The need to decide if all estrogens are intrinsically similar.

We used gene expression profiling m investigate whether the molecular effects induced by estrogens of different provenance are intrinsically similar. In this article we show that the physiologic estrogen 17[beta]-estradiol, the phytoestrogen genistein, and the synthetic estrogen diethylstilbestrol alter the expression of the same 179 genes in the intact immature mouse uterus under conditions where each chemical has produced an equivalent gravimetric and histologic uterotrophic effect, using the standard 3-day assay protocol. Data are also presented indicating the limitations associated with comparison of gene expression profiles for different chemicals at times before the uterotrophic effects are fully realized. We conclude that the case has yet to be made for regarding synthetic estrogens as presenting a unique human hazard compared with phytoestrogens and physiologic estrogens. Key words: diethylstilbestrol, estrogen, gene expression, genistein, microarray, phytoestrogen, toxicogenomics, uterus. Environ Health Perspect 112:1137-1142 (2004). doi:10.1289/ehp.7028 available via http://dx.doi.org/[Online 19 May 2004]

**********

The question of whether phytoestrogens and synthetic estrogens are toxicologically similar, or intrinsically different, presents a challenge to all involved in human hazard and risk assessments. Although there is a general concern that exposure to nanogram or microgram amounts of environmental estrogens may be associated with adverse health effects, in the public mind there is a widespread belief that foods and dietary supplements containing milligram quantities of phytoestrogens confer only health benefits. An implicit distinction therefore seems to have been drawn between synthetic and plant-derived estrogens--a belief sustained in the public mind by the assumption that natural is good and synthetic is bad--but an untested and potentially misleading notion for those involved with science-based human hazard/risk assessments.

Phytoestrogens and synthetic estrogens are generally considered separately in the literature. For example, Howdeshell et al. (1999) suggested a possible association between the advance in first estrus observed in mice exposed in utero to 2.4 [micro]g/kg of the synthetic environmental estrogen bisphenol A and reports of an increased incidence of hypospadias in boys (Paulozzi et al. 1997) and the earlier sexual maturation of girls (Herman-Giddens et al. 1997)--the implication being that synthetic estrogens present a greater hazard than the much higher levels of phytoestrogens being consumed by those same children. In contrast, there are reports of an increased incidence of hypospadias in boys born to vegetarians (North and Golding 2000), of alterations in the menstrual cycle (Cassidy et al. 1994), and of reduced breast cancer incidences (Messina 1999) among women eating diets rich in phytoestrogens. Support for these epidemiologic observations comes from experimental studies indicating that advances in sexual development in rodents can be induced by their exposure to phytoestrogens (Casanova et al. 1999; Cassidy and Fanghnan 2000; Safe et al. 2002). In contrast to these separate lines of inquiry, Newbold and colleagues have evaluated potential similarities between natural and synthetic estrogens. In seminal studies, they demonstrated that neonatal exposure of female mice to equipotent uterotrophic doses of the phytoestrogen genistein (GEN; Figure 1) or the synthetic estrogen diethylstilbestrol (DES) leads to an identical incidence of uterine adenomas at 18 months of age (Newbold et al. 2001). However, in attempting to draw parallels, or distinctions, between phytoestrogens and synthetic estrogens, it is imperative to consider growing awareness of the complexity of estrogen signaling pathway and the pleuripotential biologic activities of most organic chemicals--irrespective of their origin.

[FIGURE 1 OMITTED]

Estrogen signaling in mammalian cells is primarily mediated at the molecular level by two members of the nuclear receptor superfamily--estrogen receptors alpha (ER-[alpha]) and beta (ER-[beta]). Ligand-activated ER-[alpha] and ER-[beta] function as transcription factors, in conjunction with numerous coregulatory proteins, in order to activate or repress the transcription of ER-responsive genes (Hall et al. 2001; Moggs and Orphanides 2001). There is considerable variation in the binding affinity of ER-[alpha] and ER-[beta] among different estrogens (Kuiper et al. 1998). In the case of the chemicals studied here, the physiologic estrogen 17[beta]-estradiol ([E.sub.2]) and DES bind with a similar affinity to ER-[alpha] and ER-[beta], whereas GEN binds with approximately 20-fold higher affinity to ER-[beta] than to ER-[alpha] (Kuiper et al. 1998). Concerning nonhormonal properties of the test chemicals (most of which have only be defined in vitro), GEN inhibits a range of enzymes, including tyrosine kinases (Akiyama et al. 1987), nitric oxide synthase (Duarte et al. 1997), and topoisomerase II (Okura et al. 1988), and also decreases calcium-channel activity (Potier and Rovira 1999), lipid peroxidation (Arora et al. 1998), and diacylglycerol synthesis (Dean et al. 1989). Likewise, DES is reported to induce aneuploidy in mammalian cells (Aardema et al. 1998) and to bind to rat liver DNA (Williams et al. 1993). More recently, some phytoestrogens were reported to inhibit the aromatase-mediated conversion of testosterone to [E.sub.2] in vitro (Almstrup et al. 2002), and equol, the major circulating estrogenic metabolite associated with the dietary ingestion of phytoestrogens, is reported to selectively sequester dihydrotestosterone and thereby to act as a functional antiandrogen in vivo (Lund et al. 2004).

