Sodium fluoride (NaF, molecular weight 41.99) was procured from Sigma Chemical (St. Louis, MO, USA). Anti-Bax and anti-Bcl-2 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA) and total anti-oxidant capacity (T-AOC) test kits were purchased from NanJing Jiancheng Bioengineering Institute (Nanjing, China). All other analytical laboratory chemicals and reagents were obtained from Sigma, Invitrogen (Carlsbad, CA, USA) and Sangon Biotech Co., Ltd. (Shanghai, China). All chemicals and solvents were analytical grade. MPPP extracted and separated from maize purple plant was produced by Liaoning Dongya Seeds Co., Ltd (Shenyang, China). Zhou et al. [17] reported that MPPP used in this study contains 45.96% cyaniding-3-glucoside and 12.99% 3’,4’-dihydroxy anthocyanin-3-glucoside. MPPP was mixed in standard rodent diet, which was performed by the Shenyang Qianmin Animal Feeds Factory (Shenyang, China).

Forty male and 40 female healthy weanling Wistar rats (60–80 g) obtained from Experimental Animal Center of China Medical University (Shenyang, China) were acclimated for a week before the start of the experiment and fed common basal pellet diet and water ad libitum. Rats were assigned randomly to four groups containing 20 rats per group: Group I: Control rats receiving unfluoridated water and common basal pellet diet. Group II: Experimental rats receiving drinking water containing 100 ppm fluoride and common basal pellet diet. Group III: Experimental rats receiving drinking water containing 100 ppm fluoride and diet mixed with 5 g/kg MPPP. Group IV: Experimental rats receiving drinking water containing 100 ppm fluoride and diet mixed with 10 g/kg MPPP. Although the fluoride concentration in drinking water cannot reach 100 ppm in endemic fluorosis areas, the fluoride dose used (100 ppm) in this study was selected with the following considerations in mind: (1) The rat fluorosis model needs to be established in order to observe the protective effects of MPPP against fluorosis. (2) Rats treated for 12 weeks with 100 ppm fluoride did not show any apparent signs of toxicity in our previous experiments. (3) Species differences exist between rats and human beings.

During the course of treatment, daily water consumption, body weight gain and feed consumption were recorded periodically. All of the rats were kept in ventilated cages at 23–27 °C, with 55%–60% humidity and 12/12 h light/dark cycles. During the last exposure week, five rats per sex per group were placed in metabolic cages and 24 h urine samples were collected. After 12 weeks, exposure was stopped and rats were sacrificed under light ether anesthesia. Blood was collected from the abdominal aorta for the separation of serum. The livers and kidneys were removed immediately and weighed, and did not present any abnormality in appearance. The left external lobe of liver and cortex of left kidney were homogenized in chilled potassium chloride and centrifuged at 3,000 g for 10 min at 4 °C. The supernatant was used for biochemical analysis. Parts of middle lobe of liver and cortex of the right kidney were fixed in 2.5% glutaraldehyde for ultrastructure examination. All samples were stored at –80 °C until analysis. The experimental procedure used in this study met the guidelines of the Animal Care and Use Committee of China Medical University.

The fluoride ion (F⁻) levels in serum and urine (10 rats each group) were measured potentiometrically directly after dilution with equal volumes of total ionic strength adjustment buffer using a fluoride ion-specific electrode and results were expressed as μg/mL. The concentrations of F⁻ in livers and kidney were determined according to the method of enzyme standard instrument-fluorine reagent colorimetric method [18]. Briefly, about 50 mg of tissue sample digested with lipase and protease was dissolved in an acid mixture (nitric acid and silver nitrate) in a closed compartment. The cover of the compartment was overlaid with saturated sodium hydroxide. After neutralization for 24 h, fluorine reagent was added into the mixture, and the values were calculated from a standard curve. The amount of fluoride was expressed in microgram of fluoride per kilogram of dry tissue.

The body weight of each rat was recorded before killing. Liver and kidney were dissected out carefully, blotted free of blood and fresh weight was recorded. Organo-somatic index (OSI) was calculated by the following formula OSI = Weight of the organ / Weight of the body × 100.

