Nutrient-Gene Interactions - Semantic Scholar

Nutrient-Gene Interactions

Investigation of Lymphocyte Gene Expression for Use as Biomarkers for Zinc Status in Humans1 Karl B. Andree,* Jihye Kim,† Catherine P. Kirschke,* Jeff P. Gregg,** HeeYoung Paik,† Hyojee Joung,‡ Leslie Woodhouse,* Janet C. King,†† and Liping Huang*‡‡2

ABSTRACT A bioassay for zinc status in humans has been sought due to the importance of zinc, an essential trace metal, for many divergent functions in the human body; however, a sensitive bioassay for zinc status in humans is lacking. To address this issue, we established gene expression profiles of human lymphoblastoid cells treated with 0 or 30 ␮mol/L ZnSO4 using microarray technology. A limited number of genes were responsive to 30 ␮mol/L zinc based on the analysis of Affymetrix human genome U133A GeneChips. We also examined the gene expression patterns of zinc transporters in human lymphoblastoid cells using quantitative RT-PCR analysis. ZNT1 was upregulated in lymphoblastoid cells, whereas ZIP1 was downregulated in response to the increased zinc concentrations in the culture media. To evaluate the potential applications of using both zinc transporter genes as biomarkers of zinc status, we measured the expression levels of ZIP1 and ZNT1 in the peripheral leukocytes collected from 2 different age groups of Korean women. After administration of a zinc supplement (22 mg zinc gluconate/d for 27 d), ZIP1 expression decreased by 17% (P ⬍ 0.01) and 21% (P ⬍ 0.05) in the peripheral leukocytes collected from 15 young (20 –25 y) and 10 elderly (64 –75 y) subjects, respectively. ZNT1 expression was not affected by taking the zinc supplement. These data suggest a potential application of ZIP1 as a biomarker of zinc status in humans. J. Nutr. 134: 1716 –1723, 2004. KEY WORDS:

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zinc transporter

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zinc supplementation

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quantitative RT-PCR

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microarray

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humans

Because of the importance of zinc in maintaining physiologic functions in humans, a reliable assay for the assessment of zinc deficiency has been sought by scientists and clinicians involved in trace metal metabolism research. Different approaches have been used to develop an assay to detect zinc deficiency. Previous studies indicated that human serum zinc levels are under tight homeostatic control. Thus, they would not reflect changes in zinc intakes (18). Assessing changes in enzymatic activities of several zinc-containing enzymes including alkaline phosphatase, 5⬘-nucleotidase, and superoxide dismutase under zinc depletion conditions was inconclusive (19 – 21). Fluctuations in metallothionein (MT)3 expression in blood cells in response to variations in zinc supplementation were demonstrated. The MT transcripts are upregulated by zinc and other heavy metals through the binding of metal ions to metal-responsive transcription factor-1, which in turn is able to bind to the metal regulatory elements in the promoters of the MT genes (22). Grider et al. (23) measured MT con-

Zinc is an essential trace metal for humans (1). It plays important roles in growth and development, skin and bone metabolism, neuropsychiatric performance, immune functions, and hormonal excretions (2–5). Zinc deficiency causes retarded growth, hair loss, skin lesions, emotional disorders, intercurrent infections, and delayed puberty in adolescents (6). Severe zinc deficiency can be present in people with malnutrition, extensive burns, chronic debilitating disorders, chronic renal diseases, and genetic disorders, such as acrodermatitis enteropathica (6 – 8). Mild zinc deficiency has been reported in children and pregnant women (2,9 –15). It was shown that mild zinc deficiency affects growth and neuropsychologic performance in children (16) and may cause retarded fetal growth or shortened pregnancy in pregnant women (17). Although the symptoms of severe zinc deficiency are obvious, assessment of marginal zinc deficiency is difficult due to the lack of clinical signs and reliable laboratory indicators.

1 Supported by the U.S. Department of Agriculture (CRIS-5306 –53000 – 00800D), the USDA Specific Cooperative Agreement (58 –5306-1– 451), and the Korea Research Foundation (KRF 2000 – 042-D00103). 2 To whom correspondence should be addressed. E-mail: [email protected].

3 Abbreviations used: BACT, ␤-actin; BAG1, BCL-2 associated athanogene; BLK, B lymphoid tyrosine kinase; ERCC1, excision repair cross-complementing rodent repair deficiency, complementation group1; FBS, fetal bovine serum; MT, metallothionein; ZIP, ZRT1 and IRT1-like protein; ZNT, zinc transporter.

