The increase in expression of LFA1, an adhesion molecule with a crucial role in inflammation (Wacholtz et al., 1989), on human PBMCs with aging may contribute to the progression of age-associated diseases (Franceschi et al., 2000; Soda et al., 2005). Recent studies have shown the involvement of inflammation in the pathogenesis of many age-associated diseases (Franceschi et al., 2000). Anti-inflammatory substances and drugs seem to inhibit pathogenesis of these diseases (Demierre et al., 2005). In this study, we examined the effect of oral polyamines on age-associated pathology and the longevity of mice.

Animals

The experimental design was approved by the Institutional Review Board of Jichi Medical University, and all procedures performed on animals followed the Principles of Laboratory Animal Care (NIH Publication No. 85-23, revised 1985). Eight-week-old male Jc1:ICR mice, purchased from Saitama Doubutsu (Saitama, Japan), were used. The mice (4–5 per cage) were fed Rodent Laboratory Chow (Nippon Bio-Supply, Tokyo, Japan) and water ad libitum and allowed to adapt to the laboratory environment for approximately 2 weeks prior to initiation of experiments. The animals were then randomly divided into three groups (fed chow containing either high polyamine, normal polyamine, or low polyamine), housed in a temperature-controlled (22 C) cage supplied with high efficiency particulate-arresting (HEPA; 0.3 lm) filtered air, and maintained under a 12 h light/dark diurnal cycle. 

They were fed ad libitum Rodent Laboratory Chow until 24 weeks of age, and fed thereafter the same chow supplemented with one of three different polyamine concentrations. For the measurement of blood polyamine levels, 30 mice per group were bred. Survival was measured and pathology examinations conducted in approximately 53 mice per group. One mouse in the low polyamine group died before the switch to experimental chow; therefore, there were 52, 53, and 53 mice in the low, normal, and high polyamine groups, respectively.

Chows

Ingredients of the experimental chows were prepared and mixed by Nippon Clea Co. (Tokyo, Japan) based on formulations used for standard laboratory chow (CE-2). In order to decrease polyamine content in the low polyamine chow from the existing standard chow, soy products in which polyamine concentration is high and that exist in large amounts in standard chow, were replaced by ingredients with relatively low polyamine concentration. Thus, casein was used instead of soy cake and lard was used instead of soybean oil. The composition and raw materials of the low polyamine chow are shown in Table 1. The levels of polyamines, spermine and spermidine, were selected on the basis of the polyamine content in foods and subacute toxicity of polyamine (Bardócz et al., 1993; Okamoto et al., 1997; Sousadias and Smith, 1995; Til et al., 1997).

 High polyamine chow was prepared by adding synthetic spermine (Wako Pure Chemical Industries Ltd., Osaka, Japan), spermidine (Sigma Chemical. Co., St. Louis, MO), and putrescine, a diamine with two amino groups, to low polyamine chow (final concentration [w/w]: 0.015%, 0.06%, and 0.015%, respectively). Normal polyamine chow was prepared by adding spermine, spermidine, and putrescine to low polyamine chow (final concentration [w/w]: 0.002%, 0.008%, and 0.002%, respectively). Ingredients were mixed at 60 C to prevent evaporation of polyamines. The mixture was pelleted, and the polyamine concentrations in the three chows were confirmed by high performance liquid chromatography (HPLC). 

Data collection

For each mouse, body weight was determined every other week at 10 am, and survival was checked every other day. Food consumption was measured by subtracting the amount remaining from the amount provided three times a week, and total consumption was calculated at age 38, 44, and 50 weeks old. Blood sampling was performed in mice for the determination of blood polyamine levels at the 8th, 16th, and 26th week after start of feeding with experimental chow. At the time of blood sampling, any diseased mice, such as those that appeared debilitated or with obvious tumor(s) on the body surface or in the organs and tissues, were excluded. 

Blood was drawn under anesthesia with pentobarbital (60 lg/g body weight) from the right atrium with a 1-ml syringe fitted with a 26 G needle. Whole blood samples were kept in EDTA-coated tubes at 80 C until the assay was performed. When mice reached 88 weeks of age, the survival study was concluded, and animals were euthanized to obtain organs and tissues. Only mice with no apparent tumors or pathological abnormalities on the body surface or in the viscera were selected, and six mice each from the high polyamine chow group and the low polyamine chow group were used for the histopathological and immunohistochemical evaluations. The kidney, liver, pancreas, and gastrocnemius muscle were evaluated histologically and immunohistochemically.