Liver and kidney tissues were fixed in a solution containing 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4 °C, followed by rinsing in 0.1 M sodium cacodylate buffer. Subsequently, samples were postfixed in 1% osmium tetroxide, dehydrated through graded ethanol series, and embedded in Spurr’s resin. Resin sections of 50 nm were cut on resin microtome and ultrathin sections were stained with uranyl acetate and lead citrate, then observed and photographed using a HITACHI H-7650 (Hitachi Ltd., Tokyo, Japan) transmission electron microscope (TEM).

GSH levels and SOD activities of serum and tissues (20 rats each group) were measured with an improved 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) method and hydroxylamine assay, respectively, provided by commercial test kits. GSH-Px activity was determined following the commercial test kit method, based on the reaction between glutathione remaining after the action of GPX and 5,5-dithiobis (2-nitrobenzoic acid) to form a complex that absorbed maximally at 412 nm. T-AOC activity was measured using a commercial kit, which was based on the production of stable color of the Fe²⁺-O-phenanthroline complex. The LPO was assessed by measuring MDA level. The quantification was based on measuring formation of thiobarbituric acid reactive substances (TBARS) according to the manufacturer’s protocol. Protein concentrations of tissues were determined by the method of Bradford [19] to normalize the levels of GSH, MDA and SOD activity. The results were expressed as mg/L for GSH, U/ml for SOD, GSH-Px and T-AOC, and nmol/ml for MDA in serum, and as mg/g protein for GSH, U/mg protein for SOD and GSH-Px, and nmol/mg protein for MDA in liver and kidney tissues.

Frozen liver and kidney tissue samples (five rats from each group) weighing 0.2 to 0.5 mg were cut into shivers with scissors first and washed with cold saline to remove blood. Tissue samples were lysed in ice-cold lysis buffer by supersound, and then centrifuged at 12,000 g for 25 min at 4 °C. Total protein content in each sample was quantified using the protein assay kit. Lysates with 30 μg protein each were subjected to electrophoresis on 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA) at 12 volt 40 min. After the membranes were blocked in 5% fat-free dry milk in Tween 20 Tris-buffered saline (TBST) for 1.5 h, the membranes were incubated with primary antibodies at a dilution of 1:100 anti- Bcl-2, 1:1,000 anti- Bax and 1:1,000 polyclonal antibodies to β-actin, overnight at 4 °C. After washed with TBST, the membranes were incubated in horseradish peroxidase-conjugated secondary antibody (1:6,000) for 1 h and washed with TBST three times. Protein bands were detected by enhanced chemiluminescence reagent, and then densitometric analysis was performed with the Gel-Pro Analyzer version 3.0 software.

The data were analyzed using the SPSS 13.0 software. The results are reported as means ± standard error (SEM). Differences between treatment groups were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s test to compare means between the different treatment groups. A p < 0.05 was considered significant.

Due to the changes of water and feed consumption during rat growth, the real fluoride intake or the real MPPP intake of rats changed with the amount of water and feed consumed. According to the daily water consumption and feed consumption of rats during the course of treatment, the real average amount of MPPP intake of rats was 0.045 g MPPP/100 g body weight/day for the group of diet mixed with 5 g/kg MPPP and 0.094 g MPPP/100 g body weight/day for the group of diet mixed with 10 g/kg MPPP. The average amount of real fluoride intake of rats was 1.699 mg F/100 g body weight/day. The fluoride levels of blood, urine, liver and kidney in 100 ppm fluoride treated rats were increased significantly compared with the non-fluoride-treated group (group I), but differences were not seen between the fluoride only treated group (group II) and fluoride-mixed MPPP treated groups (group III and IV). Treatment with MPPP at the both dose levels (5 g/kg and 10 g/kg) did not alter the fluoride levels of blood, urine, liver and kidney in 100 ppm fluoride -treated rats (Table 1).