In order to advance understanding in this area, we decided to compare the genes expressed in the immature mouse uterus when it had grown in response to treatment with the estrogens [E.sub.2], DES, and GEN. The immature mouse uterus was selected for our analysis because it is a major estrogen-responsive organ and forms the basis for a reference assay of estrogenic activity (Owens and Ashby 2002), including carcinogenesis (Newbold et al. 2001). Furthermore, it expresses both ER-[alpha] and ER-[beta] (Weihua et al. 2000) and the androgen receptor (Frasor et al. 2003). We initially conducted a global analysis of gene expression in the mouse uterus at 1, 2, 4, 8, 24, 48, and 72 hr after exposure to a single high dose of either GEN (250 mg/kg) or [E.sub.2] (400 [micro]g/kg). These single high doses yielded a sustained uterotrophic response over 72 hr (Figure 2A) and were selected to avoid the complex transcriptional program that may result from the standard uterotrophic assay exposure regime in which each test compound is dosed by repeated administration on 3 consecutive days (Odum et al. 1997). Groups of 10 sexually immature mice [Alpk:APfCD-1; 19/20 days of age; maintained on RM1 diet (Special Diets Services Ltd., Witham, Essex, UK)] received a single subcutaneous injection of each compound or the test vehicle [arachis oil (AO); 5 mL/kg], and uterine RNA was isolated and pooled by group at each of the seven time points to determine gene expression levels among the 12,488 mouse genes represented on the Affymetrix MG-U74Av2 GeneChip (Affymetrix, High Wycombe, UK). Transcript profiling was performed using MG-U74Av2 GeneChip and Microarray Analysis Suite 5.0 (Affymetrix). Normalization and hierarchical clustering were performed with GeneSpring 6.0 (Silicon Genetics, Redwood City, CA, USA). MIAME (Minimum Information About a Microarray Experiment)-compliant microarray data are available as supplementary information and submitted to the Gene Expression Omnibus (GEO) database (GEO 2004). These data were analyzed using unsupervised hierarchical clustering and yielded temporal relationships between the expression profiles of 3,450 genes that were either up- or down-regulated (> 1.5-fold) by [E.sub.2] and/or GEN (Figure 2B). Each chemical induced a similar, multistage transcriptional response (Figure 2B), although it is noteworthy that we observed variations in the magnitude and timing of both early (e.g., c-fos) and late (e.g., lactotransferrin) ER-responsive genes during the uterotrophic responses induced by [E.sub.2] and GEN (Figure 2C).

[FIGURE 2 OMITTED]

A detailed description of the molecular functions of the genes affected, together with their association with physiologic changes during uterine growth, has been reported (Orphanides et al. 2003) and will be described in more detail in a future publication (Moggs et al., unpublished data).

These observations suggest that GEN does not induce "off-target" ER-independent transcriptional responses, that is, those associated with the properties of GEN other than estrogenicity. Furthermore, there was no evidence for the topoisomerase II-inhibiting properties of GEN in the bone marrow of the present mice despite demonstration of the sensitivity of that tissue to the potent micronucleus-inducing activity of the topoisomerase II inhibitor etoposide (data not shown). Together, these data led us to question whether a synthetic estrogen such as DES would also induce similar transcriptional responses in the immature mouse uterus.

In order to avoid temporal vagaries in gene expression (e.g., Figure 2C), we decided to anchor our transcript profiling data to the phenotype of the grown uterus by employing equipotent uterotrophic doses of [E.sub.2], GEN, and DES. We compared the global gene expression profiles in the uteri of intact immature mice stimulated with three daily low doses of either GEN, DES, or [E.sub.2], with an exposure regimen the same as that used in a standard 3-day uterotrophic assay (Odum et al. 1997). The route of administration and the doses of GEN and DES used were as described by Newbold et al. (2001) in their equivalent-outcome carcinogenicity bioassays of these two chemicals. Three independent replicates of four groups of sexually immature mice (Alpk:APfCD-1; 19/20 days of age; maintained on RM1 diet) received three daily subcutaneous injections of GEN (50 mg/kg), [E.sub.2] (2.5 [micro]g/kg), or DES (2 lag/kg). Control animals received the vehicle, AO (5 mL/kg). These doses elicited similar uterotrophic responses (72 hr after the initial dose; Figure 3A, Table 1) and identical histologic changes in the uteri of the treated animals (Table 1). Uterine RNA was isolated and pooled for each of the 12 groups and analyzed for changes in gene expression levels using the same Affymetrix microarray of 12,488 mouse genes. The data were analyzed using two independent statistical methods. First, unsupervised hierarchical clustering defined the global relationships (Euclidean distances) between the 12 gene expression profiles (Figure 3B). The three control groups clustered under one node, whereas the chemical treatment groups formed a separate node of compound-independent clusters, indicating equal similarity within and between the transcriptional responses induced by the three estrogens (Figure 3B). One-way analysis of variance (ANOVA), with Bonferroni (Holm 1979) correction (familywise error rate < 0.05) to minimize false positives, identified 179 genes where expression levels were altered by one or more chemical treatments (Figure 3C). Remarkably, Tukey post hoc testing revealed that all of these genes were affected in all nine compound treatment groups.

[FIGURE 3 OMITTED]

Table 2 highlights the high degree of similarity between the transcriptional responses to each of the three estrogens. These include established estrogen-responsive genes such as lactotransferrin, complement component 3, c-fos, small proline-rich protein 2A, and keratoepithelin (Hewitt et al. 2003; Naciff et al. 2003), together with many genes that have not previously been associated with estrogenicity (Table 2).

Although these three estrogens can alter the expression of some genes with different magnitudes [e.g., peptidyl arginine deiminase II is up-regulated to a lesser extent by [E.sub.2] (1.86-fold [+ or minus] 0.27) relative to GEN (9.11-fold [+ or -] 0.33) and DES (5.15-fold [+ or -] 1.53); Table 2], the present data show that the same genes are affected during equivalent uterotrophic responses. Previous studies have revealed both similarities and differences between transcriptional responses induced at a single time point after exposure to [E.sub.2] and DES in the uteri of immature ovariectomized mice (Watanabe et al. 2003) and after exposure to either GEN, bisphenol A, or 170[alpha]-ethynyl estradiol in the reproductive tract of intact adult rats (Naciff et al. 2002). We suggest that these reported differences most probably arise from dose-dependent variations in the magnitude and kinetics of gene expression (Figure 2C), rather than from the operation of distinct mechanisms of estrogenic action.

Our data indicate that estrogens of differing provenance may have in common the potential for both beneficial and adverse health effects. This highlights the need for an holistic approach to hazard assessment wherein preconceptions are replaced by an objective assessment of the likely perturbations of physiologic functions caused by combined exposures to physiologic, synthetic, and plant-derived estrogens. This need is reinforced by data showing that plasma concentrations of isoflavones in infants fed soy formula are approximately 200 times higher than for those fed human milk (Setchell et al. 1997), by the estimated daily intake of approximately 29 mg of phytoestrogens for individuals taking dietary supplements (Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment 2003), and by the demonstration that estrogens of different provenance can act additively in the rodent uterus (Tinwell and Ashby 2004).

