Activity of lysosomal exoglycosidases in submandibular glands of rats intoxicated by cadmium at doses related to human chronic environmental and occupational exposures

Work in cadmium (Cd) smelter and smoking cigarettes damages teeth and oral mucosa which are protected by tissue and salivary glycoconjugates: glycoproteins, glycolipids, and proteoglycans. We worked out a rat model imitating human “environmental” and “occupational” exposure to cadmium using 5 mg Cd and 50 mg Cd/l in drinking water, respectively. In submandibulary glands of exposed to Cd rats, we found the time and dose dependent accumulation of Cd and simultanous decrease in activity of -N-acetylhexosaminidase (HEX). In homogenates of submandibulary glands of control rats, -N-acetylhexosaminidase showed the highest activity. The activities of -mannosidase and -galactosidase were very low. None of these exoglycosidases were inhibited by Cd even at 44 mM concentration.

vary glands, we worked out an animal model (Brzóska et al., 2003).Using this model we evaluated concentrations of cadmium and changes in the activity of glycosidases in rat salivary glands after chronic exposure to 5 mg/l Cd of drinking water (imitating human environmental exposure) and 50 mg/l of drinking water (imitating human occupational exposure).

Animals.
In vivo studies were carried out on 48 male Wistar rats, 2-month-old, at an initial body mass of 180-200 g.All the animals were kept in standard conditions, had unlimited access to standard diet (LSM dry diet, Motycz, Poland) and drinking water (redistilled water with or without CdCl 2 ).The rats were allocated randomly to three experimental groups of 16 animals each.Two groups received, as the only drink, water solution of CdCl 2 at 5 mg/l (equivalent to "environmental" exposure) or 50 mg/l (equivalent to "occupational" exposure).The control group drank redistilled water (without Cd).
Tissue preparation.All the animals used for experiments were fasted for 24 h, then glandectomized under anaesthesia with enfluran and sacrificed.The glands were washed with 0.9% NaCl, trimmed of fat, weighed and homogenized at 4 o C in a Polytron type homogenizer in 10 volumes of 0.15 M KCl containig 0.2% Triton X-100.The resulting homogenate was centrifuged at 12 000 ´g for 30 min at 4°C.The supernatant was used as crude enzyme solution.
The Local Ethics Committee for animal experiments in Bialystok approved the study.Procedures involving animals and their care conformed to institutional guidelines, in compliance with national and international laws and Guidelines for the Use of Animals in Biomedical Research (Giles, 1987).
Chemicals.The substrates and standard solution of Cd were commercial preparations from Sigma Chemical Co.(St.Louis, Mo, U.S.A.).HNO 3 was purchased from Merck.All the reagents were dissolved in redistilled water (Millipore System Milli RO-Milli Q).Cadmium concentration.The submandibular glands of rats were dried and mineral-ized in an electrical muffle furnance.Cd concentration was determined by flameless atomic absorption spectrometry (Zeeman Atomic Absorption Spectrophotometer Z-5000, Hitachi) with electrothermal atomization in a graphite cuvette.The cathode lamp for Cd was operated under standard conditions using its resonance line of 228.8 nm.The Cd concentration was expressed in mg/g of wet tissue.
Exoglycosidases.Before the activity was determined, the crude supernatants were diluted with 0.15 M KCl containing Triton X-100 as follows: 100 fold for b-N-acetylhexosaminidase (HEX), 10 fold for b-galactosidase (Gal) and 20 fold for a-mannosidase (Man).Determination of enzyme activity was performed according to Zwierz et al. (1981), as follows: to 0.05 ml of the diluted supernatant, 0.1 ml of 0.1 M phosphate/citrate buffer, pH 4.7, for b-N-acetylhexosaminidase, and pH 4.3 for the other enzymes, was added.Then 0.05 ml of 20 mM solution of an appropriate substrate (a-or b-p-nitrophenyl glycoside) was added and the tubes incubated, at 37°C for 60 min.The reaction was terminated in an ice-bath by adding 1.0 ml of 0.2 M potassium tetraborate buffer, pH 9.8.The samples were mixed and centrifuged at 14 000 ´g for 6 min.The absorbance of liberated p-nitrophenol was measured at 410 nm in duplicate and appropriate standard solutions were analysed in parallel.Specific activities were expressed in kat/g of protein (katal = the amount of activity that converts one mole of substrate per second) according to the IUPAC-IUB Recommendations 1972 (Florkin & Stotz, 1973).
Direct influence of CdCl 2 on the activity of glycosidases in crude supernatants from homogenates of submandibulary glands of 2-month old rats, not exposed to cadmium, was also investigated.The reaction mixture consisted of 0.05 ml of 100´diluted supernatant, 0.175 ml of phosphate/citrate buffer, pH 4.7, and 0.025 ml of CdCl 2 at a concentration from 5 to 50 mg Cd +2 /ml.Samples were preincubated at 37°C for 20 min, then to each sample 0.15 ml of 20 mM solution of substrate was added and samples were incubated at 37°C for 60 min.Reaction was stopped by adding 1 ml of 0.2 M borate buffer, pH 9.8.After alkalization, the samples were centrifuged (20 min at 14 000 ´g), and the liberated p-nitrophenol was measured at 410 nm.The protein content was determined by the biuret method with bovine serum albumin as a standard (Gornal et al., 1949).
Statistical analysis.Statistical analysis was conducted with software package Statistica (Statsoft).As our variables had normal distribution we performed Levine's test to determine the homogeneity and analysis of variances.This revealed significant differences between groups.We used post hoc analysis calculated by the NIR test.Results were expressed as the mean ± S.D. P values of less than 0.05 were considered significant.

