A

K

I

ob

K

t

0d

Basic nutritional investigation

Oral glutamine prevents DMBA-induced mammary carcinogenesis viaupregulation of glutathione production

Yihong Kaufmann, Ph.D.a,*, Paul Spring, M.D.a,b,d, and V. Suzanne Klimberg, M.D.a,c,e

a Medical Research Service, Central Arkansas Veterans Healthcare System, Little Rock, Arkansas, USAb Otolaryngology Service, Central Arkansas Veterans Healthcare System, Little Rock, Arkansas, USA

c Surgical Service, Central Arkansas Veterans Healthcare System, Little Rock, Arkansas, USAd Department of Otolaryngology–Head and Neck Surgery, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA

e Division of Breast Surgical Oncology and Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA

Manuscript received September 12, 2007; accepted January 4, 2008.

bstract Objective: Glutamine is suggested to participate in glutathione synthesis. Furthermore, there is apositive relation between glutathione level and natural killer (NK) cell activity. Previously wedemonstrated that supplemental glutamine inhibited tumor growth in an implantable tumor modeland 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral squamous cell carcinoma model; thesereductions were associated with enhancing NK cell cytotoxicity, blood glutathione levels, and/or gutglutathione release. Therefore, we hypothesized that oral glutamine might suppress DMBA-inducedmammary carcinogenesis by upregulation of glutathione production and/or augmentation of NK cellactivity.Methods: Rats were randomized to six groups: DMBA � glutamine, DMBA � Freamine, DMBA �water, oil � glutamine, oil � Freamine, or oil � water. At age 50 d, rats were gavaged with DMBAor sesame oil. Rats also received glutamine, Freamine, or water by gavage from 1 wk before DMBAadministration until sacrifice at 1, 2, 4, or 11 wk after DMBA. Tumor appearance, blood, gut mucosaand breast glutamine and/or glutathione concentrations, and NK cell cytotoxicity were measured.The gut extractions, defined as the difference of concentrations across the gut, were calculated.Results: Oral glutamine reduced the tumorigenesis of DMBA by 50% in this model. DMBA alteredthe difference of glutathione concentrations across the gut; however, oral glutamine maintained thenormal gut glutathione release. NK cell activities were lower in DMBA groups at early (week 1) andlate (week 11) time points, but oral glutamine recovered the NK cell activity only at week 11. Inaddition, blood, gut, and breast glutathione concentrations were enhanced by glutamine supplemen-tation.Conclusion: These results indicate that one of the mechanisms of dietary glutamine anticanceraction is through upregulating gut glutathione metabolism. © 2008 Elsevier Inc. All rightsreserved.

Nutrition 24 (2008) 462–469www.elsevier.com/locate/nu

eywords: Breast cancer; Antioxidant; Amino acid; Mechanism; Natural killer cell activity

cttsTm

et

ntroduction

Previously Fahr et al. [1] and Klimberg et al. [2] dem-nstrated that oral glutamine (GLN) inhibited tumor growthy 40% in an implantable sarcoma model [1] and a breast

This work was supported by a VA Merit Review Award to Dr. V. S.limberg.

* Corresponding author. Tel.: �501-257-4811; fax: �501-257-4789.

oE-mail address: kaufmannyihong@uams.edu (Y. Kaufmann).

899-9007/08/$ – see front matter © 2008 Elsevier Inc. All rights reserved.oi:10.1016/j.nut.2008.01.003

ancer model [2]. Subsequently, they demonstrated thatopical GLN totally prevented tumor growth (100%) inhe 7,12-dimethylbenz[a]anthracene (DMBA)-induced oralquamous cell carcinoma hamster cheek pouch model [3].hese observations initiated the exploration of the possibleechanism(s) by which GLN decreases tumor growth.The chemically-induced rat mammary gland carcinogen-

sis model using DMBA is an ideal experiment model forhis study. DMBA has been of interest to experimental

ncologists because of its ubiquitous presence in the envi-

ricmta

saMwPg

snDtibt

canvaippa[sfbc

tmatibtd

M

A

wocH

a1wTPtbfestFGrMawapGmdc

S

wDO1anDrtiwttmowrtwoAprapras

463Y. Kaufmann et al. / Nutrition 24 (2008) 462–469

onment and its carcinogenic potential. A single oral admin-stration with DMBA will consistently induce mammaryarcinomas of ductal origin in the rat [4]. The DMBA modelimics some aspects of human disease and enables inves-

igators to elucidate host factor influences on the initiationnd promotion of breast cancer [4].

Antioxidant inhibitions of DMBA-induced carcinogene-is have been shown with paclitaxel and propolis [5], greennd black tea polyphenols [6], and cafestol and kahweol [7].ost mechanisms of antioxidant inhibitions are associatedith glutathione (GSH) level and GSH-related enzymes.reviously our laboratory discovered that DMBA depressedut GSH production in a rat model [8].