Table 1. Blotted uterine weights and endometrial and epithelial
cell heights (mean [+ or -] SDI after exposure to [E.sub.2],
GEN, or DES for 3 consecutive days. (a)

                Dose         Blotted uterine
Compound      (per kg)         weight (m g)

AD            5 mL         13.0 [+ or -] 2.4
[E.sub.2]   2.5 [micro]g   45.3 [+ or -] 8.6 *
GEN          50 [micro]g   39.8 [+ or -] 5.3 *
DES         2.0 [micro]g   49.8 [+ or -] 13.0 *

                          Cell height ([micro]m)

Compound           Endometrium               Epithelium

AD          159.0 [+ or -] 23.1 (11)     11.4 [+ or -] 1.1
[E.sub.2]   246.1 [+ or -] 52.4 * (9)    23.3 [+ or -] 1.4 *
GEN         273.7 [+ or -] 63.3 * (12)   23.7 [+ or -] 3.1 *
DES         273.2 [+ or -] 55.9 * (10)   22.6 [+ or -] 4.0 *

There were 12 animals/group, but not all of the histopathology
samples were suitable for analyses; numbers in parentheses
indicate the number of animals per group from which the histology
data were generated.

(a) Data were assessed for statistical significance using a
two-sided Student t-test: * p < 0.01.

Table 2. Quantitative data for 179 differentially expressed genes
(from Figure 3C) regulated in the mouse uterus by all three
estrogens ([E.sub.2], GEN, and DES). (a)

                                           Fold change in
                                          expression (mean
                                            [+ or -] SD)

                              GenBank
                             accession
Gene name                       no.           [E.sub.2]

