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Nutrition 28 (2012) 1165–1171

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Nutrition

journal homepage: www.nutr i t ionjrnl .com

Basic nutritional investigation

Fetal programming of dietary fructose and saturated fat on hepaticquercetin glucuronidation in rats

Aida Serra Ph.D. a,b, Nyssa Bryant B.S. a, Maria Jos�e Motilva Ph.D. b, Jeffrey B. Blumberg Ph.D. a,C.-Y. Oliver Chen Ph.D. a,*aAntioxidants Research Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, USAbDepartment of Food Technology, XaRTA-UTPV, Escola T�ecnica Superior d’Enginyeria Agr�aria, Universitat de Lleida, Lleida, Spain

a r t i c l e i n f o

Article history:Received 8 February 2012Accepted 18 April 2012

Keywords:QuercetinRatUDP-glucuronosyltransferaseFetal programmingFructoseSaturated fats

This study was supported by USDA ARS Cooperative Aand by the Catalan Government (Interdepartmentaland Technological Innovation).* Corresponding author. Tel.: þ617-556-3128; fax:

E-mail address: oliver.chen@tufts.edu (C.-Y. Oliver

0899-9007/$ - see front matter � 2012 Elsevier Inc. Adoi:10.1016/j.nut.2012.04.004

a b s t r a c t

Objective: Phase II biotransformation of flavonoids generates bioactive metabolites in vivo.However, data on the effect of environmental and physiologic factors and fetal programming onphase II pathways toward flavonoids are limited. We examined the effect of parental exposure toa diet high in saturated fats and fructose 1 mo before conception through lactation on in vitrohepatic uridine 50-diphosphate (UDP)-glucuronosyltransferase (UGT) activity toward quercetin inparent and offspring rats and the interaction between diet and sex.Methods: Parents were fed a diet containing 9.9% coconut fat, 0.5% cholesterol, 30% fructose, and30% glucose (SFF) or a control (C) diet containing 11% corn oil and 60% glucose. After weaning,offspring were fed the C diet for an additional 12 wk. The glucuronidation rate of microsomalUGT was determined with quercetin 30 mmol/L and 12.5 mg of protein in a total volume of 100 mLafter a 15-min incubation at 37�C. Three quercetin glucuronides (7-O-quercetin glucuronide, 40-O-quercetin glucuronide, and 30-O-quercetin glucuronide) were quantified.Results: In the parent females, the SFF diet decreased by 29% and 19% the production rate of 30- and40-O-quercetin glucuronide quercetin glucuronides, respectively, compared with the C diet (P �0.05). The production rate of 7-O-quercetin glucuronide quercetin glucuronide in the femaleoffspring rats born to C dams was 59% larger than in their male counterparts (P < 0.05), but nodifference was observed in the offspring of SFF dams.Conclusion: High dietary fructose and saturated fat decreased UGT capacity toward quercetin infemale rats and in utero exposure to the diet decreased the glucuronidation capacity of their pups.

� 2012 Elsevier Inc. All rights reserved.

Introduction

Flavonoids are ubiquitous in plant foods and products derivedfrom them, e.g., fruits, vegetables, tea, and red wine, and obser-vational and clinical evidence has shown that their intake isinversely associated with the risk of chronic diseases, e.g., certaincancers, cardiovascular disease, and neurodegenerative disorders[1,2]. Although the bioavailability, metabolism, and bioactions offlavonoids have been partly characterized, these results reveala marked interindividual variation [3], likely because of a combi-nation of environmental, physiologic, epigenetic, and genetic

greement 58-1950-7-707Commission for Research

þ617-556-3344.Chen).

ll rights reserved.

factors. Because most consumed flavonoids are extensivelytransformed by phase IImetabolic pathways, e.g., glucuronidation,sulfation, and methylation, that facilitate their elimination [4], thevariation in the capacity of phase II enzymes toward flavonoidsmay be partly responsible for this interindividual variation inbioavailability.

