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* Toxicology Research Laboratory, University of Illinois at Chicago, Chicago, IL;
Department of Pharmacy Practice, College of Pharmacy, University of Illinois at Chicago, Chicago, IL;
** National Cancer Institute, Rockville, MD;
Human Nutrition, University of Illinois at Chicago, Chicago, IL; and
Roche Vitamins Inc, Basel, Switzerland
1To whom correspondence should be addressed. E-mail: peter.j.korytko{at}pfizer.com.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, area under the curve) after the 30 and 50 mg/kg BW doses were similar. In a repeat dose study, 30 mg/(kg BW · d) administered orally to six dogs for 28 d resulted in steady-state plasma concentrations between 785 and 997 nmol/L lycopene. Apparent clearance, volume of distribution and apparent elimination half-life were 2.29 L/(h · kg), 96 L/kg and 30.5 h, respectively. Dogs were killed 1 or 5 d after the last dose and 23 tissues were collected for lycopene analysis. Lycopene concentrations were highest in liver, adrenals, spleen, lymph nodes and intestinal tissues. Liver lycopene concentrations were 66 and 91 nmol/g 1 and 5 d after cessation of treatment, respectively. Prostate lycopene concentrations were < 0.2 nmol/g both 1 and 5 d after dosing ceased (<0.4% of liver concentrations). Although 70% trans-lycopene was used in the dosing material, most of the lycopene identified in plasma and tissues was cis-lycopene.
KEY WORDS: • carotenoid • prostate • cancer • chemoprevention • canine
Lycopene is the predominant carotenoid in tomatoes and tomato products (1) as well as in some other fruits and vegetables (2). Among dietary carotenoids, lycopene is the most efficient quencher of singlet oxygen (3). This antioxidant activity is a potential mechanism by which lycopene may contribute to the prevention of a variety of cancers and other diseases. Substantial accumulation of cis lycopene isomers has been noted in humans, although trans lycopene constitutes the predominant isomer in food sources (85%) (4). It remains unclear whether this is due to in vivo isomerization or preferential absorption of cis lycopene.
The consumption of tomato products and having high concentrations of blood lycopene correlate inversely with prostate cancer risk (5–7), digestive tract cancers (8), pancreatic cancer (9), cervical intraepithelial neoplasia (10) and myocardial infarction (11), as well as cancers in many other tissues (7). Because the exact mechanism of lycopene’s protective effects has not been elucidated, it is not known whether lycopene directly prevents prostate cancer. Animal models may prove useful in evaluating the effect of lycopene as a cancer chemopreventive agent.
Canine prostate may be a useful model for studying carcinogenesis and cancer progression in human prostate (12) given the similarity between canine and human prostate cancer. Here, we evaluated the pharmacokinetics and tissue distribution of lycopene in dogs as well as the in vivo percentages of cis- and trans-lycopene isomers in plasma and tissues. These data demonstrate that orally dosed lycopene is systemically available and distributed to most tissues in dogs.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Beagle dogs (Marshall Farms, North Rose, NY), 
6–8 mo old, weighing 8.9–10.3 kg, were housed in an AAALAC Intl.-accredited facility according to NIH guidelines (13). The dogs were housed singly in runs in a temperature (18–29°C) and humidity (50 ± 20%) controlled room with a 12-h light:dark cycle. The dogs were fed 350 g of a lycopene-free dog food, 5L18 High Density Canine Diet (PMI Feeds, St. Louis, MO) each morning for 4 h and had free access to tap water. This diet contained crude protein (
27%), crude fat (16%), crude fiber (
4%), ash (7.5%) and minerals (4%). The high fat diet was used to aid lycopene absorption. Dosing for all studies occurred 2 h after initiation of feeding.
The test material, 5% lycopene (Roche Vitamins, Basel, Switzerland), was provided by the U.S. National Cancer Institute, received from McKesson Bioservices (Rockville, MD), stored desiccated under argon at 2–8°C and protected from light. The material was a fine granular reddish powder that contained white starch particles. Dose levels were based on lycopene content.
Single-dose studies.
In three separate single-dose studies, 2 dogs per dose level received a single oral dose of the test material at 10, 30 or 50 mg/kg body weight (BW) lycopene in gelatin capsules. Blood samples were collected for plasma lycopene analysis at 0 (predose), 15 and 30 min, and 1, 2, 4, 8, 12, 24, 48, 72, 96 and 120 h time points. Data from these pilot experiments were used to set dosages for the repeat-dose study. Plasma lycopene concentrations were evaluated before administration of the next dose.
Repeat-dose study.