0022-3166/04 $8.00 © 2004 American Society for Nutritional Sciences. Manuscript received 21 January 2004. Initial review completed 23 February 2004. Revision accepted 30 April 2004. 1716

Downloaded from jn.nutrition.org at USDA, National Agricultural Library on August 3, 2007

*Western Human Nutrition Research Center, Agriculture Research Service, U.S. Department of Agriculture; † Department of Food and Nutrition, Seoul National University, Seoul, South Korea; **Department of Medical Pathology, University of California Davis Medical Center; ‡The School of Public Health, Seoul National University, Seoul, South Korea; ††Division of Nutritional Genomics, Children’s Hospital Oakland Research Institute, Oakland, CA; and ‡‡Department of Nutrition and Rowe Program in Genetics, University of California at Davis, Davis, CA 95616

REGULATION OF ZINC TRANSPORTERS BY ZINC

SUBJECTS AND METHODS Cell line. A human lymphoblastoid cell line was established by Epstein-Barr virus transformation of peripheral B lymphocytes as described (42). Lymphoblastoid cells were maintained in RPMI 1640 medium with 10% (v:v) fetal bovine serum (FBS) (Invitrogen), 100 ⫻ 103 U/L penicillin G, 0.1 g/L streptomycin, and 0.25 g/L Fungizone (Invitrogen). In the zinc treatment experiments, the Chelex-treated FBS was replaced by regular FBS. The concentrations of zinc, iron, copper, manganese, magnesium, and calcium in the medium containing 10% (v:v) chelex-treated FBS were 0.032– 0.072, 0.863– 0.974, 0.045– 0.047, 0.01– 0.015, 83.06 – 87.22, and 91.33–93.54 ␮mol/L, respectively. The concentrations of zinc, iron, copper, manganese, magnesium, and calcium in the medium containing 10% (v:v) regular FBS were 1.166 –1.174, 0.76 – 0.787, 0.0865– 0.887, 0.012– 0.013, 104.84 –110.17, and 149.46 –154.11 ␮mol/L, respectively. There were no detectable cobalt or cadmium ions in the culture medium containing either Chelex-treated FBS or regular FBS.

Blood collection. Blood samples were collected from fasting subjects in syringes (5-mL syringe with EDTA) before and after zinc supplementation and were kept on ice until processing. Total RNA preparation. Human lymphoblastoid cells were treated with 0, 30, 50, or 100 ␮mol/L ZnSO4 for 24 h in RPMI 1640 media containing 10% (v:v) Chelex-treated FBS, 100 ⫻ 103 U/L penicillin G, 0.1 g/L streptomycin, and 0.25 g/L Fungizone. After ZnSO4 treatment, cells were washed 2 times with 1X PBS, pH 7.4, followed by centrifugation at 180 ⫻ g for 5 min at 4°C. Total RNA was extracted using Trizol reagent following the manufacturer’s protocol (Invitrogen, Cat. #15596-018). Blood leukocyte RNA was extracted using QiaAmp RNA blood mini kit (Qiagen). Briefly, the erythrocytes were lysed by adding the hypotonic buffer supplied in the kit to the blood, and the intact leukocytes were collected by centrifugation at 400 ⫻ g for 10 min at 4°C. The collected leukocytes were then homogenized using QIAshredder spin columns (Qiagen) under highly denaturing conditions. Total RNAs were then purified from the homogenized lysate using QIAamp spin columns (Qiagen). Microarray analysis. The cDNA used in microarray analysis was synthesized from 10 ␮g of total RNA using the SuperScript Choice system (Invitrogen). The cDNA was then transcribed in vitro in the presence of biotin-labeled nucleotides using T7 RNA polymerase after phenol-chloroform extraction and ethanol precipitation. cRNA was purified using the RNeasy mini kit (Qiagen) and fragmented at 94°C for 30 min in a buffer containing 0.2 mol/L Tris-acetate (pH 8.1), 0.5 mol/L potassium acetate, and 0.15 mol/L magnesium acetate. Fragmented cRNA was hybridized overnight at 45°C to the human genome U133A GeneChips (Affymetrix) representing ⬃22,500 transcripts. Hybridization was then detected using a confocal laser scanner (Affymetrix). Duplicate experiments and microarray assays were conducted. The expression data were generated using Microarray Suite 5.0 (MAS 5.0) Affymetrix GeneChip Software. The differentially expressed genes between mock- and ZnSO4-treated samples were identified as an average fold change of ⱖ1.5 or ⱕ1.5 between control and treatment and the P-values for the changes were ⬍0.05. Quantitative PCR. The cDNA used in quantitative PCR was synthesized from 3 ␮g of total RNA using the SuperScript FirstStrand Synthesis for RT-PCR kit (Invitrogen). The cDNA was diluted 4-fold and 2 ␮L cDNA was added to a quantitative PCR using FAM-labeled TaqMan probes purchased from Applied Biosystems. The quantitative PCR reactions were performed on a PRISM ABI 7900HT Sequence Detection System (Applied Biosystems) in triplicate, and the expression of ␤-actin (BACT) was used for normalization. Copy numbers for the zinc transporter genes were calculated using the standard curve method and normalized to the copy numbers of BACT. For other target genes that have no standard curves, changes in expression were calculated using relative quantification as follows: ⌬⌬Ct ⫽ ⌬Ctq ⫺ ⌬Ctcb, where Ct is the cycle number at which amplification rises above the background threshold, ⌬Ct is the change in Ct between 2 test samples, q is the target gene, and cb is the calibrator gene. The calibrator used in this study was BACT because it was shown to be invariant under changing zinc concentrations from microarray assays, quantitative PCR analyses, and Northern blot analysis (unpublished data) (39,43,44). Gene expression was then Ct calculated as 2⫺⌬⌬ (Applied Biosystems). Cloning of ZIP and ZNT gene fragments. The amplicons generated with the Assays-on-Demand primer sets for ACT1, ZIP1, ZIP3, ZNT1, ZNT4, ZNT5, ZNT6, and ZNT7 (Applied Biosystems) were inserted into the cloning vector pCR 2.1-TOPO following the manufacturer’s protocol (Invitrogen, Cat. #051302). The identity of all plasmids was confirmed by sequencing. Plasmids were then linearized by either an EcoRI or XhoI restriction enzyme digestion before making a series of 10-fold dilutions (103 to 108) for establishing standard curves for mRNA quantification. Human subjects. A group of 15 young women (20 –24 y) and 10 elderly women (64 –75 y) were recruited for this study through word of mouth and flyers on the campus of Seoul National University and in the neighboring areas of Seoul, South Korea (Table 1). The general health of subjects was examined and those in good health were selected for the study. Exclusion criteria included a BMI ⬍ 17 or ⬎ 28 kg/m2, smoking, chronic use of alcohol, prescription drugs, oral contraception, vitamin or mineral supplements, a hemoglobin