The capacity of phase II metabolism toward flavonoids issubject to the influence of environmental and physiologic factorsand polymorphisms. We previously demonstrated that age andsex affect hepatic and intestinal uridine 50-diphosphate (UDP)-glucuronyltransferase (UGT) activity toward flavonoids [5,6].Phytochemicals, including carotenoids, polyphenols (includingflavonoids), indoles, and allyl sulfides have also been shownmodulate the activities of phase II enzymes [7]. Although dia-betes, obesity, dietary protein, and energy have been reported tomodulate liver drug-metabolizing phase I enzyme composition[8,9], relatively little information is available regarding the impact

A. Serra et al. / Nutrition 28 (2012) 1165–11711166

of nutritional status, lifestyle, and pathologic conditions on phaseII metabolism [10]. Thus, a greater characterization is warrantedof the factors that modulate the capacity of phase II metabolismtoward flavonoids to help us understand their mechanisms ofaction in promoting health.

A growing body of evidence indicates that fetal and neonatalexposure to poor nutrition and environment can modulatemetabolic homeostasis and predispose individuals to an increasedrisk of diseases later in life through epigeneticmechanisms [11]. Toour knowledge, there have been no investigations on the impact ofnutrient exposure in utero or during early postnatal life on thecapacity of phase II metabolism. However, Guillemette et al. [12]suggested that epigenetic modifications, such as DNA methyla-tion and histone acetylation, might regulate UGT gene expression.

The typical Western diet, high in saturated fats and sugars,appears to modify fetal programming and lead to a predispositionfor the risk of obesity and metabolic abnormalities whenthe offspring reach adulthood [13,14]. Because the ontogeneticdevelopment of hepatic UGT occurs in early life [15], we investi-gated in rats the fetal programming effect of a diet high in satu-rated fats and fructose on hepatic UGT activity toward quercetin,the most abundant dietary flavonol. We also examined the impactof this diet on hepatic UGTactivity in breeding pairs of rats. In bothgenerations, the comparison between sexes on the hepatic UGTactivity toward quercetin was also investigated.

Materials and methods

Chemicals and reagents

High-performance liquid chromatographic grade acetonitrile and methanolwere obtained from Fisher Scientific (Thermo Fisher Scientific, Rockford, IL, USA).Quercetin dihydrate, UDP-glucuronic acid, alamethicin, and all other chemicaland reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Animals and diets

The detailed information about the diet can be found in our previous report[16]. Briefly, two diets, control (C) and saturated fat plus fructose (SFF), wereprepared using amino acid-defined ingredients and manufactured by Dyets Inc.(Bethlehem, PA, USA). The main difference between the two diets was 60%glucose in the C diet versus 30% glucose and 30% fructose in the SFF diet and 11%corn oil in the C diet versus 9.9% coconut fat and 0.5% cholesterol in the SFF diet.

Proven male and female Sprague-Dawley breeders (eight male and eightfemale rats) were obtained fromCharles River Laboratories (Wilmington, MA, USA).After arrival, female and male rats were assigned randomly to one of the two diets(Fig. 1). All rats were housed singly with a 12-h/12-h light/dark cycle and fed adlibitum for 4 wk. Subsequently, each female rat was mated with a male rat in thesame dietary group. During the pregnancy and lactation, dams remained ontheir designated diet. Litter size was adjusted 3 d postpartum to 8 to 10 to ensurethat each pup received comparable nutrition. At weaning (day 21), five male andfemale offspring from each group were fed with the C diet for an additional 12 wk.

Fig. 1. Study design. Rats in the F0 generation were sacrificed at weaning and in theF1 generation sacrificed at 15 wk of age. C, control; F, female; F0, parent generation;F1, offspring; M, male; SFF, fructose and saturated fats.

At weaning, parental rats (F0 generation) were sacrificed by terminal exsanguina-tion under isoflurane (Aerrane, Baxter, Cambridge, MA, USA) anesthesia. At the ageof 15 wk, all offspring (F1 generation) were sacrificed by the same protocol. Liverswere harvested and stored at�80�C until the collection of hepatic microsomes. Theprotocol was approved by the institutional animal care and use committee of theJean Mayer USDA Human Nutrition Research Center on Aging at Tufts University.