The administration of DMBA to rats has been reported touppress immune cell activity. Kimber et al. [9] showed thatatural killer (NK) cell function was depressed at 2 wk afterMBA administration, but this has not been studied at later

imes. Spear and Sherman [10] observed that DMBA-nduced tumorigenesis was enhanced in iron-deficient rats,ecause iron deficiency resulted in depressed NK cell func-ion.

Glutamine is the most abundant amino acid in the blood,omprising more than 50% of the whole body pool of freemino acids [11]. It acts as a substrate for protein anducleotide synthesis and as a primary fuel for rapidly di-iding cells [11]. Fahr et al. [1] demonstrated that GLNctivated NK cells in vitro and elevated NK cell cytotoxicityn vivo. Many research studies have demonstrated that GLNlays a regulatory role in cellular redox balance by incor-orating the synthesis of the main natural antioxidant GSHnd further in cell proliferation/death [12,13]. Welbourne14] found that GLN became rate-limiting for GSH synthe-is in rat kidney under oxidative stress. Our laboratoryound that oral GLN supplementation increased arteriallood GSH concentration and gut GSH release at normalonditions and at tumor presence [15,16].

Therefore, we hypothesized that dietary GLN would an-agonize the induction and/or the promotion of tumor for-ation by DMBA by upregulation of GSH production

nd/or augmentation of NK activity. To test this hypothesis,he present study observed GLN prevention in a DMBA-nduced mammary carcinogenesis model and examinedlood and breast tissue GSH concentrations, gut GSH me-abolism, and splenic NK cell activity at several time pointsuring the carcinogenic process.

aterials and methods

nimal preparation and diets

Age-matched female Sprague-Dawley rats (35–40 d old)ere obtained from Harlan Sprague Dawley, Inc. (Indianap-lis, IN, USA). All studies were approved by the animalare and use committee at Central Arkansas Veterans

ealthcare System. The rats were maintained in cages in the S

nimal care facility. The rats were subjected to alternate2-h periods of a dark/light cycle. During the study, all ratsere pair-fed the purified research diet TD96163 (Harlaneklad, Madison, WI, USA) and given water ad libitum.air-feeding is necessary to balance the chow intake among

he groups to ensure an isonitrogenous and isocaloric diet,ecause the tumor and/or various treatments may depressood intake. The TD96163 chow was used to avoid theffects of artificial antioxidants and variations often seen intandard chow of mineral elements and vitamins, becausehis study examined the effect of the antioxidant GSH.urthermore, TD96163 contains 1.84% of its protein asLN; thus no group is GLN deprived. In addition, rats

andomly received GLN (Sigma Chemical Co., St. Louis,O, USA) at 1 g · kg�1 · d�1 or an isonitrogenous

mount of Freamine (FA; McGaw, St. Louis, MO, USA) orater (H2O) by gavage. Animals were gavaged three timesday. Delivery three times a day kept gavage volume low,revented distress, and maintained peak blood levels ofLN, which occur at 2 to 3 h after administration. FA is aixture of essential and non-essential amino acids, which

oes not contain GLN. Diets were isonitrogenous and iso-aloric.

tudy procedure

One hundred ninety-eight female Sprague-Dawley ratsere randomized to one of six groups: DMBA�GLN,MBA�FA, DMBA�H2O, OIL�GLN, OIL�FA, orIL�H2O. Each group was further divided to 1-, 2-, 4-, and1-wk groups. In this study, the design was to examinenimals at the different time points during the entire carci-ogenic process. The week-1 group, which was 1 wk afterMBA administration, was chosen because it has been

eported that NK cell activity is suppressed by DMBA athat time [9]. The week-2 and �4 groups were selected tonvestigate the middle of the process. The week-11 groupas picked to study the endpoint of the process; by this time

he control groups completed the tumor growth. At age 50 d,he pubertal rats were gavaged with a one-time dose of 20g of DMBA (Sigma Chemical Co.) in 1 mL of sesame oil

r 1 mL of sesame oil alone. Mammary tumor developmentas assessed by weekly palpating the mammary glands of

ats in all groups and recording tumor incidence, rate, loca-ion, number, and size. Rats were sacrificed at 1, 2, 4, and 11k after DMBA administration. At sacrifice, anesthesia wasbtained with 50 mg/kg of pentobarbital sodium (Nembutal,bbott Laboratories, Stone Mountain, GA, USA) by intra-eritoneal injection. Nembutal was used because it has beeneported that it has no effect on NK cell activation [17]. Thebdomen was prepped with povidone-iodine, the aorta andortal vein were exposed through a midline incision, and theats were heparinized. Blood was withdrawn from the aortand portal vein using a 25-gauge needle attached to a 1-mLyringe. Blood was processed for GLN and GSH content.