centrations in RBC of young men and found that the MT levels did fluctuate in response to changes in dietary zinc intakes. Allan et al. (24) reported that the MT-2A mRNA level in human lymphocytes decreased in response to zinc depletion in the diet. However, MT expression levels also change in response to changes in other heavy metals, such as copper, manganese, cadmium, and cobalt in diet. Therefore, the changes in the MT expression levels would not necessarily reflect changes in the zinc intake levels (25). Zinc absorption in humans occurs at the small intestinal mucosa (26). After zinc is absorbed into absorptive enterocytes and transferred into blood, it binds to albumin and later accumulates in liver for redistribution to other organs (27). Several zinc transport proteins that appear to be specifically involved in cellular zinc homeostasis via influx, efflux, or vesicular sequestration were described in mice and humans (28 –37). These specialized zinc transporters, some of which are tissue specific, maintain intracellular zinc concentrations in a narrow physiologic range. The tight homeostatic control of cellular zinc may be achieved by a feedback mechanism by which zinc transporter expression levels change accordingly to avoid cellular zinc toxicity or deficiency when dietary zinc intakes fluctuate (38,39). Two families of zinc transporters have been identified (40,41). The ZNT family decreases cytoplasmic zinc concentrations by secretion, sequestration, or efflux, whereas the ZIP family increases cytoplasmic zinc by influx or release of stored zinc (40,41). Therefore, it is likely that the expression of the ZNT genes would be upregulated, whereas the ZIP genes downregulated when dietary zinc intakes increase. The responsiveness of these zinc transporters to various zinc levels may make them good candidates as cellular indicators of zinc status. The aim of this study was to identify potential cellular indicators of zinc status in vitro by using cultured peripheral blood cells and then to evaluate these newly identified indicators in blood samples collected from human subjects. Microarray technology was used as an initial screen for identifying genes whose expression was influenced by zinc. The microarray analysis results indicated that the ZNT1 expression level changed in response to various zinc levels in cultured lymphoblastoid cells. Following this finding, we examined the expression patterns of other zinc transporters in lymphoblastoid cells by quantitative RT-PCR analysis and identified 2 potential zinc indicators, ZIP1 and ZNT1. We then investigated their expression levels in peripheral white blood cells collected from the human subjects who had taken a zinc supplement for 27 d.