Preparation of liver microsomes

Hepatic microsomal fractions were prepared according to the method ofBolling et al. [5]. Briefly, the entire liver was pulverized in liquid nitrogen andstored at �80�C until homogenization in ice-cold sucrose homogenization bufferusing an SDT-1810 Tekman Tissumizer (Cincinnati, OH, USA) at 85% power for2 min. Microsomes were collected after centrifugation at 1000 � g for 15 min atroom temperature, 10 000 � g for 20 min at 4�C, and then at 100 000 � g for60 min at 4�C. The resulting microsomal fraction was suspended in 400 mL ofpotassium phosphate buffer (0.1 mol/L, pH 7.5, containing 20% glycerol). Themicrosomal protein content was determined with a Pierce BCA kit (ThermoFisher Scientific, Boston, MA, USA) and adjusted to 5 mg/mL with phosphatebuffer (0.1mol/L, pH 7.5). Themicrosomal suspensionwas aliquoted and stored at�80�C until use.

Glucuronidation assay

The glucuronidation assay was performed according to the method of Bollinget al. [5]. Briefly, the microsomal glucuronidation of quercetin was initiated withan incubation of microsomal protein and alamethicin at 37�C for 15 min inmicrocentrifuge tubes containing quercetin 30 mmol/L (final concentration). Thequercetin concentrationwas selected based on our previous study [5] because theconcentration was near the Km (the concentration of substrate that leads to half-maximal velocity) value of hepatic microsomal UGT in rats. A cofactor solution ofUDP-glucuronic acid was added to initiate the reaction in a final assay volume of100 mL. After incubation for the selected duration, ranging from 0 to 30 min at37�C, the reaction was terminated with 100 mL of ice-cold methanol containingdaidzein 33 mmol/L as an internal standard. The supernatant was dried undernitrogen gas at room temperature and then reconstituted in 50% methanol forhigh-performance liquid chromatographic analysis. Experiments to determinethe linearity of the glucuronidation reaction were performed with a pooledmicrosomal protein sample to establish the assayed condition of microsomalprotein concentration (5–25 mg/100 mL) and enzymatic reaction time (5–30 min).

Determination of quercetin glucuronides

Quercetin glucuronides generated from microsomal glucuronidation weredetermined using high-performance liquid chromatography, according to themethod of Bolling et al. [5]. Briefly, quercetin and its glucuronide metaboliteswere monitored at 370 nm after elution from a Phenomenex Synergi 10-mmHydro-RP80 250- � 4.6-mm column (Torrance, CA, USA). Three principal quer-cetin glucuronide metabolites were quantified, with the following retentiontimes: 11.5 min for 7-O-quercetin glucuronide (7-OH), 14.3 min for 40-O-quer-cetin glucuronide (40-OH), and 14.8 min for 30-O-quercetin glucuronide (30-OH).After normalization with the internal standard, daidzein, glucuronide metabo-lites were quantified using a standard curve of quercetin established in theabsence of uridine 50-diphospho-glucuronic acid (UDPGA).

Statistical analysis

Results are expressed as mean � standard error. The natural logarithmictransformation of data was applied before statistical analysis to normalize theunequal variance. The effects of generation, diet, and sex on the quercetinproduction rates catalyzed by UGT were assessed using a three-way analysis ofvariance (ANOVA) with interactions among the three independent variables.Because there were significant sex and diet interactions, the effects of sex anddiet and their interaction in the F0 and all groups combined were assessed usinga two-way ANOVA. When P values for the interaction between sex and diet were�0.05, post hoc analysis was performed using the Tukey honestly significantdifference test. The difference in the production rate of quercetin metabolitesbetween sexes in the F1 rats and between F0 and F1 rats was tested usingStudent’s t test. Pearson’s correlation testwas performed to assess the associationbetween production rates of quercetin metabolites. P � 0.05 was consideredstatistically significant. All statistical analyses were performed using JMP IN (SASInstitute, Cary, NC, USA).

Results

The enzymatic linearity of microsomal UGT activity towardquercetin 30 mmol/L was established in triplicate using a pooled

Table 1P-value summary of three-way analyses of variance

Source 7-OH 30-OH 40-OH TQG

Generation 0.753 �0.0001 �0.0001 0.030Sex 0.240 0.834 0.801 0.313Diet 0.055 0.083 0.210 0.053Generation � sex 0.724 0.091 0.072 0.271Generation � diet 0.630 0.884 0.651 0.860Sex � diet 0.001 0.015 0.038 0.001Generation � sex � diet 0.460 0.032 0.014 0.053

30-OH, 30-O-quercetin glucuronide; 40-OH, 40-O-quercetin glucuronide; 7-OH,7-O-quercetin glucuronide; TQG, total quercetin glucuronide metabolite