pleens were aseptically removed and assayed for NK cy-

tbt

P

G

G[m[ea

G

tGw

tr

N

ssgpatmpfm(pat(ccidaYd

2e

T

ut(

C

e0SC

R

N

wggdigTfpadgFa

T

GFoidwHwnda

464 Y. Kaufmann et al. / Nutrition 24 (2008) 462–469

otoxicity using a 51Cr release assay. Jejunum mucosa andreast tissues were processed for GSH concentration. Finalumor weight and volume were measured.

rocessing of samples/analytical procedure

LN and GSH determinationsThe preparations of whole blood or tissue for GLN and

SH determination were described in previous publications15,16]. The GLN concentration was determined by theicroanalytical method described by Bernt and Bergmeyer

18]. The GSH concentration was determined by a standardnzymatic recycling procedure, as described by Tietze [19]nd modified by Anderson [20].

ut GLN and GSH extraction calculationGut extraction is defined as the difference of concentra-

ions across the gut. Using the data of arterial and portalLN and GSH contents, the gut GLN and GSH extractionsere calculated using the following equation:

Extraction (%)

�[GLN or GSH]arterial � [GLN or GSH]portal

[GLN or GSH]arterial� 100%

A positive extraction is proportional to fractional up-ake. A negative extraction is proportional to fractionalelease.

K cell cytotoxicity determination

Aseptically removed spleens were minced using a sterilecalpel, and lymphocytes were teased from the splenic cap-ule with warm RPMI-1640 (Gibco BRL, Life Technolo-ies Inc., Grand Island, NY, USA). The resulting cells wereoured into 50-mL conical tubes, which were filled withmmonium chloride (0.83%; Sigma Chemical Co.) to lysehe red cells. The cell solution was then centrifuged for 10in at 1000 rpm. The supernatant was decanted, and the cell

ellet was Vortex-mixed with RPMI (no GLN) and centri-uged again. Next, the cells were resuspended in approxi-ately 10 mL of RPMI containing 10% fetal bovine serum

Gibco BRL) supplemented with 1% GLN. This cell sus-ension was placed in Petri dishes and incubated in 6% CO2

t 37°C for 30 min. After incubation to ensure adherence ofhe monocytes, the non-adherent lymphocytes were counted1:1 with crystal violet stain). Approximately 7 � 106 totalells from each spleen were incubated for 3 d in RPMIontaining 10% fetal bovine serum and recombinant humannterleukin-2 (500 U/mL). This cell suspension was used foretermination of NK cell cytotoxicity by a 4-h 51Cr releasessay with the NK cell–sensitive mouse tumor cell line,AC-1. The NK cytotoxicity is expressed in lytic units,

efined as the number of effector cells per 106 mediating o

0% target cell lysis. The calculation of the NK activity isxpressed as the following equation:

Percent specific lysis (%)

�Experimental release � Spontaneous release

Maximum release � Spontaneous release� 100%

umor measurementTumors were weighed and tumor volumes measured

sing Vernier calipers. Tumor volume was calculated usinghe formula for a prolate sphere: �/6 � (larger diameter)2 �smaller diameter).

alculations/statistical analysis

All data are expressed as mean � standard error. Differ-nces between means were considered significant at the P �.05 level using analysis of variance and Fisher’s exact testing.tatistical analyses were performed using StatView II (Abacusoncepts Inc., Berkley, CA, USA).

esults

utrition

There was no significant difference in the mean bodyeight among groups at the beginning of the study or at anyiven sacrifice time; however, there was an increase in eachroup over time (data not shown). All rats gained weighturing the study period. Due to the pair-feeding, chowntake was similar and not significantly different amongroups during any given study period (data not shown).otal nitrogen intake from chow and gavage was not dif-

erent between the GLN and FA groups during the gavageeriod. For these two groups, the diets were isonitrogenousnd isocaloric. The H2O-gavaged groups were added toetermine the effect of other amino acids. All H2O-gavagedroups received less nitrogen compared with GLN- andA-supplemented animals, but diets were isonitrogenousnd isocaloric among all H2O-fed groups.

umorigenesis

Over the 11-wk study period, tumor formation in theLN-gavaged DMBA-treaded rats was 50% less than in theA�DMBA or H2O�DMBA groups where all rats devel-ped tumors (Fig. 1). In addition, there was no tumor seenn any groups before week 5. The tumor growth initiallyelayed 1 wk, then kept slow, and reached significance ateek 11 in the GLN-fed group compared with the FA- and

2O-fed groups (Fig. 1). These differences were associatedith tumor weight and size differences between GLN- andon–GLN-fed animals (Table 1). There were significantecreases in tumor volume and weight in GLN-gavagednimals. Tumors in all groups of animals were less than 5%

f total body weight at sacrifice.

