The Journal of Nutrition Vol. 128 No. 10 October 1998, pp. 1650-1656

Dietary Lutein from Marigold Extract Inhibits Mammary Tumor Development in BALB/c Mice^1,2

Jean Soon Park^*, Boon P. Chew^{*, , 3}, and Teri S. Wong

^* Program in Nutrition and  Department Animal Sciences, Washington State University, Pullman, WA 99164-6351

	ABSTRACT

Abstract Introduction Methods Results Discussion References

High levels of dietary lutein can inhibit mammary tumor growth in mice. However, the antitumor effect of low levels of dietary lutein on mammary tumors is unavailable. Female BALB/c mice and the WAZ-2T (SA) mammary tumor cell line were used in two experiments. A preliminary tumor cell dose titration study (Experiment 1) was designed to determine the inoculation dose to produce ~65% tumor incidence. Mice (n = 10/dose) were inoculated with 0 to 1 × 10⁶ tumor cells in the right inguinal mammary fat pad. A tumor cell load of 2.5 × 10³ cells/inoculation produced ~65% tumor incidence. This dose was used in a subsequent study (Experiment 2) of the efficacy of dietary lutein against mammary tumor development. Mice (n = 20/treatment) were fed a semisynthetic diet containing 0, 0.002, 0.02, 0.2 or 0.4% lutein from marigold extract. After 14 d, all mice were inoculated with 2.5 × 10³ tumor cells, and tumor growth was measured daily for 70 d at which time blood, liver, spleen and tumors were obtained. Lutein + zeaxanthin uptake increased dose-dependently (P < 0.05) with dietary lutein levels from 0 to 0.02% (spleen) or 0.2% (plasma, liver and tumor). Low levels (0.002 and 0.02%) of dietary lutein lowered (P < 0.05) mammary tumor incidence, tumor growth and lipid peroxidation, and increased tumor latency, whereas higher dietary levels (0.2 or 0.4%) were less effective. Therefore, very low amounts of dietary lutein (0.002%) can efficiently decrease mammary tumor development and growth in mice.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Epidemiological studies have suggested that carotenoids may decrease the incidence of major clinical diseases such as cancer (Ziegler 1989), cardiovascular disease (Burri 1997) and age-related macular degeneration (Snodderly 1995). Consumption of carotenoid-rich diets from fruits and vegetables have been associated with a decreased risk of lung, stomach and prostate cancers (Flagg et al. 1995, Poppel and Goldbohm 1995). Optimism concerning the anticancer activity of -carotene was dampened by recent studies that showed an increased incidence of lung cancer in high-risk population of smokers and/or asbestos workers (Albanes et al. 1995, Omenn et al. 1996) and no effect on cancer and heart disease (Hennekens et al. 1996). Two other studies (Kushi et al. 1996, Verhoeven et al. 1997) reported that -carotene supplementation had no significant effect on the incidence of breast cancer. In recent years, studies on the anticancer activities of other carotenoids began to emerge. Canthaxanthin (Krinsky 1989), lycopene (Levy et al. 1995) and neoxanthin (Chang et al. 1995) have decreased cancer development or growth of cancerous cells.