Up-regulated genes
  Solute carrier family      U74079        1.8 [+ or -] 0.01
    9a3r1
  Keratin complex 2-8        X15662        2.6 [+ or -] 0.2
  Laminin beta 3             U43298        4.3 [+ or -] 0.1
  Claudin 7                  AF087825      4.5 [+ or -] 0.5
  bHLH-Zip transcription     U49507        2.6 [+ or -] 0.3
    factor
  RIKEN cDNA 1200008D14      AW208938      3.0 [+ or -] 0.3
  Basic HLH-domain           Y07836        5.9 [+ or -] 1.0
    containing, class B2
  RIKEN cDNA 9930104H07      AW122310      3.0 [+ or -] 0.3
  Fucosyltransferase 2       AF064792     27.5 [+ or -] 1.2
  Deleted in polyposis 1     U28168        1.8 [+ or -] 0.1
  Microsomal glutathione     AI843448      2.9 [+ or -] 0.2
    S-transferase 3
  Tumor-associated Ca        Y08830        4.0 [+ or -] 0.3
    signal transducer 2
  Calpain 5                  Y10656        5.5 [+ or -] 0.4
  Mitochondrial creatine     Z13969        9.7 [+ or -] 1.1
    kinase
  ATPase 6v1a1               AW123765      2.0 [+ or -] 0.1
  Tumor-associated Ca        AI563854      8.0 [+ or -] 0.4
    signal transducer 2
  Lymphocyte antigen 6       X04653        7.8 [+ or -] 0.9
    complex, locus A
  Chloride channel           AV373378     26.4 [+ or -] 3.4
    calcium-activated 3
  Small proline-rich         AJ005567     23.9 [+ or -] 1.5
    protein 21
  Oncoprotein induced        AA615075     19.0 [+ or -] 3.1
    transcript 1
  Small proline-rich         AJ005564     59.8 [+ or -] 8.4
    protein 2F
  Small proline-rich         AJ005563     12.0 [+ or -] 1.0
    protein 2E
  Mucin 1                    M84683        8.3 [+ or -] 0.6
  Lipoocalin 2               X81627      150.3 [+ or -] 15.0
  RIKEN cDNA 2210409B01      AF109906      3.5 [+ or -] 0.6
  Interferon-activated       M31418        7.9 [+ or -] 1.0
    gene 202A
  Nuclear ankyrin-repeat     AA614971      3.7 [+ or -] 0.6
    protein
  RIKEN cDNA 5730469M10      AI850090     22.0 [+ or -] 5.8
  RIKEN cDNA 1110034C02      AI837104      1.5 [+ or -] 0.1
  IMAGE cDNA 4988271         AV373294      8.0 [+ or -] 2.5
  RIKEN cDNA 5730493B19      AW122413     12.7 [+ or -] 0.3
  Peptidoglycan              AV092014     13.4 [+ or -] 1.5
    recognition protein
  Inhibin beta-B             X69620       13.6 [+ or -] 3.1
  CEA-related cell           AF101164     11.9 [+ or -] 1.6
    adhesion molecule 2
  Keratin complex 1-19       M36120        4.4 [+ or -] 0.4
  CEA-related cell           M77196       15.9 [+ or -] 2.4
    adhesion molecule 1
  SRC family-associated      AB014485      2.7 [+ or -] 0.04
    phosphoprotein 2
  Peptidoglycan              AF076482      7.7 [+ or -] 1.9
    recognition protein
  CEA-related cell           M77196       19.5 [+ or -] 3.9
    adhesion molecule 1
  CEA-related cell           X67279        6.4 [+ or -] 0.7
    adhesion molecule 1
  Spermidine N1-acetyl       L10244        8.3 [+ or -] 0.9
    transferase
  RIKEN cDNA 0610007007      AI851762      2.7 [+ or -] 0.1
  Arginase 1                 U51805       79.4 [+ or -] 9.8
  Acetyl-coenzyme A
    synthetase 2             AW125884      2.2 [+ or -] 0.2
  v-erb-b2 homolog 3         AI006228      3.4 [+ or -] 0.4
  Phospholipase D3           AF026124      2.6 [+ or -] 0.2
  RIKEN cDNA 0610031J06      AW122935      1.9 [+ or -] 0.1
  Complement component 1q    X58861        2.1 [+ or -] 0.1
  Scotin                     AW123754      2.0 [+ or -] 0.1
  CD24a antigen              M58661        3.2 [+ or -] 0.1
  Argininosuccinate          M31690        2.7 [+ or -] 0.3
    synthetase 1
  ATPase 6v1a1               U13837        2.1 [+ or -] 0.1
  Gelsolin-like actin-       X54511        3.6 [+ or -] 0.5
    capping protein
  Golgi phosphoprotein 2     AW125446      4.5 [+ or -] 0.5
  Aldolase 1A                Y00516        2.3 [+ or -] 0.2
  Cathepsin L                X06086        6.4 [+ or -] 0.8
  CD14 antigen               X13333        3.0 [+ or -] 0.1
  Decay accelerating         L41365        4.0 [+ or -] 0.1
    factor 2
  Actin-related protein      AW212775      2.1 [+ or -] 0.2
    2/3 complex 1B
  Protective protein for     J05261        2.0 [+ or -] 0.1
    [beta]-galactosidase
  Elastase 1                 M27347        2.7 [+ or -] 0.1
  Connexin 26                M81445       10.6 [+ or -] 1.0
  Ceruloplasmin              U49430       15.1 [+ or -] 2.8
  Cathepsin H                U06119        3.0 [+ or -] 0.2
  Basigin                    Y16258        1.6 [+ or -] 0.1
  Peptidylprolyl isomerase   X67809        2.2 [+ or -] 0.2
    C-associated
  Glutathione reductase 1    AI851983      2.3 [+ or -] 0.2
  START domain-              X82457        1.5 [+ or -] 0.1
    containing 3
  CD68 antigen               X68273        4.6 [+ or -] 0.6
  RIKEN cDNA E030027H19      AW211760      2.7 [+ or -] 0.3
  cDNA sequence B0004044     AI461767      3.1 [+ or -] 0.2
  E74-like factor 3          AF016294      5.1 [+ or -] 0.8
  Glutathione S-             AI843119      5.0 [+ or -] 1.1
    transferase omega 1
  Interferon-stimulated      AW122677      4.2 [+ or -] 0.1
    protein 20
  Clusterin                  D14077        3.6 [+ or -] 0.7
  Galectin 3                 X16834        7.4 [+ or -] 1.3
  Small proline-rich         AJ005559     51.1 [+ or -] 4.0
    protein 2A (b)
  Complement component       K02782       14.8 [+ or -] 1.8
    3 (b)
  Small proline-rich         AJ005561    220.3 [+ or -] 31.0
    protein 2C
  Small proline-rich         AJ005565      9.4 [+ or -] 0.9
    protein 2G
  Prominin                   AF039663      3.5 [+ or -] 0.5
  Lactotransferrin (b)       J03298       88.7 [+ or -] 18.4
  Carbonic anhydrase 2       M25944        7.9 [+ or -] 0.5
  Complement component       U47810       36.5 [+ or -] 4.4
    factor 1
  Mannosidase 2alphaB1       U87240        2.0 [+ or -] 0.2
  Small proline-rich         AJ005560     32.9 [+ or -] 3.7
    protein 2B
  Small proline-rich         AJ005559    269.8 [+ or -] 23.7
    protein 2A (b)
  RIKEN cDNA 5830413E08      AI849939      3.3 [+ or -] 0.4
  RIKEN cDNA 1110029F20      AW125508      4.1 [+ or -] 0.1
  Annexin A3                 AJ001633      2.7 [+ or -] 0.5