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TABLE 1 Physical characteristics of the young and elderly women1

n Age, y Height, cm Weight, kg BMI, kg/m2 Hemoglobin, g/L

Young

Elderly

15 22.9 ⫾ 1.2 161 ⫾ 3.6 53.4 ⫾ 6.2 20.6 ⫾ 2.4 12.9 ⫾ 0.8

10 7.4 ⫾ 3.3* 159 ⫾ 2.0* 57.2 ⫾ 5.3 22.6 ⫾ 2.3* 12.0 ⫾ 1.0*

1 Values are means ⫾ SEM. * Different from young women, P ⬍ 0.05.

RESULTS Gene expression profile in ZnSO4-treated human lymphoblastoid cells. In an effort to identify a zinc-specific yet sensitive biomarker of zinc status, we established gene expression profiles of human lymphoblastoid cells by microarray analysis using Affymetrix U133A GeneChips (Affymetrix). Total RNA samples were isolated from human lymphoblastoid cells 24 h after 0 or 30 ␮mol/L ZnSO4 treatments. The concentration selected for zinc treatment in the cell culture was based on normal human serum zinc values (9 –16 ␮mol/L) (45). The 0 ␮mol/L ZnSO4 treatment represented a deficit and 30 ␮mol/L zinc mimicked the normal condition. We used 30 ␮mol/L ZnSO4 in the experiments due to the sequestration of some of zinc ions by the Chelex-treated FBS in the culture media. Gene expression profiles were obtained with total RNA isolated from 2 independent experiments. The genes that were induced more than 50% or suppressed at least 30% by zinc treatment are listed in Tables 3 and 4, respectively. Those genes whose expression changed after zinc treatment were classified into functional groups using gene ontology. Among the 21 genes that were induced by extracellular

TABLE 2 Nutrient intakes by young and elderly women during zinc supplementation Nutrient Energy, kJ Protein, g/d Calcium, mg/d Dietary zinc, mg/d Supplementary zinc, mg/d

Young

Elderly

8122.0 ⫾ 2604.0 79.0 ⫾ 23.0 571.0 ⫾ 313.0 9.0 ⫾ 4.0 22.0

7122.0 ⫾ 1620.0 68.0 ⫾ 25.0 561.0 ⫾ 244.0 9.1 ⫾ 5.5 22.0


level ⬍ 105 g/L, and the presence of acute disease or chronic disease such as diabetes, gastrointestinal disorder, or hyperlipidemia, and a usual dietary zinc intake of ⬍5 mg/d or ⬎15 mg/d. Young women completed a 24-h recall and 2-d diet record and elderly women completed a 24-h recall before the study. The mean plasma zinc concentrations for the young and elderly women at the start of the study were 10.11 ⫾ 2.09 and 12.4 ⫾ 1.13 ␮mol/L, respectively, which were in the normal ranges for the Korean population (11.06 ⫾ 2.44 ␮mol/L) (21). All subjects gave their informed consent to participate in this study and the study protocol was reviewed and approved by the Committee on Human Research (The College of Human Ecology at Seoul National University) and the University of California, Davis Office of Human Research Protection. Study design. The women were given 22 mg supplementary zinc as zinc gluconate to take daily for 27 d while living at home. They consumed a self-selected diet during this period. The nutrient intakes during the zinc supplementation period for the subjects were calculated using a nutrient database developed by the Korean Nutrition Society (Table 2). The dietary zinc intake of the subjects was constant during the zinc supplementation period. Statistical analysis. Significant differences in physical characteristics between young and elderly women were determined using the Wilcoxon rank sum test. Results for the expression of zinc transporters (ZIP1 and ZNT1) in the blood leukocytes isolated from human subjects were analyzed using a paired Student’s t test (before and after zinc supplementation) or an unpaired Student’s t test (between young and elderly groups) with a two-tailed distribution. Differences were considered significant at P ⬍ 0.05. Data are means ⫾ SEM.