Lutein is a major carotenoid found in human blood (Parker 1989). Lutein and zeaxanthin have specific biological functions in decreasing cancer development (Chew et al. 1996, Rock et al. 1996), enhancing immune function (Chew et al. 1996) and protecting against age-related macular degeneration (Snodderly 1995). In cultured cells, lutein is more effective than -carotene in inhibiting the auto-oxidation of cellular lipids (Zhang et al. 1991) and protecting against oxidant-induced cell damage (Martin et al. 1996). In humans, high plasma lutein is correlated with greater expression of estrogen receptors in breast cancer cells and, consequently, with greater survival rates and better response to hormone therapy (Rock et al. 1996). Mice fed dietary lutein had reduced growth of a transplantable mammary tumor and enhanced lymphocyte proliferation (Chew et al. 1996). The latter study raised two questions: i) Can dietary lutein prevent breast cancer development or does it merely delay tumor latency? ii) Will much lower amounts of dietary lutein be effective against mammary cancer? We hypothesized that low levels of dietary lutein can inhibit the initiation and growth of mammary cancer cells. Therefore our objective is to study the effect of low dietary lutein on mammary tumor development in mice.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and diet.  Two experiments were conducted to test our hypothesis that feeding low amounts of lutein can inhibit mammary tumor development in mice. In both experiments, female 8-wk-old BALB/c mice were used. Animals were fed a semisynthetic diet (Park et al. 1998) and had free access to water and food. All animals (three mice/cage) were housed in a light- (12 h) and temperature- (23°C) controlled room. All procedures were approved by the Washington State University Animal Care and Use Committee (Pullman, WA).

Tumor cell culture.  The tumor cells used in all studies were from a murine mammary tumor cell line (WAZ-2T; SA) developed from a spontaneously arising mammary outgrowth in a BALB/c mouse (Ram et al. 1993). This cell line has been characterized (Danielson et al. 1980) and was previously used in similar studies with carotenoids (Chew et al. 1996). To assure consistency with the mammary tumor cells used throughout the experiment, only cell passages 34 to 36 were used.

On the day of mammary tumor challenge, tumor cells were grown to confluence, trypsinized, washed and cell number enumerated using a hemacytometer. Cell viability was assessed by trypan blue exclusion. Tumor cells were resuspended in Dulbecco's Modified Eagle's medium containing (per L) 3.7 g of sodium bicarbonate, 2.38 g of Hepes, 0.1 g of penicillin-G, 0.1 g of streptomycin sulfate, 5 mg of insulin and 100 mL of newborn bovine serum (pH 7.4; Sigma Chemical Co., St. Louis, MO). The cell suspension (50 µL) containing the desired number of tumor cells was infused into the right inguinal mammary fat pad (Chew et al. 1996). The cells were kept on ice and mixed well prior to each infusion.

Tumor cell infusion.  Animals were anesthetized with an i.p. injection of xylazine hydrochloride (Xyla-ject, Phoenix Pharmaceutical Inc., St. Joseph, MO) and ketamine hydrochloride (Vetalar, Parke-Davis, Morris Plains, NJ) (dosage: 10 and 100 mg/kg body weight, respectively) in 0.2 mL of sterile phosphate buffered saline (PBS)⁴. An incision was made along the midline previously sterilized with 70% of alcohol, and the skin was gently teased to expose the right inguinal mammary fat pad. Tumor cell infusion was accomplished using a 100-µL Hamilton glass syringe equipped with a 26-g hypodermic needle. Incisions were closed with 11-mm surgery clips. The length and width of the mammary tumors were measured daily with a pair of calipers (Mitutoya, Tokyo, Japan). To correct for skin thickness, 0.1 cm subtracted these measurements (Chew et al. 1996). Palpable tumors too small to be measured were given a diameter value of 0.2 cm. Tumor volume was calculated by using the volume of a sphere. Mice were killed when tumor diameter exceeded 1.7 cm.

Experiment 1 was designed to determine the tumor cell load that would produce ~65% of tumor incidence, an incidence level we wished to use in Experiment 2 to study the efficacy of dietary lutein in preventing mammary tumor development. Mice (n = 10/treatment) were fed nonpurified rodent food (Teklad F6 Rodent Diet, Madison, WI) and challenged with 0, 5 × 10², 1 × 10³, 5 × 10³, 1 × 10⁴, 5 × 10⁴, 1 × 10⁵, 5 × 10⁵ and 1 × 10⁶ SA mammary tumor cells. Mice were killed at 120 d postinoculation or earlier when tumor diameter exceeded 1.7 cm. Tumor incidence and latency were recorded, and mice with no palpable tumors by 120 d postinfusion were given a tumor latency of 130 d.