A. Serra et al. / Nutrition 28 (2012) 1165–1171 1167

sample with protein concentrations ranging from 5 to 25 mg ina 100-mL assay volume and incubation time from 5 to 30 min(Fig. 2). The r2 value for the two regression lines was >0.94.A decrease in the quercetin concentration was observed inaddition to an increase in microsomal protein concentration orincubation time and increases in concentrations of three quercetinmetabolites. The assay condition of 12.5 mg of protein/100 mL and15 min that fell in the middle of the linear range of the regressioncurvewas selected to test the effects of generation, diet, and sexonmicrosomal UGT activity toward quercetin 30 mmol/L.

Three quercetin glucuronide metabolites (7-, 30-, and 40-OH)were quantified. Hepatic UGT favored the production of 7-OH,followed by 30-OH. The effect of the generation on UGTactivity inthe production of 30-OH and 40-OH-quercetin glucuronides wasindependent of sex and diet (P � 0.0001; Table 1, Fig. 3). Therewere significant interactions between sex and diet on 7-OH,30-OH, and total quercetin glucuronidemetabolite (TQG), so two-way ANOVAs were performed to assess the relation between sexand diet on the production rate of quercetin UGT metabolites.Figures 4, 5, and 6 show the results of the effect of sex and diet on

Fig. 2. The linearity of UDP-glucuronosyltransferase activity toward 30 quercetinmmol/L during incubation with a microsomal protein concentration of 12.5 mg/100 mL (A) and microsomal protein content when the incubation time was set at15 min (B). The R2 values are calculated from the regression lines of quercetinmetabolites. The assay condition of 15-min incubation and microsomal protein12.5 mg/100 mL was used in all experiments. 30-OH, 30-O-quercetin glucuronide;40-OH, 40-O-quercetin glucuronide; 7-OH, 7-O-quercetin glucuronide; Gluc-Q,quercetin glucuronide metabolite.

the production rates of quercetin UGT metabolites in all rats inthe C and SFF, F0 rats, and F1 rats, respectively. In the combinedgenerations, in which the global effect of the SFF diet on UGTactivity was evaluated, the significant effect of diet on theproduction rate was dependent on sex and metabolites (Fig. 4).The P values of the sex-by-diet interaction for 7-OH and TQGproduced in the two-way ANOVAs were 0.0012 and 0.0022,respectively. For 30-OH and 40-OH glucuronides, the interactionof diet and sex was not statistically significant. The productionrates of 7-OH and TQG in C female rats were 63% and 33% largerthan in the C male rats, respectively (P � 0.05), whereas nodifference was found in the SFF rats. The diets did not affect theproduction rate of quercetin glucuronides in male rats. The SFFdiet induced decreases of 39% and 28% in the production rate of7-OH and TQG in female rats comparedwith the C diet (P� 0.05).

The magnitude of the diet and sex interaction in theproduction rate of quercetin glucuronides were more marked inthe F0 than in the F1 rats (Figs. 5, 6). The effect of the SFF diet onhepatic UGT activity was dependent on sex and quercetinmetabolite (Fig. 5), with P values (two-way ANOVA) for 7-OH,30-OH, 40-OH, and TQG of 0.0194, 0.0095, 0.0051, and 0.0063,respectively. The production rate of quercetin metabolites wasnot different between male and female rats fed the C diet. TheSFF diet led to a 22% lower production rate of 40-OH in femalethan in male rats, but not of the other metabolites. In female rats,the SFF diet resulted in 29%, 19%, and 35% decreases in theproduction rate of 30-OH, 40-OH, and TQG, respectively,compared with the C diet (P � 0.05).

In the F1 rats, the sex and parental diet interaction wassignificant for 7-OH and TQG (P¼ 0.0398 and 0.016, respectively,

Fig. 3. The UDP-glucuronosyltransferase activity of all rats toward quercetin isgeneration dependent. The production rate for 30- and 40-OH was larger in F1 ratsthan in F0 rats. * Means of the same metabolite differ (P � 0.0001, tested byStudent’s t test). 30-OH, 30-O-quercetin glucuronide; 40-OH, 40-O-quercetin glucu-ronide; 7-OH, 7-O-quercetin glucuronide; F0, parent generation; F1, offspring;T-GQ, total quercetin glucuronide metabolite.