A

fntwDwmTcl2DsOdgGG

G

u

sogaGGe

rOngarar

FGd

FDltcD

Favtv

TT

GFH

465Y. Kaufmann et al. / Nutrition 24 (2008) 462–469

rterial GLN and GSH metabolism

The GLN supplementation elevated arterial GLN levelsrom 20% to 45% over the FA-fed and H2O-fed groups inon–DMBA- or DMBA-treated animals. The similar pat-ern was found at every time point (data not shown). Thereas a significant reduction of arterial GLN level in theMBA�H2O group compared with the OIL�H2O group ateek 1 but not at late time points; however, GLN supple-entation brought it back up to the normal range (Fig. 2).hese differences in GLN levels were associated with GSHoncentration differences in arterial blood. The arterial GSHevels of the animals given GLN were increased on average0% compared with those fed FA and H2O in the non–MBA- or DMBA-treated groups over the time (data not

hown). In the DMBA-treated groups compared with theIL-treated control groups, the arterial GSH levels wereecreased from 20% to 40% in the FA- and H2O-fedroups; however, these were normal or elevated in theLN-supplemented animals and only significantly less thanLN-controls at week 11 (Fig. 3).

ut GLN and GSH metabolism

Positive gut GLN extractions (proportional to fractionalptake) were seen in all control (non-DMBA) groups; GLN

ig. 1. Percentages of tumor formation during study period are presented.LN significantly inhibits the tumor formation by DMBA. DMBA, 7,12-imethylbenz[a]anthracene; FA, Freamine; GLN, glutamine.

able 1umor volume and weight

Tumor volume (mL) Tumor weight (g)

LN 0.23 � 0.15* 0.16 � 0.11*A 2.30 � 0.61 1.32 � 0.54

2O 4.30 � 1.10 2.55 � 1.12

FA, Freamine; GLN, glutamine

g* P � 0.05, analysis of variance, GLN versus FA or H2O.

upplementation increased 30% to 50% the gut GLN uptakever FA and H2O groups (data not shown). In DMBAroups, there was a significant change at week 1. Thenimals supplemented with FA and H2O reversed the gutLN extraction from positive to negative. SupplementalLN restored normal positive extraction at week 1 and

levated it over controls at all other time point (Fig. 4).Negative gut GSH extractions (proportional to fractional

elease) were shown in all control (non-DMBA) groups.ral GLN increased the gut GSH extraction near 40% (dataot shown). Importantly, in the DMBA-treated groups, theut GSH extractions in the FA- and H2O-fed groups actu-lly reverted negative to positive early on at weeks 1 and 2,ecovered partially by week 4 when no group had tumor,nd returned to negative extraction at week 11, but onlyeached half of that seen in control groups. In contrast, the

ig. 2. Arterial blood GLN levels (micromoles per liter) at week 1 afterMBA are demonstrated. GLN-fed animals have significantly higher GLN

evels than FA-fed and H2O-fed animals in the DMBA- and non–DMBA-reated groups at this time point. DMBA administration significantly de-reases the arterial GLN level at this time. ANOVA, analysis of variance;MBA, 7,12-dimethylbenz[a]anthracene; FA, Freamine; GLN, glutamine.

ig. 3. Arterial blood glutathione (micromoles per liter) levels at week 11fter DMBA are shown. There are significant increases in GLN-fed groupsersus FA- and H2O-fed groups. There are significant decreases in DMBA-reated groups versus non–DMBA-treated groups. ANOVA, analysis ofariance; DMBA, 7,12-dimethylbenz[a]anthracene; FA, Freamine; GLN,

lutamine.

st

G

oDth

B

tamAitc

N

ctttHcchci

D

powgstgwwb

iDeoaeoaotatftcnd

sa

FptDD

Fsfwng

466 Y. Kaufmann et al. / Nutrition 24 (2008) 462–469

upplementation of GLN restored these changes and kepthe negative gut GSH extraction at all time points (Fig. 5).

ut mucosa GSH concentration

Oral GLN significantly elevated intracellular gut GSHver FA- and H2O-fed animals in the DMBA- and non–MBA-treated groups. In the initial weeks after DMBA

reatment, intracellular gut GSH levels in all groups wereigher than those in non-DMBA groups (Table 2).

reast tissue GSH concentration

Oral GLN significantly increased intracellular GSH inhe breast among all tested groups. At week 1 after DMBAdministration, each different group gavaged with DMBA re-ained the same as that of the controls without DMBA.fter week 2, DMBA significantly decreased GSH content

n FA- and H2O-treated groups. However, oral GLN main-ained GSH levels at the normal range seen in the H2Oontrol groups (Table 3).