  Peptidase 4                U51014        2.0 [+ or -] 0.1
  Laminin gamma 2            U43327        6.3 [+ or -] 1.3
  Ubiquitin-like 3           AW120725      1.5 [+ or -] 0.1
  Urate oxidase              M27695       23.8 [+ or -] 9.5
  Amiloride binding          AI197481      3.5 [+ or -] 1.0
    protein 1
  Keratin complex 1-19       AU040563      4.5 [+ or -] 1.0
  Activated leukocyte cell   L25274        3.6 [+ or -] 0.8
    adhesion molecule
  CCAAT/enhancer binding     M61007        2.3 [+ or -] 0.1
    protein [beta]
  Peptidyl arginine          AB013848      8.6 [+ or -] 0.7
    deiminase, type I
  Enolase 1 [alpha]          AI841389      2.5 [+ or -] 0.3
  p53 apoptosis effector     AI854029      2.9 [+ or -] 0.3
    related to Pmp22
  [beta]-Glucuronidase       M19279        1.9 [+ or -] 0.1
  Leucine-rich               AW230891      9.3 [+ or -] 1.1
    [alpha]-2-glycoprotein
  Quiescin 06                AW123556      3.7 [+ or -] 0.2
  GADD45a                    U00937        1.9 [+ or -] 0.2
  Alkaline phosphatase 2     J02980        9.2 [+ or -] 0.4
  Immediate early            X67644        5.5 [+ or -] 0.8
    response 3
  Progressive ankylosis      AW049351      2.2 [+ or -] 0.1
  RAS p21 protein            AA163960      6.8 [+ or -] 0.9
    activator 4
  Tumor-associated calcium   M76124        2.1 [+ or -] 0.2
    signal transducer 1
  Hydroxysteroid (17-beta)   AA822174      1.9 [+ or -] 0.1
    dehydrogenase 11
  Platelet-activating        U57746        1.9 [+ or -] 0.1
    factor acetylhydrolase
    1ba1
  Branched chain             U42443        2.4 [+ or -] 0.2
    aminotransferase 1
  RIKEN cDNA 2400004E04      AI846720      1.7 [+ or -] 0.1
  Myeloblastosis oncogene    M12848        2.8 [+ or -] 0.4
  [K.sup.+] conductance      AF042487      3.1 [+ or -] 0.2
    calcium-activated
    channel N4
  ATPase 6v1b2               AI843029      1.7 [+ or -] 0.1
  Cystic fibrosis            M60493        3.4 [+ or -] 0.5
    transmembrane
    regulator
  RIKEN cDNA 1110008P14      AI839839      4.3 [+ or -] 0.4
  Fused toes                 Z67963        2.6 [+ or -] 0.2
  Solute carrier family      AW124340      3.5 [+ or -] 0.5
    39a8
  Cytochrome b-561           AI846517      2.2 [+ or -] 0.2
  Secreted                   X13986       30.2 [+ or -] 3.3
    phosphoprotein 1
  Ion transport regulator    X93038        5.3 [+ or -] 0.4
    Fxyd3
  Janus kinase 3             L40172        2.1 [+ or -] 0.2
  Cytochrome b-245alpha      AW046124      2.9 [+ or -] 0.4
  RIKEN cDNA A430096B05      AI465965      6.3 [+ or -] 1.0
  Small proline-rich         AJ005568      8.6 [+ or -] 2.1
    protein 2J
  Cathepsin B                M65270        2.2 [+ or -] 0.1
  RIKEN cDNA 1600025H15      AI842734      2.2 [+ or -] 0.1
  c-fos oncogene (b)         V00727        3.2 [+ or -] 0.4
  Guanine nucleotide         AI843937      1.6 [+ or -] 0.1
    binding protein
    [gamma]5
  Serine palmitoyl-          U27455        1.6 [+ or -] 0.1
    transferase Ic2
  Cystatin B                 U59807        1.5 [+ or -] 0.1
  Villin 2                   X60671        1.9 [+ or -] 0.2
  RIKEN cDNA 0610010012      AI849011      1.9 [+ or -] 0.1
  Matrix metallo-            L36244       47.8 [+ or -] 18.6
    proteinase 7
  RIKEN cDNA 4930422J18      AV376312      2.0 [+ or -] 0.3
  RIKEN cDNA 1700017B05      AW049360      1.6 [+ or -] 0.1
  Galactosidase beta 1       M57734        1.8 [+ or -] 0.1
  Cathepsin C                U74683        2.6 [+ or -] 0.1
  Interferon-stimulated      X56602        3.6 [+ or -] 0.6
    protein 15
  MAP kinase--interacting    Y11092        1.8 [+ or -] 0.1
    kinase 2
  Glutathione S-             X98056        3.5 [+ or -] 0.4
    transferase theta 2
  Homeobox B6                M18401        1.5 [+ or -] 0.02
  Procollagen Vlalpha 3      AF064749      2.1 [+ or -] 0.2
  Interferon regulatory      U73037       11.7 [+ or -] 0.9
    factor 7
  Scavenger receptor class   AB008553      2.7 [+ or -] 0.1
    B2
  Polyimmunoglobulin         AB001489      8.1 [+ or -] 0.7
    receptor
  Proteasome subunit         Y10875        2.1 [+ or -] 0.04
    [beta]10
  RIKEN cDNA 0610010E05      AV312736      2.9 [+ or -] 0.3
  RIKEN cDNA 0610010E05      AI854839      3.7 [+ or -] 0.5
  Xanthine dehydrogenase     X75129       12.2 [+ or -] 2.2
  Prominin                   AF039663      3.5 [+ or -] 0.2
  Interferon-induced         U43084       17.5 [+ or -] 2.9
    protein IFIT1
  Interferon-induced         U43086        8.1 [+ or -] 2.3
    protein IFIT3
  Proteasome subunit         U22033        2.0 [+ or -] 0.1
    [beta]8
  RIKEN cDNA 1600023A02      AW121336      1.9 [+ or -] 0.1
  Small proline-rich         AF057156     11.1 [+ or -] 3.7
    protein 1A
  MAP kinase-interacting     AI845732      2.0 [+ or -] 0.1
    kinase 2
  Lymphocyte antigen 6       U47737        2.0 [+ or -] 0.01
    complex, locus E
  Guanylate nucleotide       AJ007970      3.0 [+ or -] 0.1
    binding protein 2
  Peptidyl arginine          D16580        1.9 [+ or -] 0.3
    deiminase, type II (b)
Down-regulated genes
  Solute carrier family      AI838274      2.0 [+ or -] 0.2
    29a1
  Lymphocyte specific 1      D49691        1.6 [+ or -] 0.1
  Claudin 5                  U82758        2.0 [+ or -] 0.2
  Potassium channel td12     AI842065      1.6 [+ or -] 0.04
  Zinc finger homeobox 1a    D76432        1.5 [+ or -] 0.1
  Monoamine oxidase A        AI848045      2.3 [+ or -] 0.2
  Histidine decarboxylase    X57437        4.8 [+ or -] 0.8
  [alpha]-2 Adrenergic       M97516        3.0 [+ or -] 0.3
    receptor
  Transcription factor 21    AF035717      1.8 [+ or -] 0.2
  Homeobox D8                X56561        2.2 [+ or -] 0.1
  Carboxypeptidase X2        AF017639      4.1 [+ or -] 0.7
  RIKEN cDNA A230106A15      AI848841      3.8 [+ or -] 0.2
  Reduced expression 3       AA790008      3.1 [+ or -] 0.2
  TGF-[beta] binding         AA838868      1.8 [+ or -] 0.1
    protein 4
  Keratoepithelin (b)        L19932       11.5 [+ or -] 2.5
  GLI-Kruppel family         AB025922     11.6 [+ or -] 0.8
    member GLI