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Fig. 4. The dietary effect on hepatic UDP-glucuronosyltransferase activity toward quercetin in all parent and offspring rats is sex and metabolite dependent: (A) 7-OH, (B) 30-OH, (C) 40-OH, (D) T-QG. Means with different letters in each panel differ (P � 0.05, tested by two-way analysis of variance followed by Tukey–Kramer highly significantdifference multicomparison). 30-OH, 30-O-quercetin glucuronide; 40-OH, 40-O-quercetin glucuronide; 7-OH, 7-O-quercetin glucuronide; C, control diet; F, female; M, male;SFF, fructose and saturated fat diet; T-GQ, total quercetin glucuronide metabolite.

A. Serra et al. / Nutrition 28 (2012) 1165–11711168

from two-way ANOVA; Fig. 6). Neither parental diet nor sexaffected the production rate of 30- and 40OH quercetin glucuro-nides. Because statistical significance was not found with theTukey multicomparison test, Student’s t test was performed toevaluate whether the means within each dietary group were

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Fig. 5. The dietary effect on hepatic UDP-glucuronosyltransferase activity toward quercetT-QG. Means with different letters in each panel differ (P � 0.05, tested by two-way acomparison. 30-OH, 30-O-quercetin glucuronide; 40-OH, 40-O-quercetin glucuronide; 7-Osaturated fat diet; T-GQ, total quercetin glucuronide metabolite.

different. The production rates of 7-OH quercetin glucuronideand TQG in the female rats born to C dams were 59% and 28%larger than in their male counterparts (P ¼ 0.035 and �0.05,respectively). No such difference was observed in the offspringborn to SFF dams.

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in in parent rats is sex and metabolite dependent: (A) 7-OH, (B) 30-OH, (C) 40-OH, (D)nalysis of variance followed by Tukey–Kramer highly significant difference multi-H, 7-O-quercetin glucuronide; C, control diet; F, female; M, male; SFF, fructose and

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Fig. 6. The effect of diets on hepatic UDP-glucuronosyltransferase activity toward quercetin in offspring rats is sex and metabolite dependent: (A) 7-OH, (B) 30-OH, (C) 40-OH,(D) T-QG. * Means in each panel differ (P � 0.05, tested by Student’s t test). 30-OH, 30-O-quercetin glucuronide; 40-OH, 40-O-quercetin glucuronide; 7-OH, 7-O-quercetinglucuronide; C, control diet; F, female; M, male; SFF, fructose and saturated fat diet; T-GQ, total quercetin glucuronide metabolite.

A. Serra et al. / Nutrition 28 (2012) 1165–1171 1169

A series of Pearson correlation tests was performed toprovide some insight into the impact of diet on UGT isoen-zymes. The magnitudes of the correlation between theproduction rates of quercetin metabolites follow: in all rats,TQG and 7-OH (r ¼ 0.91, P � 0.0001), TQG and 30-OH (r ¼ 0.83,P � 0.0001), TQG and 40-OH (r ¼ 0.62, P � 0.0001), 7-OH and30OH (r ¼ 0.53, P ¼ 0.0005), and 30-OH and 40-OH (r ¼ 0.89,P � 0.0001); in the F0 rats, TQG and 7-OH (r ¼ 0.96, P � 0.0001),TQG and 30-OH (r ¼ 0.89, P � 0.0001), TQG and 40-OH (r ¼ 0.68,P ¼ 0.004), 7-OH and 30OH (r ¼ 0.73, P ¼ 0.0011), and 30-OH and40-OH (r ¼ 0.89, P � 0.0001); and in the F1 rats, TQG and 7-OH(r ¼ 0.95, P � 0.0001), TQG and 30-OH (r ¼ 0.86, P � 0.0001),TQG and 40-OH (r ¼ 0.77, P ¼ 0.004), 7-OH and 30-OH (r ¼ 0.67,P ¼ 0.0004), 30-OH and 40-OH (r ¼ 0.86, P � 0.0001), and 7-OHand 40-OH (r ¼ 0.56, P ¼ 0.0046).