K cell cytotoxicity

The NK cell cytotoxicities were examined over time. NKell cytotoxicities were significantly lower in the DMBA-reated groups at weeks 1 and 11 after DMBA adminis-ration. However, NK cell activities were elevated inhe GLN�DMBA group over the FA�DMBA and

2O�DMBA groups at these time points but only signifi-antly so at week 11. At week 2, there was a rebound of NKell cytotoxicity in DMBA-treated animals, which wasigher than in non-DMBA controls. At weeks 4 and 11, NKell activities were increased in control non-DMBA animals

ig. 4. Gut GLN extractions (percentage) of DMBA groups over specificeriods are presented. Under a stress state, which is DMBA treatment inhis study, GLN remains and even elevates gut GLN uptake, althoughMBA changes the uptake early on. ANOVA, analysis of variance;MBA, 7,12-dimethylbenz[a]anthracene; FA, Freamine; GLN, glutamine.

ncluding all GLN-, FA-, and H2O-fed groups (Fig. 6). D

iscussion

Over the 11-wk study period, we demonstrated that sup-lemental oral GLN significantly inhibited the developmentf the DMBA-induced mammary carcinoma as comparedith tumor growth in the FA- and H2O-supplementedroups, where all rats developed tumors. As expected, theignificantly decreased tumor size and weight were seen inhe GLN-fed group compared with the FA- and H2O-fedroups. There was a trend toward decreased tumor size andeight in the FA group, but it did not reach significance,hich indicated that other amino acids did not reduce tumorurden.

One mechanism of DMBA carcinogenesis is that DMBAs metabolized through an oxidative process that damagesNA. DMBA can be metabolized to an electrophilic diol

poxide, a radical cation, or a reactive and redox active-quinone that is incorporated into DNA to form stablend/or depurination DNA adducts [21], which cause annhanced early expression of mutated c-Ha-ras and c-erb Bncogene later [22,23]. Therefore, the administration ofntioxidants may retard the processes. Several mechanismsf these antioxidants have been implicated [24]. Most ofhem are the antioxidations by upregulation of GSH contentnd GSH-related enzymes [5–7,25]. GSH is a ubiquitoushiol-containing tripeptide and has many diverse biologicunctions. GSH as an antioxidant participates in detoxifica-ion and protects the organism from oxidative injuries byonjugating electrophilic/oxidizing substances. GSH as aon-antioxidant modulates diverse cell redox signal trans-uction, cell proliferation, and immune response [26,27].

The present study has shown that GLN supplementationignificantly elevated the arterial GLN levels in the DMBAnd non-DMBA groups at every time point and even main-

ig. 5. Gut glutathione extractions (percentage) of DMBA groups arehown. DMBA reverses the gut GSH extraction from negative to positiveor up to 2 wk, returns to negative at week 4, and stays low negative untileek 11. In contrast, oral GLN keeps negative extraction and reaches theormal range. The dashed line across the plot represents changes in gutlutathione extraction over the time points. ANOVA, analysis of variance;

MBA, 7,12-dimethylbenz[a]anthracene; FA, Freamine; GLN, glutamine.

tewlsFbwiGFmiebtsrnittbbsctl

Dt(

ntIGsGmitTeatrwse

torku

TG

G

GFH

c

contr

TB

GFH

i

467Y. Kaufmann et al. / Nutrition 24 (2008) 462–469

ained arterial GLN levels at a normal range that was low-red by DMBA at week 1. Increases in arterial GLN levelsere associated with significant increases in arterial GSH

evels seen in all GLN-fed groups over that time. DMBAignificantly reduced the arterial GSH concentrations in theA- and H2O-fed groups all the time; however, oral GLNrought the GSH levels back up to the normal range, whichas seen in the control groups. At week 11, the GSH level

n GLN�DMBA group was lower than that in theLN�OIL group but still higher than those in theA�DMBA and H2O�DMBA groups, because some ani-als in this group had tumor at this time. In addition, the

ntracellular GSH concentration in target tissue (breast) wasxamined. GLN supplementation significantly increased thereast GSH concentration at every time point. The inhibi-ion of DMBA on GSH concentration in breast tissue washown 2 wk after the DMBA dose. However, oral GLNestored GSH concentration to the normal level seen in theon-DMBA controls. These results indicate that DMBA andts induced tumor deplete arterial and breast GSH concen-rations, which can be restored by oral GLN supplementa-ion. Furthermore, the increased difference of GSH contentetween the GLN- and the FA- or the H2O-treated groups inreast tissue was much more than that in arterial blood. Thisuggests that GLN affects GSH more in local tissue than inirculating blood. These findings support our hypothesishat the carcinogenic effect of DMBA may be mediated, ateast in part, by oxidation. In addition, GLN may reduce the