In Experiment 2, mice (n = 30/treatment) were fed a semisynthetic diet (Chew et al. 1996) containing 0, 0.002, 0.02, 0.2 and 0.4% of lutein from marigold extract (INEXA C. A., Quito, Ecuador). Details on the composition of the lutein from marigold extract and on the preparation of the diets are as described in a companion paper (Park et al. 1998). Food intake was measured daily and body weight recorded weekly.

Tumor cell challenge.  Two weeks after the initiation of dietary treatments, mice were inoculated with 2.5 × 10³ tumor cells in the right mammary fat pad as described in Experiment 1. This tumor load was selected because Experiment 1 showed that this challenge dose would produce ~65% of tumor incidence on d 50 postinoculation. Mammary glands were palpated daily and tumor size measured with a pair of calipers when mammary tumors became palpable (approximately on d 25 after inoculation). All mice were killed on d 70 after inoculation. Mice with no palpable tumor on d 70 postinoculation were given a latency period of 80 d.

Sampling.  On d 70 postinoculation, blood was collected from anesthetized animals into heparinized tubes by heart puncture. Liver, spleen and tumors were excised, weighed, snap-frozen and stored at 80°C under a layer of nitrogen. All tumors were solid tumors.

Thiobarbituric acid (TBA) assay.  Lipid peroxidation activity in tumors was measured by the TBA procedure (Uchiyama and Mihara 1978). The solid tumors were rinsed with PBS, minced and about 1 g was homogenized in 9 mL of cold KCl (11.5 g/L). Duplicate aliquots of 0.5 mL of homogenate were pipetted, and 3 mL of phosphoric acid (10 g/L) plus 1 mL of TBA solution (6 g/L) were added. The mixture was heated for 45 min in a boiling water bath. After cooling, 4 mL of n-butanol was added and vortexed vigorously. The mixture was centrifuged at 1,000 × g for 10 min, and absorbance of the aqueous phase was read at 532 nm. Tetramethoxypropane was used as the external standard. The thiobarbituric acid reactive substance (TBARS) values were expressed as µmol of malondialdehyde (MDA)/g tumor tissue and also as the difference in optical density between 532 and 520 nm. Because identical trends were observed with both methods, only values expressed as µmol of MDA/g tumor tissue are presented.

View larger version (71K): [in this window] [in a new window]   Fig 1. Tumor incidence on d 120 postinoculation in mice challenged with 0 to 1 × 106 mammary tumor cells/inoculation. Tumor incidence (%) was calculated by expressing the number of mice with measurable tumors as a percentage of the total number of mice (n = 10/challenge dose). The correlation coefficient (r2) between tumor incidence and challenge dose is 0.896.

Lutein extraction and high performance liquid chromatography (HPLC).  The extraction and HPLC quantification of lutein and zeaxanthin in plasma, liver, spleen and tumors are described in a companion paper (Park et al. 1998). Again, because lutein and zeaxanthin could not be clearly resolved, results are presented as lutein + zeaxanthin.

Statistical analysis.  Changes in food intake, body weight and tumor volume were analyzed by repeated measures analysis of variance using the General Linear Models Procedure of SAS (SAS 1991). The statistical model was: Yij = µ + treatmenti + mice(treatmenti)(error A) + periodj + treatment * periodij + eij (error B). Treatment differences in lutein + zeaxanthin concentrations in plasma and tissues were compared using the student's t test. Tumor incidence was calculated by expressing the number of mice with measurable tumors as a percentage of total number of mice and differences were compared using Chi square (Steel and Torrie 1980).

	RESULTS

Abstract Introduction Methods Results Discussion References

Experiment 1

View larger version (39K): [in this window] [in a new window]   Fig 2. Tumor latency in mice 120 d after being inoculated with 0 to 1 × 106 mammary tumor cells (n = 10/challenge dose). Tumor latency represents the number of days postinoculation when mammary tumors were first palpable. Mice with no palpable tumor on d 120 postinoculation were given a latency of 130 d. The correlation coefficient (r2) between tumor incidence and challenge dose is 0.887.

