Male rats of the Wistar strain (CLEA Japan, Tokyo, Japan), 3 weeks of age, were used. Throughout the experiment, all rats were housed under controlled conditions (24 ± 0.5°C and 12 hours of light from 6 A.M. to 6 P.M.). The animals were permitted free access to normal commercial feed (AIN-76A, Oriental Yeast, Tokyo, Japan), which was a purified diet based on casein as a sole source of protein, and tap water ad libitum at the animal research center of the Kagawa Nutrition University (Saitama, Japan). All experimental procedures conformed to the regulations described in the Guide to the Care and Use of Laboratory Animals of the U.S. National Institutes of Health (NIH).

At 4 weeks of age, the animals were divided into two groups of 10 rats each, i.e. a control group and an experimental group. The animals in the control and experimental groups were each fed 10 g per day of the control diet (AIN-76A) and the low-zinc diet (F2ZnDD, Oriental Yeast) 3, respectively, and weighed every 7 days. Five animals out of each group were given a single intravenous injection of bromodeoxyuridine (BrdU; 10 ml/kg body weight, Cell proliferation kit, Amersham, RPN 20, Lot 315827A, Pack 318198, UK) into the tail vein, 2 hours before autopsy. Each rat was bled, at the age of 10 weeks, by cardiac puncture under deep anesthesia (1.5 g urethane/kg body weight, Merck, Darmstadt, Germany), and the spleen, liver, kidney, adrenals, testis, ventral prostate and femur were removed and weighed. Each prostate obtained was immediately fixed in a 10% formaldehyde buffer solution (pH 7.2) to evaluate the S-phase cells in the prostate. Examinations of hemograms, plasma levels of hormones, and biochemical markers were performed later (SRL, Inc., Tokyo, Japan).

The levels of BrdU incorporated into the cellular DNA was determined by a monoclonal anti-BrdU antibody; the measurement proceeded according to the manufacture's instructions. Six sections of each prostate were randomly chosen and BrdU-immunoreactive cells were counted among 400 cells per section. The results are expressed in term of BrdU-immunoreactive (S-phase) cells as a percentage of total cells.

Thymidylate synthase (TS; EC 2.1.1.45) and thymidine kinase (TK; EC 2.7.1.21) catalyze the formation of deoxythymidine monophosphate (dTMP) by the methylation of deoxyuridine monophosphate (dUMP) with the concomitant conversion of N⁵,N¹⁰-methylenetetrahydrofolic acid to 7,8-dihydrofolic acid via the de novo pathway and the phosphorylation of thymidine via the salvage pathway, respectively 4. RT-PCR was performed for quantitative analysis of TS and TK mRNA levels in the prostate. Total RNA was extracted from each prostate sample with a QuickPrep™ Total RNA Extraction Kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). Reverse transcription was performed using oligo (dT) primers [0.5 ml oligo (dT)_12–18 primers (1.0 μg/ml) (GIBCO BRL, Gaithersburg, MD, USA)] with a SUPERSCRIPT™ Preamplification System (GIBCO BRL) according to the supplier's instructions. Once the cDNA copy had been created using the mRNA template, the PCR was conducted immediately, as outlined below. Alternatively, the cDNA was stored at -20°C until use. The PCR was performed with recombinant Taq DNA polymerase (Nippon Gene, Tokyo) according to the manufacture's instructions. The RNA (1.0 μg) was subjected to RT-PCR using the primers for TS and TK cDNA for 34 cycles (each cycle consisted of denaturing at 94°C for 40 seconds, annealing at 55°C for 40 seconds and extension at 72°C for 40 seconds) in a Gene Amp PCR System 2400(Perkin Elmer, Branchburg, NJ, USA). RT-PCR was carried out with three sets of primers(TS: 5'-TGAATGGGGAGCTATCTTGCCA-3' and 5'-TCGTTGGATGTGG-ATTATACCC-3'; TK: 5'-TAGCACAGGCGGCACACGGAGT-3' and 5'-TGCTCCGCG-ATGTGACCCAGGA-3'; and β-actin: 5'-AGGCCCAGAGCAAGAGAGGCAT-3' and 5'-CATGGCTGGGGTGTTGAAGGTC-3'). The levels of TS mRNA and TK mRNA were determined by densitometry from photographs taken with an image analyzer (AE6920-MF Densitograph, ATTO, Tokyo), and are expressed as a ratio of the mRNA level of β-actin as an internal standard.

All parameters were expressed as the mean ± standard error (SEM). Statistical analyses were performed using the unpaired t-test and one-way analysis of variance (ANOVA). The p values less than 0.05 were considered to be statistically significant.

There were no differences in mean body weight between the two groups for 2 weeks after the start of the study, i.e. at age 4 and 5 weeks. However, 3 weeks into the study, i.e. at the age of 6 weeks, mean body weight in the low-zinc diet group was less than 90% of the control value (p < 0.01), and as the experiment continued, it dropped to approximately 80% (p < 0.01) (data not shown).

Although there was little difference in the wet weight of the adrenals, the wet weights of the spleen, liver, kidney, testis, ventral prostate and femur were markedly lower in the low-zinc diet group than the control group (p < 0.01) (data not shown).

Concentrations of hemoglobin (Hb) and hematocrit (Ht), and numbers of erythrocytes (RBC), leukocytes (WBC) and platelets (Pt) were lower in the low-zinc diet group than the control group (p < 0.01 and 0.05) (data not shown).

Although plasma levels of glucose, free fatty acid and triglyceride were not affected by the low-zinc diet, plasma level of total protein, albumin, total cholesterol and phospholipid were lowered compared with the control (p < 0.01 and 0.05) (Table 1). The activities of glutamic-oxaloacetic transaminase and lactate dehydrogenase were altered by the low-zinc diet though the differences were not statistically significant. Alkaline phosphatase activity in animals fed the low-zinc diet was markedly reduced to less than 60% of that in the controls (p < 0.01). Although there were no differences in plasma concentrations of sodium (Na), chloride (Cl), potassium (K), calcium (Ca) and inorganic phosphorus (iP) between the groups, the zinc (Zn) concentration in rats fed the low-zinc diet was markedly reduced to less than 60% of that in the controls (p < 0.01). There were no differences in plasma levels of growth hormone and corticosterone between the groups. However, plasma levels of luteinizing hormone and testosterone were lowered by the low-zinc diet (p < 0.01).

Although BrdU-immunoreactive cells amounted to 3.19 ± 0.69% of the total cells in the prostate of the normal control (Fig. 1C), that percentage decreased to only 1.31 ± 0.48% in the prostate of rats fed the low-zinc diet (p < 0.05) (Fig. 1Z).

The mRNA expression levels of prostatic thymidylate synthase (p < 0.05) and thymidine kinase (p < 0.01) were markedly reduced to 50% of the normal control by the low-zinc diet (Fig. 2).