able 2ut mucosa tissue GSH concentration

SH (�mol/g tissue) DMBA

Week 1 Week 2

LN 1.88 � 0.18* 1.76 � 0.13*A 1.14 � 0.22† 0.85 � 0.15

2O 1.21 � 0.24‡ 0.79 � 0.26

DMBA, 7,12-dimethylbenz[a]anthracene; FA, Freamine; GLN, glutami* P � 0.05, analysis of variance, GLN versus FA or H2O in DMBA we

ontrol.† P � 0.05, analysis of variance, FA in DMBA week 1 versus FA in c‡ P � 0.05, analysis of variance, H2O in DMBA week 1 versus H2O in

able 3reast tissue GSH concentration

GSH (nmol/g tissue) DMBA

Week 1 Week 2

LN 320.3 � 34.9* 75.9 � 5.3*A 158.1 � 18.8 21.1 � 6.1†

2O 95.7 � 20.4 15.2 � 7.6‡

DMBA, 7,12-dimethylbenz[a]anthracene; FA, Freamine; GLN, glutami* P � 0.05, analysis of variance, GLN versus FA or H2O in DMBA we

n DMBA week 1 and control.† P � 0.05, analysis of variance, FA in DMBA weeks 2, 4, and 11 ver

‡ P � 0.05, analysis of variance, H2O in DMBA weeks 2, 4, and 11 versus H

MBA-induced carcinogenesis by increasing GSH levels inhe circulating bloodstream (arterial blood) and target tissuebreast).

The gut is recognized not only by its traditional role inutrient absorption and digestion but also as having impor-ant metabolic, immunologic, and barrier functions [28,29].nterestingly our laboratory discovered that the gut was aSH producer under normal conditions and tumor-bearing

tate, and the gut’s production of GSH was increased byLN supplementation [15,16]. The gut GSH metabolismay play an important role in prevention of chemically-

nduced carcinogenesis. It controls the supply of GSH forhe liver where most DNA adducts occur or are activated.herefore, in this study we examined the gut GLN and GSHxtractions in all groups over time. We made the samessumption as previously, that the positive or negative ex-raction represents fractional uptake or fractional release,espectively, because it is unlikely that GLN or FA dietsould have had any effect in the blood flow. A previous

tudy has shown no difference in blood flow between GLN-nriched and GLN-free animals [30].

The gut GLN extraction results from this study indicatehat the gut took up GLN under normal conditions and thatral GLN increased gut GLN uptake. DMBA dramaticallyeversed GLN uptake only at week 1. Oral GLN not onlyept GLN uptake at that time but also kept the higher GLNptake for the remaining time.

Oil (control)

Week 4 Week 11

1.29 � 0.09* 1.01 � 0.05* 1.02 � 0.08*0.60 � 0.09 0.66 � 0.09 0.63 � 0.080.78 � 0.22 0.64 � 0.11 0.53 � 0.07

H, glutathione, 2, 4, and 11 and control, GLN in DMBA weeks 1 and 2 versus GLN in

ol.

Oil (control)

Week 4 Week 11

80.3 � 10.3* 135.6 � 16.6* 278.5 � 38.6*18.1 � 3.1† 24.5 � 6.1† 138.7 � 19.917.7 � 2.6‡ 26.6 � 5.8‡ 107.7 � 23.9

H, glutathione2, 4, and 11 and control, GLN in DMBA weeks 2, 4, and 11 versus GLN

in DMBA week 1 and control.

ne; GSeks 1

ontrol.

ne; GSeks 1,

sus FA

2O in DMBA week 1 and control.

scrptnnptmbmbtp

wbttcDsaa

nsnaNahi

tstsiaDnfttTng

C

icttic

A

os

FDGA FA, Fr

468 Y. Kaufmann et al. / Nutrition 24 (2008) 462–469

The gut GSH extraction results from this study demon-trate that normally the gut released GSH. Oral GLN in-reased this gut GSH fractional release. However, DMBAeversed the gut GSH extraction from normal negative toositive extraction to totally block the gut GSH release upo 4 wk after the DMBA dose, then gradually returned toegative extraction, but continued to be lower than theormal controls, which was thought to be caused by theresence of tumor. This block leads to a decreased supply ofhe important antioxidant GSH to the liver at a time when itay be crucial for prevention of oxidative damage produced

y DMBA and at a place where most DMBA metabolismay be activated and further result in DNA adducts in the

reast. In contrast, the supplementation of oral GLN main-ained the normal GSH release and restored the gut GSHroduction at all time points.