Tumor latency period decreased linearly with increasing tumor load (Fig. 2). None of the mice inoculated with 5 × 10² cells developed tumors by d 120 postinoculation when the study was terminated. Even though tumor incidence was maximal with tumor cell challenge doses greater than 5 × 10⁴ cells, tumor latency period continued to decrease with higher inoculation doses through the highest dose used (1 × 10⁶ cells).

Experiment 2

Body weight and feed intake.  Body weight was not significantly different among treatment groups during the study period (Table 1). However, there were significant treatment differences in the weights of livers and spleen, with mice fed the control diet having heavier organs than those fed 0.002% of lutein (P < 0.05, Table 1). These differences in organ weight reflected tumor development rather than the level of lutein supplementation. For instance, disregarding dietary treatments, mice with tumors had liver weights 40% heavier (P < 0.05) than mice without tumors (Table 2). Similarly, spleen weights averaged 26-fold in mice that developed tumors compared to those without tumors. No differences in liver or spleen weights were observed when comparing dietary treatments in mice with tumors, and likewise in mice without tumors.

There were no significant treatment differences in total food intake throughout the experimental period (Table 1). Total lutein intake among the treatment groups reflected the level of lutein supplementation (P < 0.05, Table 1). Therefore, the level of dietary lutein did not significantly affect body weight, food intake, or liver and spleen weight.

Lutein uptake.  Uptake of lutein + zeaxanthin by plasma, liver, spleen and tumor at the termination of the study (d 70 after inoculation) is illustrated. Lutein + zeaxanthin was not detectable in plasma and tissues of unsupplemented mice (Fig. 3, Table 3). Concentrations of plasma lutein + zeaxanthin increased (P < 0.05) in a dose-dependent manner through dietary lutein supplementation levels of 0.2% (Fig. 3). Dietary lutein supplementation of 0.4% did not increase plasma lutein + zeaxanthin concentrations further.

View larger version (21K): [in this window] [in a new window]   Fig 3. Lutein + zeaxanthin concentrations in (A) plasma (B) liver, (C) spleen and (D) tumors of mice fed diets containing 0, 0.002, 0.02, 0.2 and 0.4% of lutein for 84 d. Values are means ± pooled SEM (n = 20/group). Lutein was not detectable in unsupplemented mice and a value of 0.06 µmol/L (plasma) or 0.001 nmol/g (tissues) was given, which is the detection limit. Different letters associated with each bar denote significant difference (P < 0.05).

The concentration (Fig. 3) and total uptake (Table 3) of lutein + zeaxanthin by the liver increased dose-dependently in mice fed 0 to 0.2% of dietary lutein. Like plasma, there was no further increase in liver lutein + zeaxanthin concentrations in mice fed 0.4% of lutein.

In the spleen, the concentration (Fig. 3) and total content (Table 3) of lutein + zeaxanthin increased dose-dependently (P < 0.05) in mice fed up to 0.02% of lutein, with no further increase observed with dietary lutein above 0.02%.

The uptake of lutein + zeaxanthin by tumors was similar to that observed with liver although the concentration of lutein + zeaxanthin in liver was about five-fold higher than that of tumors. There was a dose-related (P < 0.05) increase in tumor lutein + zeaxanthin concentrations (Fig. 3) and in total lutein + zeaxanthin uptake (Table 3) with dietary supplementation of up to 0.2% of lutein.