zinc, MT genes responded the most dramatically (Table 3). Notably, the zinc transporter 1 gene (ZNT1, SLC30A1) is among the genes significantly upregulated by zinc (Table 3). Only 9 genes were downregulated by the zinc treatment. The largest decrease in expression among the 9 genes was the gene encoding fatty acid binding protein 5, a protein involved in fatty acid uptake, transport, and metabolism (Table 4). To confirm the alterations in mRNA levels observed by the microarray assays, the genes including ZNT1, ERCC1 (excision repair cross-complementing rodent repair deficiency, complementation group 1), BLK (B lymphoid tyrosine kinase), and BAG1 (BCL-2 associated athanogene) were selected for verification by quantitative RT-PCR analysis. These genes were selected due to their functional importance in zinc homeostasis and in cell development. The expression levels of these selected genes in the lymphoblastoid cells treated with 0, 50, and 100 ␮mol/L ZnSO4 were examined by quantitative RTPCR using TaqMan probes specific to each gene (Applied Biosystems). The patterns of induction of ZNT1 and ERCC1 were similar to the results of the microarray assays. However, the magnitude of the induction was more prominent in the quantitative RT-PCR assays (Fig. 1). The high magnitude of the induction detected by the TaqMan probes may reflect the fact that higher concentrations of zinc were used in the quantitative RT-PCR experiments. In addition, ZNT1 and ERCC1 showed dose-responsive inductions under the influence of zinc sulfate concentrations used in the experiments (Fig. 1). The changes in the expression of BLK (100% induction) and BAG1 (34% suppression) were also confirmed by quantitative RT-PCR analysis, even though there was no additional change observed when 100 ␮mol/L ZnSO4 was used (Fig. 1). Expression of zinc transporters in human lymphoblastoid cells. Although the Affymetrix U133A GeneChip contains most zinc transporter genes, such as ZIP1, ZIP2, ZIP4, ZNT1, ZNT3, ZNT4, and ZNT5, several other zinc transporter genes including ZIP3, ZNT2, ZNT6, and ZNT7 were not represented on the chip. To assess whether the expressions of these genes were regulated by zinc in human lymphoblastoid cells, we performed quantitative RT-PCR analysis to examine their mRNA expression levels in the lymphoblastoid cells grown in medium containing 0 or 30 ␮mol/L ZnSO4 for 24 h using TaqMan probes specific to each gene (Applied Biosystems). In an effort to compare the expression levels of all zinc transporter genes, we also included the zinc transporter genes that were represented on the U133A GeneChip in our quantitative RT-PCR analysis. The expression results were obtained from 2 independent experiments and each experiment was performed in triplicate. Our quantitative RT-PCR results showed that there was little to no mRNA expression of ZIP2, ZIP4, ZNT2, and ZNT3 genes in human lymphoblastoid cells (data not shown). Among those zinc transporters expressed in lymphoblastoid cells, the ZNT7 gene was expressed at the highest


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TABLE 3 Genes upregulated by zinc treatment in human lymphoblastoid cells Functional group/UniGene names

Symbol

SLC30A1

Fold increase1

7779

2.83

933

1.52

MYST1

84148

2.00

ERCC1

2067

2.30

PTGES2

80142

1.52

1EMU1

129080

1.74

ACADS

35

1.52

5230

1.52

CD22

PGK1 MT1E MT1E-like MT1F MT1G MT1H MT1H-like MT1L MT1X MT2A

4493 AL0316022 4494 4495 4496 AF3333882 4500 4501 4502

4.92 2.14 3.72 2.83 4.00 4.00 4.29 6.96 4.00

UNC119

9094

1.62

MAL BLK

4118 640

1.62 2.00

ZNT9

7555

1.52

1 P ⬍ 0.005. 2 Accession numbers.

level (369 molecules/104 BACT transcripts in 0 ␮mol/L ZnSO4-treated cells and 399 molecules/104 BACT transcripts in 30 ␮mol/L ZnSO4-treated cells), and ZNT6 was expressed at the lowest level (7 molecules/104 BACT transcripts in 0 ␮mol/L ZnSO4-treated cells and 8 molecules/104 BACT transcripts in 30 ␮mol/L ZnSO4-treated cells) (Fig. 2). The ZNT1 gene was the only one regulated in the presence of 30 ␮mol/L zinc sulfate (3.1-fold increase) (Fig. 2). In addition, the expression level of ZIP3 was ⬃39% higher than that of ZIP1, and both ZIP genes had higher expression than the ZNT genes except for ZNT7 in lymphoblastoid cells (Fig. 2). To examine the mRNA expression of zinc transporters under conditions that closely mimic the dietary zinc supplementation in humans, the lymphoblastoid cells were treated with 0, 50, or 100 ␮mol/L zinc sulfate for 24 h. Total RNA was purified and subjected to quantitative RT-PCR analysis. Two zinc transporters, ZIP1 and ZNT1, appeared to be regulated by excess zinc (Fig. 3 and data not shown). The upregulation of ZNT1 expression by zinc was consistent with the previous results (Fig. 1 and 2). Again, ZNT1 expression showed a dose-responsive induction under the influence of zinc sulfate concentrations used in the culture media (Figs. 1 and 3). The relative fold changes in mRNA expression of ZNT1 were 3.4 and 8.9 in 50 and 100 ␮mol/L ZnSO4-treated cells, respectively, compared with the expression levels in untreated cells (Fig. 3). It is interesting to note that although ZIP1 mRNA expression was downregulated 19% (50 ␮mol/L Zn) and 26% (100 ␮mol/L Zn) in response to the excess zinc in the culture