Six dogs were dosed orally with 30 mg/(kg BW · d) lycopene once daily for 28 d using gelatin capsules 2 h after feeding. Blood samples were collected on d 1 and 28 at the same time points as in the single-dose studies. Each week, dogs were weighed and subjected to a complete physical examination, and food consumption was quantified. Dogs were also examined daily for clinical signs of toxicity. To determine the tissue distribution 1 and 5 d after the last dose, three dogs were killed and necropsied 24 h after the final dose; the remaining dogs were killed and necropsied 120 h after the final dose. Dogs were killed by sodium pentobarbital anesthesia and exsanguination. From each dog, tissues and organs were systematically collected, frozen in liquid N2, and stored at -80°C. Gastrointestinal tissues were washed before freezing.
Plasma and tissue lycopene analysis.
The total lycopene concentration in all serum samples was measured by HPLC (14). The detection limit for lycopene was 10 nmol/L of serum, or 0.1 ng/injection. The laboratory that completed the bioanalytical measurements is a reference laboratory for the National Institute of Standards and Technology (Gaithersburg, MD) quality assurance program for carotenoids (15).
Tissue samples were homogenized with 5–10 volumes of methanol/water (1:1, v/v, with 10 g/L pyrogallol). The volume of medium was carefully measured and the amount of tissue per gram of homogenate calculated. Whenever possible, triplicate aliquots of the homogenate (1 g) were weighed and saponified by addition of 0.2 mL of 60% KOH, followed by 1 h incubation at 70°C. The saponified mixture was extracted 3 times with 2 mL hexane (containing 0.01% BHT). Hexane layers were combined, evaporated completely, and the residue reconstituted with stabilized ethyl ether (1 part) and 3 parts of HPLC mobile phase. The total reconstituted volume of the sample was carefully assessed. Either 10 or 20 µL of this final extract was injected and analyzed by the HPLC method as described. Tissue concentrations of lycopene were reported as nmol/g of tissue.
Lycopene isomers were separated in the same extracts using a Suplex pKb-100 C18 column with methanol/acetonitrile/isopropanol (54:44:2, v/v/v) isocratic elution (16). This method has been used for the separation of lycopene isomers in resected prostate tissue from prostate cancer patients (17).
Analysis of lycopene in test material.
The lycopene content of the test material was determined by the HPLC method described. The granules were dispersed in warm distilled water, and 1 mL of this stock mixture was extracted by the addition of 1 mL ethanol and 2 mL toluene. The extraction with toluene was repeated until all color was transferred to the upper phase. Toluene extracts were combined, transferred quantitatively to a volumetric flask and adjusted to 100 mL with ethanol.
Pharmacokinetic Analysis.
For the single-dose study, initial pharmacokinetic estimates of the plasma concentration vs. time data were obtained using the method of residuals with the microcomputer program WinNonlin (Version 1.1, Scientific Consulting, Apex, NC). These estimates were used to fit the data to a one-compartment model by nonlinear iterative least-squares regression with the same microcomputer program. A one-compartment model with lag time and first-order absorption and elimination (Model 4, WinNonlin) best fit the plasma concentration vs. time data of all dogs. The inverse of the observed concentration was used as the weighting in the pharmacokinetic modeling. Compartmental pharmacokinetic parameters were estimated using microconstants.
For the multiple dose study, all plasma concentration vs. time data obtained over 28 d were used to determine the pharmacokinetic parameters of lycopene after multiple daily doses. The final pharmacokinetic parameters of lycopene obtained from the single-dose study were used as initial estimates in the same one-compartment model and microcomputer program. The inverse of the observed concentration was used as the weighting in the pharmacokinetic modeling.
Statistical analysis.
Comparisons between serum and tissue isomers, and hourly changes in serum isomer proportions were analyzed by paired t test, using SigmaStat version 2.0 (SPSS, Chicago, IL). Differences with P < 0.05 were considered significant. Values in the text are means or means ± SD.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Oral administration of lycopene to male dogs did not cause any observable adverse effects as measured by food consumption, body weight changes, clinical signs and clinical observations. Red material was observed in feces after dosing, but this was interpreted to be unabsorbed lycopene.
Plasma lycopene concentrations after single doses of 10, 30 and 50 mg/kg BW lycopene peaked 15 h after dosing (Table 1, Fig. 1). The individual concentration vs. time curves suggest that absorption and distribution were ongoing processes during the first 24 h after drug administration. The terminal elimination phase of lycopene was best characterized with the concentration vs. time data after absorption and distribution were completed. The apparent half-life of lycopene in plasma of dogs following a single dose was 36 h. Increasing dose levels did not produce a consistent increase in maximum plasma concentration or area under the concentration vs. time curve (AUC0-). Because these data suggest that similar plasma concentrations may be observed with daily doses of either 30 or 50 mg/kg BW, 30 mg/(kg BW · d) was chosen as the dose level for the multiple dose study.