Mammary tumor growth.  Dietary lutein inhibited the growth and establishment of mammary tumors in BALB/c mice inoculated with 2.5 × 10³ SA mammary tumor cells. The incidence and latency of mammary tumors in unsupplemented mice (Table 4) were similar to that observed in Experiment 1. Mice not supplemented with lutein reached a maximal tumor incidence of 70% by d 50 postinoculation whereas only 20 to 37% of mice fed 0.02 to 0.4% of lutein produced tumors (P < 0.05, Fig. 4). Interestingly, none of the mice fed 0.002% of lutein had palpable tumors prior to d 50 postinoculation. Tumor incidence in mice fed lutein gradually increased until the end of the study period. However, final tumor incidence on d 70 (termination of the experiment) was higher (P < 0.05) in unsupplemented mice than in mice fed 0.002 or 0.4% of lutein (Table 4).

View larger version (28K): [in this window] [in a new window]   Fig 4. Tumor incidence in mice fed 0, 0.002, 0.02, 0.2 and 0.4% of lutein and challenged with 2.5 × 103 mammary tumor cells/inoculation. Tumor incidence (%) was calculated by expressing the number of mice with measurable tumors as a percentage of the total number of mice (n = 20/treatment). Treatment differences, indicated by no letters in common at a time point, were identified using Chi-square.

Tumor latency averaged 15 d longer (P < 0.05) in lutein-supplemented mice than in control mice (Table 4). Mice fed 0.002% of lutein had a longer tumor latency period than all other groups.

Although mammary tumor incidence in control mice reached a maximal level of 70% by d 50 (Fig. 4), tumor volume continued to increase until animals were terminated on d 70 (Fig. 5). Final tumor volume and tumor weight were greater (P < 0.05) in control mice than in mice fed lutein (Table 4). Tumor growth throughout the study period was slowest in mice fed 0.002% of lutein.

View larger version (24K): [in this window] [in a new window]   Fig 5. Tumor volume (calculated by using the volume of a sphere) in mice fed 0, 0.002, 0.02, 0.2 and 0.4% of lutein and challenged with 2.5 × 103 mammary tumor cells/inoculation. All mice (n = 20/group) were included in the calculation, with mice not bearing tumors given a tumor volume value of zero. Values are means ± pooled SEM (repeated measures analysis of variance). Means at a time point with no letters in common differ significantly, P < 0.05.

TBARS.  Lipid peroxidation activity in mammary tumors as measured by the TBARS assay decreased (P < 0.05) in a linear fashion in mice fed from 0 to 0.02% of lutein (Fig. 6). However, mice fed higher levels of lutein (0.2 or 0.4%) had TBARS values that did not differ from that of the control group. The changes in TBARS activity (lowest in mice fed 0.02% of lutein) did not reflect the uptake of lutein by tumors which increased in a dose-dependent manner through 0.2% of lutein. Lipid peroxidation activity, expressed as the difference in optical density between 532 and 520 nm, showed similar trends (data not shown).

View larger version (41K): [in this window] [in a new window]   Fig 6. Lipid peroxidation as measured using thiobarbituric acid reactive substance (TBARS) in tumors from mice fed 0, 0.002, 0.02, 0.2 and 0.4% of lutein. Values are means ± SEM (n = 8-14/group). Means with no letters in common differ significantly, P < 0.05.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Chew et al. (1996) reported that dietary lutein inhibited the growth of mammary tumors in mice. Their study used very high amounts of supplemental lutein (0.1 and 0.4%) and a high mammary tumor challenge dose (1 × 10⁶ SA tumor cells). In fact, 100% of the mice in all treatment groups developed tumors and consequently did not permit the assessment of the possible effects of lutein in preventing the establishment of mammary tumor cells inoculated into the mammary fat pad. The present study was designed to refine these findings by i) selecting a more optimal tumor challenge dose to assess the inhibitory action of lutein against the establishment of mammary tumor cells prior to the promotional stage and ii) using lower amounts of dietary lutein. A dose-titration study (Experiment 1) showed that 65% of mice developed tumors when challenged with 2.5 × 10³ SA mammary tumor cells. Therefore, a challenge dose of 2.5 × 10³ cells was used in Experiment 2 to study the activity of dietary lutein in inhibiting the development and growth of mammary tumors.