TABLE 4 Genes downregulated by zinc treatment in human lymphoblastoid cells Functional group/UniGene names

Symbol

Locus linker

Decrease1 %

Amino acid metabolism 6-Pyruvoyltetrahydropterin synthase Apoptosis BCL2-associated athanogene Cell cycle arrest Growth arrest and DNA-damageinducible, ␣ Fatty acid (FA) metabolism FA binding protein 5 (psoriasisassociated) FA desaturase 1 Microtubule motor Dynein, cytoplasmic, heavy polypeptide 1 RNA binding protein Sjogren syndrome antigen A2 Unknown function Chromosome 6 open reading frame 11 HT021 1 P ⬍ 0.01.

PTS

5805

42.5

573

34.2

GADD45A

1647

38.3

FABP5 FADS1

2171 3992

50.0 42.5

DNCH1

1778

38.3

SSA2

6738

34.2

9277 57415

42.5 34.2

BAG1

C6orf11 HT021


Carrier Solute carrier family 30, member 1 (zinc transporter) Cell adhesion CD22 antigen Chromatin structure and dynamics MYST histone acetyltransferase DNA repair Excision repair cross-complementing rodent repair deficiency, complementation group 1 Electron transporter Prostaglandin E synthase 2 Extracellular matrix Emilin and multimerin-domain containing protein Fatty acid metabolism Acyl-Coenzyme A dehydrogenase Glycolysis Phosphoglycerate kinase 1 Heavy metal binding protein Metallothionein 1E Metallothionein 1E-like Metallothionein 1F Metallothionein 1G Metallothionein 1H Metallothionein 1H-like Metallothionein 1L Metallothionein 1X Metallothionein 2A Phototransduction Unc-119 homolog (C. elegans) Signal transduction Myelin and lymphocyte protein, T-cell differentiation protein B lymphoid tyrosine kinase RNA binding protein Zinc finger protein 9

Locus linker

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medium, there were no changes in ZIP1 mRNA levels between the cells treated with 0 and 30 ␮mol/L ZnSO4 (Figs. 2 and 3). These data suggest that regulation of ZIP1 mRNA expression is less sensitive and extensive than that of ZNT1 in lymphoblastoid cells. Expression of ZIP1 and ZNT1 in blood leukocytes from human subjects. Our in vitro mRNA expression study using human lymphoblastoid cells suggested that ZIP1 and ZNT1 may serve as specific biomarkers of zinc status. To test this possibility, we collected peripheral leukocytes from human subjects before and after zinc supplementation in a dietary zinc

FIGURE 3 Quantification of the ZIP1 and ZNT1 expression in human lymphoblastoid cells treated with 0, 50, or 100 ␮mol/L ZnSO4 for 24 h. Total RNA was isolated. The gene expressions of ZIP1 and ZNT1 were normalized to the expression of BACT and the gene expression levels are presented as copy numbers/104 BACT transcripts. Values are means ⫾ SEM, n ⫽ 3.

supplementation study in South Korea. Total RNA was isolated from the peripheral leukocytes and subjected to quantitative RT-PCR assays using TaqMan probes specific to the ZIP1 and ZNT1 genes (Applied Biosystems). The means for the ZIP1 mRNA molecules in the blood leukocytes of 15 young women collected at baseline and post-zinc supplementation were 45 and 37/104 BACT molecules, respectively (Fig. 4). The reduction in ZIP1 expression (17%) was significant (P ⬍ 0.01). The means for the ZIP1 mRNA molecules in the blood leukocytes collected from 10 elderly women before and after zinc supplementation were 41 and 26/104 BACT molecules, respectively (Fig. 4). The reduction in the ZIP1 expression (21%) after zinc supplementation was significant (P ⬍ 0.05). Although the expression of ZIP1 was suppressed after 27 d of zinc supplementation in both young and elderly women, there were no changes in the mRNA levels of ZNT1 (Fig. 4). ZIP1 expression levels did not differ between young and elderly women at baseline (45 transcripts/104 BACT transcripts vs. 41 transcripts/104 BACT transcripts, respectively (P ⬎ 0.05). Interestingly, greater percentage decreases in ZIP1 mRNA levels occurred in elderly women after zinc supplementation (Fig. 4). The difference between young and elderly women in ZIP1 expression after zinc supplementation was significant (P ⬍ 0.05). In addition, ZNT1 expression levels did not differ between young and elderly women before or after zinc supplementation (Fig. 4). DISCUSSION

FIGURE 2 Quantification of mRNA expression for the ZIP and ZNT genes in human lymphoblastoid cells treated with 0 or 30 ␮mol/L ZnSO4 for 24 h. Total RNA was isolated. The gene expression was normalized to the expression of BACT and the gene expression levels are presented as copy numbers/104 BACT transcripts. Values are means ⫾ SEM, n ⫽ 3.