Because DMBA dramatically changed GSH metabolism,e expected intracellular gut GSH in the DMBA group toe decreased. However, in the initial weeks after DMBAreatment, the intracellular gut GSH concentration was ac-ually significantly elevated over that of the non-DMBAontrol group. This result supports the above finding thatMBA had a prolonged block in gut GSH production and

uggests that DMBA could affect the GSH transporternd/or GSH synthesis-related enzyme. Further experimentsre needed to elucidate this suggestion.

Another possible mechanism for DMBA-induced carci-ogenesis is by suppression of NK cell activity. NK cells, aubpopulation of cytotoxic lymphocytes that is present inormal individuals, are capable of spontaneous cytolyticctivity against a variety of tumor cells [31]. Unlike T cells,K cells cause non-specific tumor cell lysis spontaneously

t first contact [32]. Activated NK cells participate in otherost functions through their production of cytokines such as

ig. 6. Natural killer cell cytotoxicity (lytic units) for all groups over tMBA-treated group early on. In addition, there is significantly low NK cLN increases this activity and remains in the normal range. A lytic unit iNOVA, analysis of variance; DMBA, 7,12-dimethylbenz[a]anthracene;

nterferon-� [33]. d

In this study, NK cell activities were examined overime. The suppression of NK cell activity by DMBA waseen at weeks 1 and 11. GLN elevated NK cell activity overhe FA- and H2O-fed groups at these time points but onlyignificantly so at week 11, when tumor burden was signif-cantly higher in the DMBA-treated controls. At week 2, wectually observed a rebound of NK cell cytotoxicity inMBA-treated animals, which was even higher than in theon-DMBA control groups. Therefore, DMBA only af-ected NK activity at an early stage after DMBA adminis-ration and at a late stage. Supplemental oral GLN reversedhe decrease in NK cell activity by DMBA only at week 11.his indicated that the change of NK activity over time didot best correlate with the inhibition of DMBA’s carcino-enesis by GLN.

onclusion

Dietary GLN reduced DMBA-induced mammary tumor-genesis by 50%. This reduction was related to increasedirculating and target tissue GSH concentrations. Impor-antly, it most correlated with differences in gut GSH frac-ional release, which DMBA disturbed while GLN normal-zed. This finding provides important information forlarifying the mechanism of GLN anticancer action.

cknowledgments

The authors thank Dr. Jacki Kornbluth in the Departmentf Pathology at St. Louis University School of Medicine forharing her knowledge of natural killer cell cytotoxicity

presented. There is significantly low natural killer cell activity in theivity in the DMBA-treated group at the end of the study, and at this timeed as the number of effector cells per 106 mediating 20% target cell lysis.eamine; GLN, glutamine; LU, lytic units.

ime isell act

s defin

etermination.

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

469Y. Kaufmann et al. / Nutrition 24 (2008) 462–469

eferences

[1] Fahr MJ, Kornbluth J, Blossom S, Schaeffer R, Klimberg VS. Glu-tamine enhances immunoregulation of tumor growth. JPEN 1994;18:471–6.

[2] Klimberg VS, Kornbluth J, Cao Y, Dang A, Blossom S, Schaeffer F.Glutamine suppresses PGE2 synthesis and breast cancer growth.J Surg Res 1996;63:293–7.

[3] Lim V, Korourian S, Cao Y, Hanna E, Klimberg VS. Glutamineprevents DMBA-induced squamous cell cancer. Paper presented atthe 31st Annual Association for Academic Surgery; Dallas, Texas;November 1997.

[4] Huggins C, Grand LC, Brillantes FP. Mammary cancer induced by asingle feeding of polynuclear hydrocarbons, and its suppression.Nature 1961;189:204–7.

[5] Padmavathi R, Senthilnathan P, Chodon D, Sakthisekaran D. Thera-peutic effect of paclitaxel and propolis on lipid peroxidation andantioxidant system in 7,12 dimethyl benz[a]anthracene-inducedbreast cancer in female Sprague Dawley rats. Life Sci 2006;78:2820–5.

[6] Mohan KV, Subapriya R, Hara Y, Nagini S. Enhancement of eryth-rocyte antioxidants by green and black tea polyphenols during 7,12dimethyl benz[a]anthracene-induced hamster buccal carcinogenesis.J Med Food 2006;9:373–7.

[7] Cavin C, Holzhaeuser D, Scharf G, Constable A, Huber WW, SchilterB. Cafestol and kahweol, two coffee specific diterpenes with anticar-cinogenic activity. Food Chem Toxicol 2002;40:1155–63.

[8] Johnson AT, Kaufmann Y, Luo S, Babb K, Hawk R, Klimberg VS.Gut glutathione metabolism and changes with 7,12-DMBA and glu-tamine. J Surg Res 2003;115:242–6.

[9] Kimber I, Jones K, Vignali DA. The influence of DMBA on NK cellfunction in rats. J Clin Lab Immunol 1986;20:193–8.