In this study, dietary lutein did not significantly influence body weight and feed intake. Liver and spleen weights were generally higher in control mice than in those fed lutein. However, differences in liver and spleen weights reflected mammary tumor development rather than dietary treatment. Within a treatment group, mice bearing tumors had larger liver and spleen than those not bearing tumors. This is expected because hepatomegaly and splenomegaly are conditions that result from cancer development. These observations suggest a lack of adverse effects of dietary lutein from marigold extract and is in agreement with other reports (CARIG 1996, Chew et al. 1996, Gartner et al. 1996).

Measuring the uptake of lutein by the tumor, liver and spleen allowed us to determine the tissue that correlates with the antitumor activity of lutein. The antitumor activity of lutein could indeed be mediated through immune modulation that involves other organs. Uptake of dietary lutein + zeaxanthin increased in a dose-dependent manner in plasma, liver and tumor in mice fed 0 through 0.2% of lutein. In contrast, uptake of lutein + zeaxanthin by the spleen was maximal in mice fed 0.02% lutein. A previous study using similar experimental conditions showed very rapid uptake of lutein + zeaxanthin by plasma, liver and spleen (Park et al. 1998). In that study, plasma lutein + zeaxanthin was saturated by d 7 in mice fed 0.05 to 0.4% of lutein. In contrast, lutein + zeaxanthin concentrations in the liver and particularly in the spleen generally continued to increase through the 28-d study period. In addition, uptake of lutein + zeaxanthin by the spleen was generally dose-dependent through 28 d of feeding. Mice fed the highest dose of lutein (0.4%) had the highest accumulation of lutein + zeaxanthin (0.015 nmol/g spleen) in the spleen (Park et al. 1998). In the present study, mice were fed lutein for 84 d. Uptake of lutein + zeaxanthin by the spleen was dose-dependent with dietary lutein levels of up to 0.02%. Feeding doses higher than 0.02% did not result in greater accumulation of lutein + zeaxanthin in the spleen, thereby indicating maximal lutein + zeaxanthin uptake by the spleen with dietary lutein of 0.02%. It is entirely possible that results on lutein + zeaxanthin uptake by the spleen in this study would be similar to that reported by Park et al. (1998) if the feeding period (28 d) had been the same. Overall, the concentration of lutein + zeaxanthin in the tumor in this study was 50-80% lower than in the spleen and liver, respectively.

Results of the present study showed that dietary lutein reduced mammary tumor growth and development. Tumor incidence, weight and volume generally were lower and tumor latency longer in lutein-fed mice. The highest antitumor activity was observed in mice fed 0.002% of lutein. Chew et al. (1996) similarly showed decreased tumor growth in mice fed lutein. In the latter study, very high amounts of lutein were fed (0.1 and 0.4%) and a very high challenge dose (1 × 10⁶ cells) was inoculated. Results from the present study are in general agreement with Chew et al. (1996) for mice fed similar amounts of lutein. However, as expected, mammary tumor growth and development differed between the two studies due to a much lower challenge dose (1 × 10⁶ vs. 2.5 × 10³ tumor cells) used in the present study. Because the lower tumor challenge dose was expected to produce a tumor incidence of about 65% on d 50 postinoculation, it permitted us to study if dietary lutein could prevent the initial establishment of the tumor cells rather than inhibit tumor growth. Indeed, mice fed lutein, especially those fed 0.002% of lutein, had a lower incidence of mammary tumors on d 70 postinoculation. This suggests, for the first time, that lutein is not only capable of inhibiting mammary tumor growth but possibly of preventing tumor initiation. Krinsky (1989) suggested that carotenoids, specifically -carotene and canthaxanthin, exert their effects by interfering with the promotional phase of carcinogenesis. Therefore, it is likely that the latter carotenoids (-carotene and canthaxanthin) may exert different biological actions than that of lutein. Alternatively, definitive studies on the possible action of -carotene and canthaxanthin in inhibiting carcinogenesis prior to the promotional stage of tumor development have not been conducted.