                                           Fold change in
                                    expression (mean [+ or -] SD)

Gene name                            GEN                   DES

Up-regulated genes
  Solute carrier family        2.0 [+ or -] 0.1      2.0 [+ or -] 0.2
    9a3r1
  Keratin complex 2-8          3.1 [+ or -] 0.2      3.1 [+ or -] 0.3
  Laminin beta 3               5.5 [+ or -] 1.1      5.3 [+ or -] 0.7
  Claudin 7                    6.5 [+ or -] 1.0      5.8 [+ or -] 0.6
  bHLH-Zip transcription       3.1 [+ or -] 0.3      2.9 [+ or -] 0.1
    factor
  RIKEN cDNA 1200008D14        3.5 [+ or -] 0.1      3.3 [+ or -] 0.3
  Basic HLH-domain             6.6 [+ or -] 0.9      6.6 [+ or -] 0.8
    containing, class B2
  RIKEN cDNA 9930104H07        3.2 [+ or -] 0.4      3.3 [+ or -] 0.1
  Fucosyltransferase 2        34.6 [+ or -] 8.5     36.7 [+ or -] 5.5
  Deleted in polyposis 1       2.0 [+ or -] 0.02     2.0 [+ or -] 0.1
  Microsomal glutathione       3.3 [+ or -] 0.6      3.3 [+ or -] 0.1
    S-transferase 3
  Tumor-associated Ca          4.6 [+ or -] 0.9      4.6 [+ or -] 0.3
    signal transducer 2
  Calpain 5                    6.3 [+ or -] 1.0      6.6 [+ or -] 0.6
  Mitochondrial creatine      12.2 [+ or -] 2.1     13.1 [+ or -] 1.8
    kinase
  ATPase 6v1a1                 2.1 [+ or -] 13.2     2.1 [+ or -] 0.2
  Tumor-associated Ca          9.2 [+ or -] 1.0      8.5 [+ or -] 0.4
    signal transducer 2
  Lymphocyte antigen 6         8.8 [+ or -] 0.3      8.5 [+ or -] 0.4
    complex, locus A
  Chloride channel            26.7 [+ or -] 1.0     26.2 [+ or -] 3.9
    calcium-activated 3
  Small proline-rich          24.7 [+ or -] 1.3     23.6 [+ or -] 1.6
    protein 21
  Oncoprotein induced         20.0 [+ or -] 1.2     18.9 [+ or -] 2.5
    transcript 1
  Small proline-rich          65.8 [+ or -] 1.1     60.6 [+ or -] 2.6
    protein 2F
  Small proline-rich          12.9 [+ or -] 0.8     12.1 [+ or -] 0.9
    protein 2E
  Mucin 1                      8.6 [+ or -] 0.3      8.5 [+ or -] 0.5
  Lipoocalin 2               175.7 [+ or -] 10.5   162.8 [+ or -] 6.5
  RIKEN cDNA 2210409B01        4.0 [+ or -] 0.3      3.8 [+ or -] 0.8
  Interferon-activated         9.8 [+ or -] 2.5      8.8 [+ or -] 0.7
    gene 202A
  Nuclear ankyrin-repeat       4.3 [+ or -] 0.7      4.1 [+ or -] 0.9
    protein
  RIKEN cDNA 5730469M10       30.5 [+ or -] 9.1     27.0 [+ or -] 6.5
  RIKEN cDNA 1110034C02        1.6 [+ or -] 0.1      1.6 [+ or -] 0.03
  IMAGE cDNA 4988271          10.6 [+ or -] 1.6      9.2 [+ or -] 1.1
  RIKEN cDNA 5730493B19       19.0 [+ or -] 4.1     15.7 [+ or -] 0.9
  Peptidoglycan               18.3 [+ or -] 3.0     14.5 [+ or -] 2.1
    recognition protein
  Inhibin beta-B              19.4 [+ or -] 4.7     16.0 [+ or -] 1.4
  CEA-related cell            17.8 [+ or -] 5.3     14.2 [+ or -] 1.7
    adhesion molecule 2
  Keratin complex 1-19         5.5 [+ or -] 1.1      4.8 [+ or -] 0.5
  CEA-related cell            23.9 [+ or -] 5.9     19.0 [+ or -] 3.8
    adhesion molecule 1
  SRC family-associated        3.2 [+ or -] 0.4      2.9 [+ or -] 0.3
    phosphoprotein 2
  Peptidoglycan               10.4 [+ or -] 2.7      9.0 [+ or -] 2.0
    recognition protein
  CEA-related cell            30.4 [+ or -] 8.8     22.2 [+ or -] 2.7
    adhesion molecule 1
  CEA-related cell             8.4 [+ or -] 1.1      7.1 [+ or -] 1.1
    adhesion molecule 1
  Spermidine N1-acetyl        11.2 [+ or -] 0.8      9.3 [+ or -] 0.6
    transferase
  RIKEN cDNA 0610007007        3.0 [+ or -] 0.3      2.8 [+ or -] 0.1
  Arginase 1                 131.9 [+ or -] 20.0    99.6 [+ or -] 14.5
  Acetyl-coenzyme A
    synthetase 2               2.0 [+ or -] 0.1      2.2 [+ or -] 0.2
  v-erb-b2 homolog 3           3.1 [+ or -] 0.6      3.4 [+ or -] 0.4
  Phospholipase D3             2.4 [+ or -] 0.2      2.6 [+ or -] 0.2
  RIKEN cDNA 0610031J06        1.8 [+ or -] 0.1      1.8 [+ or -] 0.1
  Complement component 1q      2.0 [+ or -] 0.1      2.0 [+ or -] 0.2
  Scotin                       2.0 [+ or -] 0.2      2.0 [+ or -] 0.2
  CD24a antigen                3.1 [+ or -] 0.1      3.3 [+ or -] 0.3
  Argininosuccinate            2.7 [+ or -] 0.3      2.8 [+ or -] 0.4
    synthetase 1
  ATPase 6v1a1                 2.1 [+ or -] 0.2      2.2 [+ or -] 0.2
  Gelsolin-like actin-         3.7 [+ or -] 0.5      3.7 [+ or -] 0.2
    capping protein
  Golgi phosphoprotein 2       4.6 [+ or -] 0.5      4.7 [+ or -] 0.1
  Aldolase 1A                  2.3 [+ or -] 0.1      2.4 [+ or -] 0.1
  Cathepsin L                  6.3 [+ or -] 1.1      6.9 [+ or -] 0.4
  CD14 antigen                 2.8 [+ or -] 0.1      3.1 [+ or -] 0.2
  Decay accelerating           3.8 [+ or -] 0.8      3.8 [+ or -] 0.2
    factor 2
  Actin-related protein        2.1 [+ or -] 0.1      2.1 [+ or -] 0.2
    2/3 complex 1B
  Protective protein for       2.0 [+ or -] 0.1      2.0 [+ or -] 0.1
    [beta]-galactosidase
  Elastase 1                   2.5 [+ or -] 0.2      2.6 [+ or -] 0.1
  Connexin 26                  9.8 [+ or -] 0.5     10.4 [+ or -] 0.8