Discussion

Dietary polyphenols are subject to extensive phase IIbiotransformation, which make them more hydrophilic andreadily excreted. Most absorbed polyphenols are found in circu-lation and tissues, predominately in conjugated forms, e.g., glu-curonates, sulfates, methylates, or a combination of these forms,although some may be present in aglycone and/or their parentplant forms. Therefore, the variation in the capacity of phase IImetabolism in humans may partly account for the markedlyvaried pharmacokinetics and bioefficacy of these nutrients [3].Court [17] indicated that age, sex, enzyme inducers, and geneticpolymorphisms have been implicated as sources of variability inUGT capacity. We previously observed that the in vitro produc-tion rate of flavonoid metabolites catalyzed by the hepaticmicrosomal UGT of F344 rats was dependent on age, UGTisoenzymes, and flavonoid structure [5]. We also observed that

advanced age modulates the in vitro production rate of quercetinglucuronides catalyzed by the microsomal UGT present in thesmall intestine of F344 rats, with the degree and direction ofchanges dependent on the intestinal segment examined [6]. Theeffects of drug, nutrient, or dietary pattern on phase II metabo-lism have been recognized [7,8]; for example, Navarro et al. [18]noted that the consumption of cruciferous vegetables was asso-ciated with a decrease in the excretion of salicylic acid glucuro-nide. In addition, Iwuchukwu et al. [19] reported that resveratrol,curcumin, and chrysin induce UGT1A1 mRNA expression inCaco-2 cells. Our study provides another line of evidence thathigh dietary fructose and saturated fat may modulate the UGTactivity toward quercetin.

Quercetin is biotransformed extensively by glucuronidation,sulfation, and methylation. We previously characterized eightquercetin phase II metabolites in the liver of F344 rats fed a 0.45%quercetin (w/w) diet for 6 wk and noted that 92% of the quercetindetected in the liver was metabolites with 67.6% being conjugatedwith a glucuronide [20]. Consistent with the results of Boersmaet al. [21], our in vitro microsomal UGT reaction generated threequercetin metabolites, with 7-OH being dominant. However, theproduction of quercetin glucuronides by UGT could vary becausethe capacity of phase II enzymes is subject to the modulation ofenvironmental and chemopreventive agents and nutritionalstatus in the context of the totality of genetic background [22].Osabe et al. [10] reported that the mRNA and protein of hepaticUGT1A1 and 1A6 increased in male rats fed a diet containing 10%lard and 60% sucrose, but not in females, ascribing the increases tothe dietary modulation of the constitutive androstane receptorand peroxisome proliferator-activated receptor-a. In contrast tothe report of Osabe et al. [10], our study showed that a diet con-taining 9.9% coconut fat, 0.5% cholesterol, 30% fructose, and 30%glucose decreased the in vitro hepatic UGT activity toward

A. Serra et al. / Nutrition 28 (2012) 1165–11711170

quercetin in the F0 female rats compared with the C diet con-taining 11% corn oil and 60% glucose. Interestingly, we did not findthis change in the F0 male rats. It is also worth noting that theimpact of diet appears to be dependent on the UGT isoenzymebecause decreaseswere found only in the production rate of 30-OHand 40-OH quercetin glucuronides. Based on the high correlationcoefficients between these two metabolites, it is possible theywere produced by overlapping UGT isoenzymes. The differencebetween these two studies may be due to the rodent species(Wistar versus F344), methodology (protein and gene expressionversus enzyme activity), and diet (sucrose and lard [long-chainsaturated fats] versus fructose, glucose, and coconut oil[medium-chain saturated fats]). Further, because quercetinglucuronides were generated through the collective effort of allmicrosomal UGT isoenzymes, we were not able to identifya change in individual UGT isoenzymes. Boersma et al. [21] foundthat when using human recombinant UGT, several UGT isoen-zymes metabolized quercetin with different conversion rates,with UGT1A9, 1A1, 1A3, 1A8, and 2B7 being the most efficient.