Determination of polyamine concentrations

Whole blood samples in EDTA-coated tubes were stored at 80 C. To measure polyamine, each sample was twice thawed and sonicated for 5 min and centrifuged at 18,000g for 10 min. Each of the resulting supernatant (100 ll) was transferred to a new microfuge tube containing 20% trichloroacetic acid (100 ll) with 20 lM N-(3-aminopropyl) cadaverine as an internal standard. After centrifugation at 18,000g for 10 min, each supernatant was collected and stored at 20 C until measurement by HPLC. 

The HPLC conditions were as follows: column, cation exchange resin, JEOL LC-R-2, 4.6 mm 8 cm; elution buffer, a mixture of 12.5% (v/v) of methanol and 87.5% (v/v) of 0.28 M sodium citrate buffer, pH 5.5, containing 2.0 M sodium chloride; flow rate 0.5 ml/min; column temperature, 65 C; post-column reagent solution, 6 mM o-phthaldialdehyde in 0.4 M potassium borate buffer, pH 10.4, containing 0.2% mercaptoethanol, 0.1% Brij 35; flow rate, 0.25 ml/min at 65 C; fluorescence detection, excitation 345 nm, emission 450 nm.

Histological and immunohistochemical examinations

The tissues were collected and fixed in 10% formaldehyde, embedded in paraffin, and cut into serial 3-lm sections. One of the sections was stained with hematoxylin and eosin (H&E). Histological features in the high polyamine group were compared to those in the low polyamine groups and those in young mice (age, 20 weeks). Sections were stained with a rabbit polyclonal antibody to rat SMP-30 (1:150; Senescence Marker Protein-30; Shima Laboratories, Tokyo, Japan), and the staining was visualized using a ChemMate Evision/peroxidase complex kit (DAKO Japan, Kyoto, Japan). Intensity of immunohistochemical staining was graded relative to the level of background staining and quantified on a scale of 0–5 (Table 4). The histological and immunohistochemical examinations were conducted by two veterinary pathologists, and their evaluations were peer reviewed.

Statistical analysis

Data are expressed as means ± SD. Comparisons of three groups of data were analyzed by the Kruskal–Wallis test, and comparisons among two groups of data were analyzed by the Mann–Whitney test. A p value less than 0.05 was considered to be statistically significant. The survival times were estimated using the Kaplan–Meier method. Comparison of survival between groups was tested using a generalized Wilcoxon analysis. 

Food consumption and changes in body weight

The amount of consumption of chow was different among the three groups of mice (p = 0.043, Kruskal–Wallis test). Average food consumption of mice fed the low polyamine chow was similar to that of mice fed normal polyamine chow (6.37 ± 1.08 g/day/mouse [low polyamine] vs. 6.94 ± 0.30 g/day/mouse [normal polyamine]; p = 0.354 by Mann–Whitney test). Mice in the high polyamine group (7.70 ± 1.11 g/day/mouse) consumed significantly more chow (p = 0.031: vs. low polyamine chow group, and p = 0.047: vs. normal polyamine chow group). Nevertheless, the body weight in all three groups was similar (Fig. 1) from age 11 to 55 weeks. 

Polyamine levels in mouse whole blood

After 8 and 16 weeks of consuming the experimental chows, the mean spermine concentrations in the blood from six mice per group did not significantly differ between the three groups (Table 2). On the 26th week of feeding high polyamine chow (when the mice were 50 weeks old), the mean blood spermine concentration increased with very wide inter-individual variability (range = 3.4– 22.7 lmol/L). The mean spermine concentration from the high polyamine chow group tended to be higher, but not statistically different, from the normal polyamine group (p = 0.085) and the low polyamine group (p = 0.058) (Table 2). 

Similar to blood spermine concentrations, the mean blood spermidine concentrations from six mice per group did not differ between the three groups after 8 and 16 weeks of consuming the experimental chows. On the 26th week of feeding, consumption of high polyamine chow increased blood spermidine concentrations in some animals and increased the variability of spermidine concentration (range = 29.1–100.3 lmol/L). The mean blood spermidine concentration in the high polyamine group was higher than that in the normal polyamine group (p = 0.038) and the low polyamine group (p = 0.004) (Table 2).

Discussion

The enzymatic activities of spermine/spermidine synthases (which catalyze the synthesis of spermine from spermidine and spermidine from putrescine) decrease gradually with aging and are not under regulatory and rate limiting control (Morgan, 1998). Therefore, intracellular de novo synthesis of polyamines, spermine and spermidine, decreases with aging (Das and Kanungo, 1982; Morgan, 1998). However, polyamines in foods have been shown to be absorbed quickly from the intestinal lumen and distributed to organs and tissues in the body (Bardocz et al., 1990; Uda et al., 2003).