AM again induced a transitory decrease. However, the mean food efficiency among the FX-treated animals was significantly lower than those of the others (Table IIGo).


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Table II. Food efficiency
 
Body composition
Body composition analyses are shown in Table IIIGo. At 4 weeks post-dosing, mean fat mass increased in all groups when compared with the baseline. However, when compared with control animals, all three drugs tended to have a lower fat mass at 4 weeks, while only the AM-treated group continued to show this effect at 10 weeks.


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Table III. Body composition
 
Organ weights
BAT weights corrected for body weight are shown in Table IVGo. Although relative BAT weight declined as expected over the 10 week experimental period for all drug groups, the FX-treated retained the most while the AM-treated retained the least BAT weight. These BAT weights for FX- and AM-treated animals were inversely related to food efficiency (Table IIGo), as expected.


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Table IV. BAT weight
 
Table VGo shows relative liver weights. FX-treated rats had the highest relative liver weights at both times of death, while the AM-treated animals had the lowest, especially at 10 weeks.


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Table V. Liver weight
 
Plasma T3 and T4
Plasma concentrations of T3 and T4 are given in Table VIGo. T3 concentrations tended to decrease and T4 to increase over time for all animals. T3 and T4 levels were consistently lower in the drug treatment groups at both times, when compared with the respective control groups, with the levels for the FX-treated animals being the lowest. However, there were no drug-related effects on T3:T4 ratios.


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Table VI. Plasma T3 and T4a
 
Liver histopathological quantification
Table VIIGo presents the results of hepatocellular GGT+ foci development after 4 and 10 weeks drug administration. Before giving the drug treatment (week 0), GGT+ cells were sparse and widely scattered over the whole liver section. Only 15 GGT+ foci were observed among 120 liver sections from 15 rats. At the end of the 4 and 10 week treatment periods, all 15 rats in each group, except for one FN-treated rat following the 4 week treatment, developed GGT+ hepatic foci, obviously in response to the AFB1 treatment. After 4 weeks of drug administration, no significant difference (P > 0.05) was found in the relevant foci parameters of number or fraction of liver occupied by foci.


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Table VII. Hepatocellular GGT+ focia,b
 
At the end of 10 weeks, FN, like its d-isomer (1), significantly increased foci number per cm2 and per cm3. FX treatment also increased both of these indices, although only the increase in foci number per cm2 and mean fractional volume of liver occupied by foci were statistically significant. In contrast, AM slightly, but non-significantly, suppressed these foci indices at 4 and 10 weeks.

Table VIIIGo shows the histopathological scores for H&E and reticulin stained liver sections. FX- and AM-treated rats have more severe fibrosis and lipidosis (fat vacuoles) than those in other groups. FX also appeared to promote pseudo-lobulation and clear cell foci (glycogen deposition). FN enhanced the clear cell foci.


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Table VIII. Histopathological studya
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is clear that the doses of these anorectic drugs used in this study were sufficient to cause their intended effects on food consumption, energy utilization and body weight gain. However, these effects were not associated with any obvious signs of toxicity.

However, these two categories of anorectic drugs (serotoninergic, FX and FN; dopaminergic, AM) behaved rather differently in their effects on energy utilization. Weight reduction induced by FX was mainly due to a reduction in lean body mass (Table IIIGo), whereas weight suppression induced by AM preferentially related to a reduction in fat mass. Data from this study (Table IVGo) and others (2527) have indicated that increased energy expenditure induced by FN and FX may be achieved by stimulating diet-induced thermogenesis, as measured by the relative weight of BAT tissue in this study, the activity of GDP-binding proteins in BAT mitochondria (1,28) and/or by increased oxygen consumption (29,30). In contrast, AM appears to augment energy expenditure mainly through increased locomotor activity (31,32). Having a significantly lower BAT tissue weight (Table IVGo), and thus less thermogenesis, AM-fed animals were generally more active in the cages and dumped food cups more frequently than the other groups. Although both FN and FX activate the serotonergic system, they do not act in exactly the same way in terms of their pharmacological profiles, metabolism and pharmacokinetics (33). These differences may also account for some of the differences observed in the current experiment.