  Ceruloplasmin               15.0 [+ or -] 5.5     14.5 [+ or -] 2.1
  Cathepsin H                  3.0 [+ or -] 0.3      3.0 [+ or -] 0.3
  Basigin                      1.5 [+ or -] 0.1      1.7 [+ or -] 0.1
  Peptidylprolyl isomerase     2.2 [+ or -] 0.1      2.4 [+ or -] 0.3
    C-associated
  Glutathione reductase 1      2.3 [+ or -] 0.1      2.6 [+ or -] 0.3
  START domain-                1.4 [+ or -] 0.03     1.5 [+ or -] 0.01
    containing 3
  CD68 antigen                 4.2 [+ or -] 0.6      5.0 [+ or -] 0.7
  RIKEN cDNA E030027H19        2.7 [+ or -] 0.2      2.9 [+ or -] 0.1
  cDNA sequence B0004044       3.4 [+ or -] 0.1      3.8 [+ or -] 0.5
  E74-like factor 3            5.9 [+ or -] 0.5      6.5 [+ or -] 0.5
  Glutathione S-               4.6 [+ or -] 0.6      4.1 [+ or -] 0.7
    transferase omega 1
  Interferon-stimulated        4.3 [+ or -] 0.7      3.4 [+ or -] 0.4
    protein 20
  Clusterin                    3.9 [+ or -] 0.9      3.4 [+ or -] 0.4
  Galectin 3                   8.7 [+ or -] 0.7      6.9 [+ or -] 0.4
  Small proline-rich          78.3 [+ or -] 15.7    44.2 [+ or -] 5.2
    protein 2A (b)
  Complement component        18.8 [+ or -] 1.0     14.8 [+ or -] 0.8
    3 (b)
  Small proline-rich         340.5 [+ or -] 37.1   214.1 [+ or -] 41.1
    protein 2C
  Small proline-rich          11.0 [+ or -] 0.3      9.6 [+ or -] 0.6
    protein 2G
  Prominin                     3.6 [+ or -] 0.6      3.4 [+ or -] 0.3
  Lactotransferrin (b)        99.2 [+ or -] 13.9    76.9 [+ or -] 21.8
  Carbonic anhydrase 2         8.2 [+ or -] 0.9      7.3 [+ or -] 0.4
  Complement component        38.4 [+ or -] 5.6     32.9 [+ or -] 4.2
    factor 1
  Mannosidase 2alphaB1         2.0 [+ or -] 0.1      1.9 [+ or -] 0.1
  Small proline-rich          39.2 [+ or -] 1.9     30.7 [+ or -] 5.0
    protein 2B
  Small proline-rich         329.1 [+ or -] 42.9    59.1 [+ or -] 40.8
    protein 2A (b)
  RIKEN cDNA 5830413E08        3.3 [+ or -] 0.5      3.0 [+ or -] 0.3
  RIKEN cDNA 1110029F20        4.1 [+ or -] 0.4      3.7 [+ or -] 0.1
  Annexin A3                   4.2 [+ or -] 0.7      3.2 [+ or -] 0.6
  Peptidase 4                  2.9 [+ or -] 0.3      2.3 [+ or -] 0.2
  Laminin gamma 2             17.3 [+ or -] 6.8     10.2 [+ or -] 1.0
  Ubiquitin-like 3             1.8 [+ or -] 0.1      1.7 [+ or -] 0.03
  Urate oxidase              143.9 [+ or -] 62.9    43.8 [+ or -] 17.7
  Amiloride binding           10.1 [+ or -] 0.8      6.0 [+ or -] 1.2
    protein 1
  Keratin complex 1-19         7.2 [+ or -] 1.0      5.6 [+ or -] 0.4
  Activated leukocyte cell     5.1 [+ or -] 0.9      4.4 [+ or -] 0.5
    adhesion molecule
  CCAAT/enhancer binding       2.8 [+ or -] 0.3      2.6 [+ or -] 0.1
    protein [beta]
  Peptidyl arginine           15.8 [+ or -] 3.1     12.0 [+ or -] 1.7
    deiminase, type I
  Enolase 1 [alpha]            3.2 [+ or -] 0.5      3.0 [+ or -] 0.3
  p53 apoptosis effector       4.1 [+ or -] 0.8      3.7 [+ or -] 0.5
    related to Pmp22
  [beta]-Glucuronidase         2.3 [+ or -] 0.2      2.2 [+ or -] 0.1
  Leucine-rich                17.6 [+ or -] 4.1     14.5 [+ or -] 2.6
    [alpha]-2-glycoprotein
  Quiescin 06                  5.5 [+ or -] 1.2      4.8 [+ or -] 0.7
  GADD45a                      2.6 [+ or -] 0.3      2.3 [+ or -] 0.1
  Alkaline phosphatase 2      22.6 [+ or -] 6.2     15.8 [+ or -] 2.0
  Immediate early             10.8 [+ or -] 2.2      8.9 [+ or -] 2.0
    response 3
  Progressive ankylosis        2.9 [+ or -] 0.4      2.8 [+ or -] 0.4
  RAS p21 protein             14.2 [+ or -] 2.5     12.1 [+ or -] 1.7
    activator 4
  Tumor-associated calcium     2.7 [+ or -] 0.3      2.6 [+ or -] 0.2
    signal transducer 1
  Hydroxysteroid (17-beta)     2.3 [+ or -] 0.3      2.4 [+ or -] 0.1
    dehydrogenase 11
  Platelet-activating          2.2 [+ or -] 0.2      2.3 [+ or -] 0.1
    factor acetylhydrolase
    1ba1
  Branched chain               3.4 [+ or -] 0.2      3.4 [+ or -] 0.01
    aminotransferase 1
  RIKEN cDNA 2400004E04        2.4 [+ or -] 0.2      2.3 [+ or -] 0.2
  Myeloblastosis oncogene      5.6 [+ or -] 1.1      5.0 [+ or -] 0.2
  [K.sup.+] conductance        4.2 [+ or -] 0.6      3.5 [+ or -] 0.8
    calcium-activated
    channel N4
  ATPase 6v1b2                 1.8 [+ or -] 0.1      1.8 [+ or -] 0.1
  Cystic fibrosis              4.5 [+ or -] 0.8      3.9 [+ or -] 0.3
    transmembrane
    regulator
  RIKEN cDNA 1110008P14        6.0 [+ or -] 1.0      5.1 [+ or -] 0.2
  Fused toes                   3.2 [+ or -] 0.3      2.8 [+ or -] 0.1
  Solute carrier family        4.5 [+ or -] 0.7      3.8 [+ or -] 0.3
    39a8
  Cytochrome b-561             2.5 [+ or -] 0.2      2.3 [+ or -] 0.2
  Secreted                    47.4 [+ or -] 7.4     31.1 [+ or -] 2.7
    phosphoprotein 1
  Ion transport regulator      6.8 [+ or -] 1.1      5.5 [+ or -] 0.6
    Fxyd3
  Janus kinase 3               2.5 [+ or -] 0.2      2.2 [+ or -] 0.2
  Cytochrome b-245alpha        3.6 [+ or -] 0.4      2.9 [+ or -] 0.2
  RIKEN cDNA A430096B05        8.6 [+ or -] 0.03     6.3 [+ or -] 0.6
  Small proline-rich          13.8 [+ or -] 3.2      8.5 [+ or -] 0.7