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The highest concentration of lycopene was in liver in dogs 1 and 5 d after the 28-d dosing period (Table 3). The mean liver lycopene concentrations were 65.6 and 91.0 nmol/g in dogs killed 1 or 5 d after the last dose, respectively. Lycopene concentrations were also relatively high in adrenals (18.5–26.4 nmol/g), spleen (7.0–10.6 nmol/g), and lymph node (5.16–5.38 nmol/g). Lycopene concentrations in the intestinal tissues ranged from 0.38 to 3.04 nmol/g. Tissue lycopene concentrations were usually greater 5 d, compared with 1 d, after cessation of dosing. This may be due to variation among a small number of dogs or to the transfer of lycopene from plasma to tissues. All other tissues had concentrations generally < 0.7 nmol/g. Lycopene concentrations in prostate were 0.143 and 0.181 nmol/g, 1 and 5 d after cessation of treatment, respectively.
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The distribution of lycopene isomers in dog plasma was different from the proportion present in the test material (70% trans, 30% cis lycopene). During the first 120 h of the repeat-dose study [30 mg/(kg BW · d)], the percentage of trans lycopene was greater at 4 h (41.4 ± 0.90%), 8 h (42.0 ± 1.76%) and 12 h (39.8 ± 1.86%) compared to 1 h (35.3 ± 0.50%) after dosing (Fig. 4). Thereafter, the percentage of the trans isomer remained around 35% of total lycopene.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Single-dose studies characterized the pharmacokinetic parameters for lycopene after oral exposure and were used to set dose levels for the 28-d repeat-dose study. Plasma systemic lycopene levels (defined as AUC0-) did not differ substantially after single doses of 30 or 50 mg/kg BW lycopene. Repeat oral administration of 30 mg/kg BW/d for 28 d resulted in steady-state plasma concentrations between 785 and 997 nmol/L lycopene. These steady-state plasma concentrations in dogs were similar to human serum lycopene concentrations (664–1300 nmol/L) after 3 wk of dietary lycopene supplementation of only 30 mg/d in human subjects (18). These data indicate that dog plasma lycopene concentrations can be elevated to concentrations similar to those in humans, but dogs require substantially more dietary supplementation with lycopene to attain these plasma concentrations. This may be due to low absorption in dogs because visible quantities of lycopene were regularly observed in feces after dosing. In rats, 55% of dietary lycopene supplement was found in feces (19). Given potential interspecies differences for lycopene metabolism, additional studies comparing human and canine metabolism of lycopene are warranted.
At 1 and 5 d after the 28-d dosing period, lycopene concentrations in tissue samples were highest in liver, adrenals, spleen, lymph nodes and intestinal tissues (in descending order). Liver lycopene concentrations were 65.6 and 91 nmol/g 1 and 5 d, respectively, after cessation of treatment. Lycopene was not detected in brain but was detectable in abdominal fat. The presence of lycopene in abdominal fat but not in brain indicates that lycopene may not cross the blood brain barrier. Prostate lycopene concentrations (0.14–0.18 nmol/g 1 and 5 d after cessation of dosing) were generally lower than in most other tissues. Although dose concentrations and administration routes were different from published studies, the tissue distribution patterns from the current study concur with previous tissue distribution studies using lycopene and [14C]lycopene in rats and rhesus monkeys (19,20) in which lycopene concentrations were highest in liver. In humans, dietary supplementation with 30 mg/d lycopene for 3 wk increased prostate lycopene concentrations from 0.28 and 0.82 nmol/g (17). Our data indicate that lycopene dosing in dogs results in lycopene concentrations in dog prostate that are of the same order of magnitude as those in human prostate.
A substantial accumulation of cis lycopene isomers has been noted in vivo in humans, although trans lycopene constitutes the predominant isomer in food sources (85%) (4). This greater accumulation of cis isomers has been attributed to their efficient absorption and incorporation into micelles, preferred uptake into tissues, or isomerization reactions, although the exact mechanisms remain unclear. In the present study, although the lycopene dose was composed of 30% cis and 70% trans isomers, the proportion of trans isomers was much lower than that of cis isomers (23–40% trans) in all tissues. Moreover, the percentage of trans isomers in many tissues was significantly lower than in the plasma, suggesting either a greater uptake of cis lycopene in these tissues or increased isomerization of trans to cis lycopene. The percentage of trans lycopene in the liver (28.1 ± 6.1%), kidney (25.9 ± 2.8%), adrenals (31.0 ± 0.8%) and testes (31.0 ± 0.8%) of dogs was much less than the proportions in the same tissues in humans (liver, 45.5%; kidney, 48%; adrenals, 43%; testis, 65%) (21). This indicates possible differences between dogs and humans in lycopene isomer metabolism and/or uptake. However, the percentage of cis isomers in prostate tissue of dogs (71.1 ± 1.2%) was similar to the proportion in human prostate tissue, 79–88% (4,22).
In summary, these data demonstrate that oral administration of lycopene to dogs at nontoxic doses can increase the lycopene concentration in plasma and tissues, specifically in prostate, to concentrations that are similar to those in humans. Therefore, the dog may be a useful model for evaluating lycopene for cancer chemopreventive activity in prostate and other tissues.
Manuscript received 17 April 2003. Initial review completed 14 May 2003. Revision accepted 20 June 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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