There was no significant difference in body weight (weight gain) recorded between the control and treatment groups (data not shown). No significant change in the OSI of liver of any treatment group was observed as compared with the control group. A significant decrease (p < 0.05) in the OSI of kidney was found in fluoride alone treated rats (group II). The treatment of fluoride along with MPPP significantly attenuated the decrease induced by fluoride in the OSI of kidney (p < 0.05), and the OSI of kidney of Group IV was higher than that of Group II (p < 0.05), see Table 2. A protective effect of MPPP against the decrease of OSI of kidney caused by fluoride can be concluded.

Transmission electron microscope (TEM) analysis of the ultrastructure of liver and kidney in rats is shown in Figure 1. In the control group, liver and kidney exhibited normal integral structures, and the liver cells showed one or more oval nuclei with visible clear nucleoli and double nuclear membranes, abundant mitochondria, endoplasmic reticulum, and ribosomes in the cytoplasm of the liver cells (Figure 1a).

In fluoride alone treated rats, liver cells showed cytomorphosis, vague cell boundaries, shrinkage of nuclear and cell volume, swollen mitochondria, broken or disappeared ridges, swollen endoplasmic reticulum, and apoptotic bodies (Figure 1b). In the rats treated with fluoride and MPPP (groups III and IV), liver cells still showed swollen mitochondria, but the numbers of cells with swollen mitochondria was decreased compared to group II, and pathological nuclear changes were attenuated (Figure 1c,d). The kidney cells in the control group were nearly round, with regular shape and clear cell boundaries, and the nucleus, mostly round or oval, was located in the center of the cells (Figure 1e). Kidney proximal tubule cells of fluoride-treated rats showed swollen mitochondria, vague or disappeared ridges, vacuole formation, and cell nuclei were pushed towards the basement membrane (Figure 1f). The swollen mitochondria of kidney proximal tubule cells in the fluoride plus MPPP groups showed a moderate decrease compared with the fluoride alone group (Figure 1g,h).

In the fluoride alone treated group, the MDA levels in serum and liver tissue were significantly higher than those of the control group (p < 0.05). The presence of 5 g/kg MPPP reduced the elevation of MDA levels in blood and liver of fluoride alone -treated rats. No statistically significant changes in MDA levels in kidney tissue were observed for all groups (Table 3, Figure 2).

The antioxidative status was assessed by measuring the activities of SOD and GSH-Px, and GSH levels in blood, liver and kidney, as well as T-AOC activity of blood. The results showed that there were no significant differences for SOD activities in blood and liver among groups. SOD activity in kidney of rats co-treated with 5 g/kg MPPP and fluoride was significant higher than that of fluoride alone -treated rats (p < 0.01). However, SOD activity in kidney of rats co-treated with 10 g/kg MPPP and fluoride decreased and was significant lower than that of control group and fluoride plus 5 g/kg MPPP group. The GSH-Px activities in the blood and kidney were significantly increased in the fluoride plus 5 g/kg MPPP group compared with the fluoride alone-treated group, and decreased in the fluoride plus 10 g/kg MPPP group compared with the fluoride plus 5 g/kg MPPP group (p < 0.01). No significant changes were observed in liver for GSH-Px activity among the groups. The increases of GSH levels in liver and kidney are shown in Figure 2. The opposite was observed in the 5 g/kg MPPP and fluoride co-treated groups. Lower dose (5 g/kg) MPPP induced a significant increase in GSH levels compared to the control group and fluoride alone-treated group (p < 0.05) in liver and kidney. Higher dose (10 g/kg) MPPP also increased GSH levels compared with the control group and the fluoride alone-treated group in liver (p < 0.01), but this was not found in kidney. There were no significant changes in blood GSH level and T-AOC activity among all groups (p > 0.05).

Expressions of Bax and Bcl-2 in liver and kidney were measured by western blot analysis, as shown in Figure 3. The protein expressions of Bax in liver and kidney dramatically increased in fluoride alone-treated rats compares with the control group rats, and decreased in 5 g/kg and 10 g/kg MPPP groups compared with fluoride alone-treated group.

On the contrary, Bcl-2 protein expressions of liver and kidney in the fluoride alone-treated group decreased significantly compared with the control group rats, whereas Bcl-2 protein of liver in 5 g/kg and 10 g/kg MPPP groups was elevated compared with the fluoride alone-treated group.