Lutein may exert its antitumor activity through its antioxidant function. In vitro, lutein protects human liver cells against oxidant-induced damage (Martin et al. 1996). Lutein is capable of quenching singlet oxygen even though its activity is lower than that of -carotene (Di Mascio et al. 1989). While lipid peroxidation may be positively related to tumor growth, Chew et al. (1996) reported that the TBARS activity of tumors from mice fed 0.1 or 0.4% of lutein was not influenced even though dietary lutein inhibited tumor growth. Results from the present study showed lower TBARS levels in tumors from mice fed 0.002 or 0.02% of lutein compared to unsupplemented mice. However, TBARS activity increased in mice fed higher levels of dietary lutein (0.2 and 0.4%) even though total accumulation of lutein in tumors increased dose-dependently. Tumor growth inhibition also was higher in mice fed the lower levels of dietary lutein (especially those fed 0.002% of lutein) compared to those fed 0.2 or 0.4% of lutein. Therefore, these studies suggest that tumor lipid peroxidation activity may be positively correlated with tumor growth only with lower levels of dietary lutein supplementation, whereas higher dietary lutein levels (0.02%) may result in increased lipid peroxidation activity and lower tumor growth inhibition. Indeed, the lack of positive correlation between lipid peroxidation activity and tumor growth reported by Chew et al. (1996) may be due to the high (0.1 to 0.4%) levels of supplemental lutein. High levels of dietary lutein, and therefore, high concentrations of lutein in tissues may indeed create a pro-oxidant instead of an antioxidant environment (Crabtree and Adler 1997). These findings may be relevant to two recent studies that reported negative effects of supplemental -carotene on lung cancer. The ATBC study (Albanes et al. 1995) showed an 18% increase in the incidence of lung cancer among heavy smokers given 20 mg of -carotene daily for 5 to 8 y. The CARET study (Omenn et al. 1996) reported 28% more lung cancer cases and 17% more deaths among heavy smokers and asbestos-exposed workers given 30 mg of -carotene plus 25,000 of IU retinyl palmitate daily. The possibility remains that the level of -carotene supplementation in these human studies may be too high (coupled with a long supplementation period), thereby producing an adverse instead of a beneficial effect. Indeed, the antitumor and antilipid peroxidation activity of dietary lutein observed in the present study was diminished or even abolished in mice fed higher levels of lutein.

The orientation of the various carotenoids in the membrane lipid bilayer may explain their protection against free radical-induced damage. For example, zeaxanthin is more effective than -carotene because the zeaxanthin molecule is anchored in both polar sides of the membrane bilayer, thereby providing an exceptionally good condition for the rigidifying effect of zeaxanthin in the hydrophobic part of the membrane (Gabrielska & Gruszecki 1996). Aside from its potential antioxidant function, lutein may act through other mechanisms. These include immunomodulation (Chew 1993), cell-to-cell communication (Bertram and Bortkiewicz 1995) and prostaglandin production (EIAttar and Lin 1991).

Previous studies have demonstrated anticancer activities of carotenoids other than -carotene. For example, canthaxanthin is more effective than -carotene in delaying skin tumor growth in mice (Krinsky 1989), while lycopene is more active than - and -carotene in inhibiting human cancer cell proliferation (Levy et al. 1995). Neoxanthin is effective as a chemopreventative agent in reducing cancer in hamster buccal pouch (Chang et al. 1995). Our studies (this study and Chew et al. 1996) have similarly demonstrated that the oxycarotenoid, lutein, can inhibit the growth of mammary cancer.

In summary, low amounts of dietary lutein from marigold extract can inhibit the development and growth of mammary tumors in mice.

	FOOTNOTES

Manuscript received 16 December 1997. Initial reviews completed 10 March 1998. Revision accepted 13 July 1998.

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