Sexual dimorphism is apparent in rodent models, with thebiotransformation of endobiotics and xenobiotics through thephase II pathway, but equivocal in humans [19,23]. The diver-gence in UGT activity and protein and gene expression betweensexes depends on the substrate, species, tissue, and UGT isoen-zyme [24,25]. For example, hepatic Ugt1a5, 1a8, 2b1, and 2b2mRNA levels in female rats are about 35%, 130%, 50%, and 60%larger, respectively, than those in male rats [26]. Mazur et al. [27]noted that the hepatic microsomal glucuronidation capacitytoward bisphenol appeared to be larger in female than in malerodents, and Osabe et al. [10] found that female rodentsexpressed more mRNA and protein of certain UGT isoenzymes.However, Dai et al. [28] found that overall in vivo acetaminophenglucuronidation was lower in female than in male mice.Regarding glucuronidation capacity in humans, Court [17] didnot find a sex-dependent effect on the in vitro transformation ofdrugs using 55 human livers. In this study, we found a gender--dependent effect on in vitro hepatic quercetin glucuronidation inadult F1 rats born to C dams and in all F0 and F1 rats in the Cgroup. It is worth noting that such an effect of sex was significantonly in the production of 7-OH quercetin glucuronide and TQGs,but not of 30-OH and 40-OH metabolites. Because the regiose-lectivity of UGT isoenzymes dictates what biotransformedproducts are produced [29], our results may be a consequence ofthe differential influence of sex on individual UGT isoenzymes.Thus, our results suggest the bioavailability and bioefficacy ofquercetin or other polyphenols may be sex dependent. Thus,further in vivo studies examining the pharmacokinetics ofquercetin and its functional endpoints are warranted.

During the past decade, the potential impact of fetalprogramming on molecular and biochemical phenotypes andthe subsequent risk for chronic disease have become widelyappreciated. However, little is known of the effect of fetalprogramming on the developmental plasticity of detoxificationmechanisms in later life. In an ontogeny study of hepatic UGT ofinbred Gunn and Wistar rats, Bustamante et al. [30] detectedthe activity of UGT1A1 on day 22 of gestation found it reachedits highest level at adult life. Thus, it is plausible that thecapacity of detoxification mechanisms such as UGT in adult lifecould be predetermined by dietary factors in early life. Usinga guinea pigs, Smith et al. [31] found that intermittent in uteroexposure to morphine enhanced the gene expression of hepaticUGT 2A3 by w150% in 7-d-old female offspring compared withuntreated controls. The present shows that exposure to a highfat and fructose diet during gestation and lactation decreases

UGT activity toward quercetin in female adult offspring. Thisfetal programming effect elicited by the diet might be limited tocertain UGT isoenzymes, as suggested by the unchangedproduction rate of 30- and 40-OH quercetin glucuronides.Because UGT activity is dependent on tissue type, isoenzyme,species, substrate, and, potentially, sexual hormones, ourresults help inform a new direction to our understanding of theunderlying mechanisms for the marked interindividual varia-tion in polyphenol pharmacokinetics. Because phase II path-ways are crucial to transforming exogenous and endogenouslipophilic compounds, including environmental carcinogens,into more readily excreted hydrophilic products, our data haveimplications on how nutritional exposure in early life mightprogram the risk for chronic diseases later in life; for example,the capacity of phase II enzymes has been show to associate tocancer susceptibility [32].

There are a few limitations in our study. First, only oneconditionwas used to assess the hepaticmicrosomal UGTactivitytoward quercetin (quercetin 30 mmol/L, microsomal protein 12.5mg/100 mL, and 15-min incubation). The protein amount andincubation time were selected based on our linearity experi-ments. The quercetin concentration was selected based on ourprevious rat study [5] so that the concentration was close to theKm (the concentration of substrate that leads to half-maximalvelocity) value of hepatic UGT. Thus, our results may be gener-alizable to other experimental conditions. Second, our resultsmight not be readily applicable to the in vivo pharmacokineticsand bioefficacy of quercetin and other flavonoids because of thecomplexity of quercetin biotransformation with its concurrentreactions of glucuronidation, sulfation, and methylation in thesmall intestine, liver, and kidney [20].

Conclusion

The biotransformation of polyphenols by phase II enzymesmay contribute to the marked interindividual variation in theirpharmacokinetics. Our observation that a diet high in saturatedfat and fructose decreases the in vitro UGT activity towardquercetin in female rats suggests a potential change in the bio-efficacy of quercetin because the catechol moiety in the B ringplays an important role in its antioxidant capacity [33]. Fetalprogramming by specific dietary factors in utero can decreasethe capacity of selected hepatic UGT isoenzymes toward quer-cetin in female adults.

Acknowledgments

The authors thank Carly Zampariello and Kendra French fortheir excellent technical support.

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