    protein 2J
  Cathepsin B                  2.6 [+ or -] 0.2      2.2 [+ or -] 0.1
  RIKEN cDNA 1600025H15        2.7 [+ or -] 0.3      2.2 [+ or -] 0.2
  c-fos oncogene (b)           4.7 [+ or -] 0.9      3.7 [+ or -] 0.8
  Guanine nucleotide           1.8 [+ or -] 0.1      1.6 [+ or -] 0.03
    binding protein
    [gamma]5
  Serine palmitoyl-            2.0 [+ or -] 0.2      1.7 [+ or -] 0.1
    transferase Ic2
  Cystatin B                   1.7 [+ or -] 0.1      1.5 [+ or -] 0.02
  Villin 2                     2.4 [+ or -] 0.3      1.9 [+ or -] 01
  RIKEN cDNA 0610010012        2.5 [+ or -] 0.3      1.9 [+ or -] 0.03
  Matrix metallo-            208.6 [+ or -] 83.3    48.2 [+ or -] 11.9
    proteinase 7
  RIKEN cDNA 4930422J18        2.9 [+ or -] 0.3      2.0 [+ or -] 0.2
  RIKEN cDNA 1700017B05        2.0 [+ or -] 0.1      1.6 [+ or -] 0.1
  Galactosidase beta 1         2.0 [+ or -] 0.1      1.7 [+ or -] 0.1
  Cathepsin C                  3.3 [+ or -] 0.2      2.3 [+ or -] 0.3
  Interferon-stimulated        2.0 [+ or -] 0.2      3.8 [+ or -] 0.7
    protein 15
  MAP kinase--interacting      1.4 [+ or -] 0.1      1.9 [+ or -] 0.1
    kinase 2
  Glutathione S-               2.9 [+ or -] 0.4      3.6 [+ or -] 0.2
    transferase theta 2
  Homeobox B6                  1.5 [+ or -] 0.1      1.6 [+ or -] 0.03
  Procollagen Vlalpha 3        1.9 [+ or -] 0.2      2.1 [+ or -] 0.02
  Interferon regulatory        8.1 [+ or -] 0.7     12.6 [+ or -] 1.5
    factor 7
  Scavenger receptor class     2.4 [+ or -] 0.3      2.6 [+ or -] 0.2
    B2
  Polyimmunoglobulin           6.2 [+ or -] 0.8      7.9 [+ or -] 1.0
    receptor
  Proteasome subunit           1.9 [+ or -] 0.04     2.1 [+ or -] 0.1
    [beta]10
  RIKEN cDNA 0610010E05        2.5 [+ or -] 0.2      2.7 [+ or -] 0.4
  RIKEN cDNA 0610010E05        3.0 [+ or -] 0.1      3.4 [+ or -] 0.3
  Xanthine dehydrogenase       8.9 [+ or -] 1.2     10.1 [+ or -] 1.5
  Prominin                     2.9 [+ or -] 0.2      3.1 [+ or -] 0.3
  Interferon-induced           9.9 [+ or -] 1.5     14.0 [+ or -] 2.1
    protein IFIT1
  Interferon-induced           4.7 [+ or -] 0.2      6.7 [+ or -] 0.6
    protein IFIT3
  Proteasome subunit           1.8 [+ or -] 0.2      2.1 [+ or -] 0.1
    [beta]8
  RIKEN cDNA 1600023A02        1.7 [+ or -] 0.1      2.0 [+ or -] 0.04
  Small proline-rich           8.6 [+ or -] 0.8     14.7 [+ or -] 0.8
    protein 1A
  MAP kinase-interacting       1.7 [+ or -] 0.1      2.0 [+ or -] 0.2
    kinase 2
  Lymphocyte antigen 6         1.7 [+ or -] 0.1      2.0 [+ or -] 0.1
    complex, locus E
  Guanylate nucleotide         2.0 [+ or -] 0.2      2.6 [+ or -] 0.1
    binding protein 2
  Peptidyl arginine            9.1 [+ or -] 0.3      5.2 [+ or -] 1.5
    deiminase, type II (b)
Down-regulated genes
  Solute carrier family        2.9 [+ or -] 0.3      2.5 [+ or -] 0.1
    29a1
  Lymphocyte specific 1        2.3 [+ or -] 0.2      1.8 [+ or -] 0.1
  Claudin 5                    2.8 [+ or -] 0.1      2.7 [+ or -] 0.5
  Potassium channel td12       2.0 [+ or -] 0.04     2.1 [+ or -] 0.1
  Zinc finger homeobox 1a      1.7 [+ or -] 0.1      1.8 [+ or -] 0.01
  Monoamine oxidase A          2.7 [+ or -] 0.2      2.5 [+ or -] 0.4
  Histidine decarboxylase      7.1 [+ or -] 0.6      5.4 [+ or -] 0.8
  [alpha]-2 Adrenergic         4.2 [+ or -] 1.1      3.6 [+ or -] 0.3
    receptor
  Transcription factor 21      2.2 [+ or -] 0.2      2.0 [+ or -] 0.1
  Homeobox D8                  2.8 [+ or -] 0.03     2.5 [+ or -] 0.2
  Carboxypeptidase X2          5.2 [+ or -] 1.0      4.2 [+ or -] 0.2
  RIKEN cDNA A230106A15        4.7 [+ or -] 0.5      4.2 [+ or -] 0.7
  Reduced expression 3         3.5 [+ or -] 0.3      3.2 [+ or -] 0.4
  TGF-[beta] binding           2.1 [+ or -] 0.1      1.8 [+ or -] 0.2
    protein 4
  Keratoepithelin (b)         12.6 [+ or -] 0.9      9.7 [+ or -] 3.6
  GLI-Kruppel family          12.2 [+ or -] 2.6      8.2 [+ or -] 2.6
    member GLI

Abbreviations: CEA, carcinoembrionary antigen; SRC, steroid receptor
coactivator; TGF, transforming growth factor.

(a) Gene names (derived from the NetAffx database; Liu et al. 2003),
GenBank accession numbers (GenBank 2004), and mean ([+ or -] SDI fold
induction/repression of gene expression are shown in the same order
as the gene cluster in Figure 3C. (b) Genes mentioned in the text.

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Jonathan G. Moggs, John Ashby, Helen Tinwell, Fei Ling Lim, David J. Moore, Ian Kimber, and George Orphanides

Syngenta CTL, Alderley Park, Cheshire, United Kingdom

Address correspondence to J.G. Moggs, Syngenta CTL, Alderley Park, Cheshire, SK10 4TJ UK. Telephone: [phone]. Fax: [phone]. E-mail: [e-mail]

We thank A. Soames for the histopathology data, I. Kupershmidt and E. Hunter (Silicon Genetics) for advice on statistical analysis of microarray data, and T. Barlow (Food Standards Agency) for critical comments.

This work was partially supported by the U.K. Food Standards Agency.

The authors declare they have no competing financial interests.

Received 12 February 2004; accepted 19 May 2004.