10] Spear AT, Sherman AR. Iron deficiency alters DMBA-induced tumorburden and natural killer cell cytotoxicity in rats. J Nutr 1992;122:46–55.

11] Bergstrom J, Furst P, Noree LO, Vinnars E. Intracellular free aminoacid concentration in human muscle tissue. J Appl Physiol 1974;36:693–7.

12] Klimberg VS. Glutamine, cancer, and its therapy. Am J Surg 1996;172:418–24.

13] Amores-Sanchez MI, Medina A. Glutamine, as a precursor of gluta-thione, and oxidative stress. Mol Genet Metab 1999;67:100–5.

14] Welbourne TC. Ammonia production and glutamine incorporationinto glutathione in the functioning rat kidney. Can J Biochem 1979;57:233–7.

15] Cao Y, Feng Z, Hoos A, Klimberg VS. Glutamine enhances gutglutathione production. JPEN 1998;22:224–7.

16] Kaufmann Y, Klimberg VS. Effect of glutamine on gut glutathionefractional release in the implanted tumor model. Nutr Cancer 2007;

59(2):199–206.

17] Markovic SN, Murasko DM. Anesthesia inhibits poly I:C inducedstimulation of natural killer cell cytotoxicity in mice. Clin ImmunolImmunopathol 1990;56:202–9.

18] Bernt E, Bergmeyer HU. L-glutamate UV-assay with glutamate de-hydrogenase and NAD. In: Bergmeyer HU, editor. Methods of enzy-matic analysis. Volume 4. 2nd English ed. Deerfield Beach, FL:Verlag Chemie International; 1974, p. 1704–8.

19] Tietze F. Enzymatic method for quantitative determination of nano-gram amounts of total and oxidized glutathione: applications to mam-malian blood and other tissues. Ann Biochem 1969;27:502–22.

20] Anderson ME. Enzymatic and chemical methods for the determina-tion of glutathione. In: Dolphin D, editor. Glutathione. Volume 1(83).New York: John Wiley & Sons; 1983, p. 340–65.

21] Penning TM, Burczynski ME, Hung C-F, McCoull KD, Palackal NT,Tsuruda LS. Dihydrodiol dehydrogenases and polycyclic aromatichydrocarbon activation: generation of reactive and redox activeo-quinones. Chem Res Toxicol 1999;12:1–18.

22] Husain Z, Fei YB, Roy S, Solt DB, Polverini PJ, Biswas DK.Sequential expression and cooperative interaction of c-Ha-ras andc-erb B genes in vivo chemical carcinogenesis. Proc Natl Acad SciUSA 1989;86:1264–8.

23] Chakravarti D, Penlling JC, Cavalieri EL, Rogan EG. Relating aro-matic hydrocarbon-induced DNA adducts and c-H-ras mutations inmouse skin papillomas: the role of apurinic sites. Proc Natl Acad SciUSA 1995;92:10422–6.

24] Slaga TJ. Inhibition of the induction of cancer by antioxidants. AdvExp Med Biol 1995;369:167–74.

25] Desai VG, Casciano D, Feuers RJ, Aidoo A. Activity profile ofglutathione-dependent enzymes and respiratory chain complexes inrats supplemented with antioxidants and treated with carcinogens.Arch Biochem Biophys 2001;394:255–64.

26] Locigno R, Castronovo V. Reduced glutathione system: role in cancerdevelopment, prevention and treatment. Int J Oncol 2001;19:221–36.

27] Dickinson DA, Forman HJ. Glutathione in defense and signaling:lessons from a small thiol. Ann NY Acad Sci 2002;973:488–504.

28] Wilmore DW, Smith RJ, O’Dwyer ST, Jacobs DO, Ziegler TR, WangXD. The gut: a central organ after surgical stress. Surgery 1988;104:917–23.

29] Schneeman B. Food factors and gastrointestinal function: a criticalinterface. Biofactors 2004;21(1–4):85–8.

30] Klimberg VS, Souba WW, Salloum RM, Plumley DA, Cohen FS,Dolson DJ, et al. Glutamine-enriched diets support muscle glutaminemetabolism without stimulating tumor growth. J Surg Res 1990;48:319–23.

31] Herberman RB, Ortaldo JR. Natural killer cell: their role in defensesagainst disease. Science 1981;214:24–30.

32] Trinchieri GB, Penussia B. Human natural killer cells: biologic andpathologic aspects. Lab Invest 1984;50:489–513.

33] Kasahara T, Djeu J, Dougherty S, Oppenheim J. Capacity of humanlarge granular lymphocytes (LGL) to produce multiple lymphokines:interleukin-2, interferon and colony stimulating factor. J Immunol

1983;131:2379–85.