The lack of a sensitive and specific biomarker of zinc status has restricted early detection of zinc deficiency in both clinical and public health practices. In an effort to search for a molecular biomarker of zinc status, we used Affymetix GeneChips to investigate the mRNA expression profiles of cultured human lymphoblastoid cells treated with 0 or 30 ␮mol/L zinc


FIGURE 1 Relative gene expression for ZNT1, ERCC1, BLK, and BAG1 in human lymphoblastoid cells treated with 0, 50, or 100 ␮mol/L ZnSO4 for 24 h. Total RNA was isolated. Gene expression was normalized to the expression of BACT. The fold changes were calculated relative to the expression of each gene in 0 ␮mol/L ZnSO4-treated lymphoblastoid cells using the comparative CT method. Values are means ⫾ SEM, n ⫽ 5.


sulfate, which closely mimic the zinc-deficient and zinc-adequate conditions in vivo. We found a limited number of genes whose expression levels were altered by 30 ␮mol/L zinc treatment. The lack of massive changes in gene expression may reflect the effectiveness of intracellular zinc homeostasis due to the importance of zinc for many biological functions. Zincresponsive genes were found in several functional groups in lymphoblastoid cells, in agreement with previous reports demonstrating that zinc has broad range of effects on various metabolic processes including DNA repair, signal transduction, glycolysis, and metabolism of fatty acids and amino acids (46 –50). Metallothioneins including the MT1 and MT2 subtypes responded most dramatically among the genes that were induced by zinc in lymphoblastoid cells. Previous studies demonstrated that MT expression is induced by zinc via the metal responsive elements in the promoter regions (51,52). The percentage change in ZNT1 expression induced by zinc was comparable to that of MT expression, suggesting a similar mode of transcriptional regulation for these genes in humans. Indeed, a previous study demonstrated that the metal response elements in the mouse ZnT1 promoter were required for the induction of ZNT1 expression by zinc, suggesting that the zinc-specific metal responsive elements and cofactors may also underlie the regulation of ZNT1 in humans (53–55). The spectrum of mRNA expression in human lymphoblastoid cells was examined in this study with regard to the expression of zinc transporters that are known to be tissue- and cell type–specific (29,34). ZIP2, ZNT2, ZNT3, and ZIP4 were undetectable in lymphoblastoid cells cultured under either zinc-depleted or -repleted conditions. Among the zinc transporters expressed in lymphoblastoid cells, ZNT7 was the most abundantly expressed; however, its expression was not influenced by extracellular zinc. The function of the ZNT family is to decrease the cytoplasmic zinc concentration by sequestration, secretion, or efflux (40), and this is done in opposition to the ZIP family (41). Thus, it would be likely that the expres-