These results provide new evidence that FN and FX, like d-FN, are able to promote the development of AFB1-induced hepatic preneoplastic GGT+ foci through an increasing number of foci per unit area and per volume of liver (Table VIIGo). In contrast, AM treatment did not enhance foci development; if anything, foci development was reduced. Foci number and volume in the control animals tended to decrease from the end of week 4 to that at the end of week 10, although these difference are not statistically significant. Foci activity over time depends on the presence of promotional stimuli (3436). In general, the preneoplastic foci exhibit greater cell proliferation and a higher cell turnover rate than that of normal hepatocytes (37); overall, focal lesions rely on the equilibrium between rate of cell growth and cell death in foci (38). In the case of FN- and FX-treated animals, it appeared that cell proliferation may be predominant in the first 4 weeks, as indicated by formation of more, larger foci. Of more significance is the comparison of treatment (FN or FX) versus control effects for two time points, for three measurements (i.e. a total of six measurements). For all six measurements, foci activity of the drug-treated groups was greater than the respective control values, with some of these comparisons being statistically significant. Thus these anorectic serotonergic drugs would appear to increase the formation of preneoplastic foci.

Restricting caloric intake or increasing energy expenditure has long been shown to prevent tumor development in different tumor models (3944). Enhanced energy expenditure was also hypothesized by our group as one of the mechanisms responsible for inhibition of AFB1-induced liver preneoplastic and neoplastic lesions in low protein-fed animals (2), thus the original testing of d-FN as a probe to selectively enhance energy expenditure in rats fed the tumor-promoting casein diet. The findings of this study, however, suggest that even though thermogenesis and anorexia may combine to restrict physiologically available energy required for foci development, FN and FX nonetheless still appear to be able to enhance the carcinogenic process.

The mechanisms for this effect remain unclear. GGT+ focus development may be influenced by many factors, such as environmental chemicals (45,46), hormones (47), nutrition (48,49), age (50), sex (51), tissues (51) and animal strain (46,52,53). Thus, mechanisms by which these two drugs promote focus development could be exceedingly complex. However, the observations from this and our previous study (1), as well as that of Brandes et al. (13), give the impression that this promotional effect is more related to stimulation of the serotonergic system (FN and FX) than of the dopaminergic system (AM). Indeed, increased dopamine activity has previously been shown to be associated with decreased hepatic and mammary tumorigenesis (2,15,54).

Serotonin per se is able to stimulate DNA synthesis and cell division in many tissues and certain neoplasms (55,56). Besides serotonergic nerves, serotonin is also present in enterochromaffin cells of the gastrointestinal tract and, to a lesser extent, in blood platelets (57,58). Administration of FN and FX has been reported to interfere with serotonin re-uptake or efflux from these cells (5962). As a result, more serotonin would be available in the circulation and be delivered to the liver, where serotonin is inactivated (63). Serotonin has been shown to be essential for the lodging of neoplastic cells in liver tissue (64). However, it remains uncertain whether serotonin alone could have induced the formation of hepatic foci in this model, since elevation of serotonin in the body may induce a series of changes at many tissue sites (55,56).

Reducing energy intake and weight gain and/or increasing energy expenditure tend to inhibit tumorigenesis, which may also partially contribute to the lower focus formation in the AM-exposed animals, especially since serotonin activity is not altered. However, these diet-induced effects upon tumor inhibition were not sufficient to prevent the more dominant promotional effect induced by FN and FX. These findings are suggestive of tumor-enhancing effects by serotonergic drugs, at least upon hepatic tumor development. Whether such effects also apply to other tissues is not yet clear.


    Acknowledgments
 
The authors thank Drs Amy J.Lanou and Banoo Parpia for their advice and help. This work was supported by a grant (399-8359-N126) from the Cancer Research Foundation of America.


    Notes
 
2 Present address: Box 2619, Duke University Medical Center, Durham, NC 27710, USA Back

3 To whom correspondence should be addressed at: DUMC—2619/MSRB, Durham, NC 27710, USA Email: lin00026{at}mc.duke.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 30, 1998; revised March 12, 1999; accepted May 5, 1999.





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