sion of the ZIP genes is downregulated by zinc and the ZNT genes upregulated. Indeed, in our quantitative RT-PCR study, the expression of ZIP1 in lymphoblastoid cells was downregulated in response to the excess extracellular zinc (Fig. 3). However, the change in ZIP1 expression in lymphoblastoid cells in the presence of physiologic zinc concentrations was very small. In contrast, the regulation of ZNT1 expression by zinc in lymphoblastoid cells was more sensitive to the changes in extracellular zinc concentrations (Figs. 2 and 3). Because the zinc transporter genes are directly involved in zinc metabolism, our first choice for a cellular zinc biomarker was among the ZIP and ZNT genes. On the basis of our microarray and quantitative RT-PCR results, ZIP1 and ZNT1 were chosen as candidate biomarkers of zinc status for further study in the human subjects recruited. We excluded the MT genes as candidate biomarkers of zinc status because the expression of MT is also inducible by other heavy metals. In the present study, we found no significant difference between the 2 age groups of Korean women for ZNT1 expression in peripheral leukocytes before or after zinc supplementation. The expression levels of ZIP1 before zinc supplementation did not differ between the 2 age groups. The expression levels of ZIP1 were suppressed post-zinc supplementation in both young and elderly groups. However, the reduction in the expression of ZIP1 was greater in the elderly group than in the young group. The inverse relationship between zinc supplementation and the ZIP1 expression was significant in both age groups (P ⬍ 0.01 for young and P ⬍ 0.05 for elderly groups). The degrees of reduction in the ZIP1 expression (17% in young women and 21% in elderly women) in response to the dietary zinc supplementation were consistent with those using cultured lymphoblastoid cells (19% reduction). In our zinc supplementation study, the mean decrease in ZIP1 mRNA expression was 4% larger in the elderly women than in the young women. This finding suggests that elderly women may retain more zinc in their bodies after zinc supplementation. Studies in experimental animals and humans showed that aging caused an increase in intestinal zinc absorption and a decrease in endogenous zinc excretion during zinc supplementation, which may lead to a slightly higher zinc accumulation in elderly adults (56,57). Our study on the effect of dietary zinc supplementation on ZIP1 expression agrees with the previous findings. Although many individuals had an increase in ZNT1 expression with zinc supplementation, we did not find a clear relation between ZNT1 expression and increased dietary zinc in blood samples collected from either age group. However, we observed that ZNT1 expression increased sharply for some individuals after zinc supplementation (data not shown). This may be explained by differences in the subjects’ differential white blood cell counts. Whitney et al. (58) demonstrated by cDNA microarray analysis that differences displayed by subgroups of blood cells, such as lymphocyte, neutrophil, and reticulocyte subcomponents, accounted for specific features of interindividual variation in gene expression patterns. Because zinc transporters are expressed in a tissue- and cell type– specific manner, the variation in the relative proportions of specific blood cell subsets in the peripheral blood could affect the sensitivity of detecting the changes in ZNT1 expression. The high variability of ZNT1 expression profiles in our subjects is likely due to the varying proportions of blood cell subtypes between individuals. Because our in vitro gene expression study indicated that ZNT1 is the most sensitive gene responding to the changes in zinc concentrations besides the MT genes in lymphoblastoid cells, ZNT1 may yet be a useful biomarker of zinc status if a cell-sorting protocol is applied


FIGURE 4 The mRNA expression of ZIP1 and ZNT1 in the peripheral leukocytes collected before and after zinc supplementation in young (YW) and elderly Korean women (EW). The gene expressions of ZIP1 and ZNT1 were normalized to the expression of BACT and the gene expression levels are presented as copy numbers/104 BACT transcripts. Values are means ⫾ SEM, n ⫽ 15 (YW) and n ⫽ 10 (EW). *Different from baseline in young women, P ⬍ 0.01 group; #different from baseline in elderly women, P ⬍ 0.05; †different from young women, P ⬍ 0.05.

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ACKNOWLEDGMENTS We thank Erik Gertz and the staff in the Bioanalytical Support Laboratory, Western Human Nutrition Research Center, and staff in the Department of Pathology, School of Medicine, University of California at Davis, for the technical support. We thank Jane Gitschier and Barbara Levinson at University of California at San Francisco for the gift of human lymphoblastoid cell line. We thank Lei Zhang for critical reading of the manuscript.

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before expression analysis (59,60). The approach we are investigating will lend itself to the rapid examination of gene expression in B lymphocytes from whole blood in a large-scale population study for marginal zinc deficiency if a fully automated system has been developed. The test, however, requires a large sample of blood (5 mL of whole blood for isolation of 4 – 8 ⫻ 105 B lymphocytes) and requires particularly careful handling soon after collection. B lymphocytes proliferate in bone marrow and mature in peripheral lymphatic tissues, such as lymph notes and spleen. The fully matured B lymphocytes later enter the circulation with a life span ranging from a few days to 2 wk (61). The changes in the expression of zinc transporters (ZNT1 and ZIP1) in B lymphocytes would likely reflect the changes of zinc contents in bone marrow, peripheral lymph nodes, spleen, and blood. Therefore, detection of the changes in expression of ZNT1 and ZIP1 in the circulating B lymphocytes would be expected to reflect the combination of acute and chronic changes in zinc intakes. In conclusion, this is the first attempt to explore differential gene expression of cellular zinc indicators identified in in vitro assays in blood samples collected from human subjects before and after zinc supplementation. We demonstrated that ZIP1 gene expression is downregulated post-zinc supplementation regardless of age in Korean women. This provided an important measure of changes in zinc intakes in humans. Although the changes in the ZIP1 mRNA expression levels by zinc supplementation are relatively small, the expression of ZIP1 should serve as a useful reference for further study of biomarkers of zinc status in humans.


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