RESULTS
Cadmium concentrations in submandibular glands of rats exposed for 6 or 24 weeks to "environmental" or "occupational" doses of cadmium are presented in Fig. 1.In submandibular glands of rats exposed to "occupational" doses of Cd for 24 weeks, the Cd concentration was significantly increased, in comparison to the rats exposed to "environmental" doses for the same period, as well as to those exposed for 6 weeks to the same Cd doses.However, "occupational" exposure for 24 weeks caused significant twofold increase in concentration of Cd in the submandibulary gland, in comparison to 6 weeks exposure (Fig. 1).
Figure 2 presents the high activity of HEX in the rat submandibular glands homogenate.Activities of Man and Gal were 12 and 36 times lower than HEX, respectively.Cd at concentrations from 4.4-44 mM had no effect neither on activities of HEX, nor Man or Gal (Fig. 2).HEX activities in submandibular glands of rats exposed to the "environmental" dose of Cd for six weeks and to the "ocupational" dose for 24 weeks are presented in Fig. 3. Very evident decrease in HEX activity was observed, both when rats were exposed to "occupational" dose for 24 or 6 weeks.However, there was no change in HEX activity after exposure to the "environmental" dose for 6 weeks.

DISCUSSION
Toxic effects of cadmium on kidneys (Brzóska et al., 2003), bones, and the respiratory tract are well documented (Mitsumori et al., 1998), but the influence of cadmium on the salivary gland was not studied so far.We elaborated a rat model related to conditions in human (Brzóska et al., 2003).Using this model we measured cadmium concentration in rat submandibulary gland, after chronic exposure to 5 mg/l of Cd drinking water (related to human "environmental" exposure) and 50 mg/l (related to human "occupational" exposure).We found accumulation of cad-

Concentration of Cd
2+ in submandibulary glands of control rats (C); after "environmental" exposure -5 mg Cd/l drinking water (E); after "occupational" exposure -50 mg Cd/l drinking water (O).P # 0.05; P # 0.001.The activity was determined as described in Materials and Methods.Other details as in Fig. 1. mium in submandibulary glands of rats dependent on the dose and time of "environmental" and "occupational" exposure (Fig. 1).The small increase of Cd concentration after 24 weeks of "environmental" exposure (in comparison to 6 weeks) suggests, that rat's defense mechanisms are able to protect salivary glands against accumulation of cadmium applied chronically at the "environmental" dose.Our results also might suggest that during "occupational" chronic exposure, the ability of the submandibulary glands similarly as kidneys (Brzóska et al., 2004) to excrete absorbed cadmium was exceeded, and progressive accumulation of cadmium in submandibulary glands occured (Fig. 1).Similar results were also presented when kidneys were used (Brzóska et al., 2003).
The direct treatment of soluble fraction of submandibular glands of control group of rats with even high concentrations of Cd did not inhibit none of the investigated exoglycosidases (HEX,Man,Gal) ( Fig. 2).Despite of these results we decided to follow the changes in HEX activity in submandibular glands, of rats treated with Cd for different time and doses ( Fig. 3).Our results might suggest that cadmium might damage the biosynthesis of N-acetyl-b-glucosaminidase in submandibulary glands of rats.It is known, that HEX in the submandibulary gland of rats is synthesized by mucous and convoluted tubular cells (Pugh & Walker, 1961;Hayashi, 1967).We have no data on changes in cell structure of submandibulary gland of rats chronically exposed to cadmium.However, we observed an increase in expression of proliferating cell nuclear antigen (PCNA) and absence of Ki-67 expression in salivary glands of rats exposed to the "environmental" and "occupational" doses of cadmium for 24 weeks.The increase in PCNA and the absence of Ki-67 antigen, suggest the predominance of DNA repair rather than proliferation mechanism (Czykier et al., 2003).In our previous investigation we found damage to epithelial cells of main renal tubules (proximal convoluted tubules and straigh tubules) after "environmental" exposure to cadmium."Occupational" exposure of rats for 6 weeks blurred the structure of the renal tubular epithelium, caused atrophy of the brush border and partial fragmentation of cells (Brzóska et al., 2003).Cadmium in the submandibulary gland may cause similar disturbances as in kidneys, what may explain the observed decrease of HEX activity in this tissue, as cadmium even at a high concentration has no direct effect on the activity of soluble enzyme preparation (Fig. 2).
Of the three exoglycosidases tested in submandibular glands of rats chronically exposed to cadmium, we found highest changes in HEX activities.N-acetylglucosamine and N-acetylgalactosamine are abundant components of salivary glycoproteins (Wu et al., 1994;Zalewska et al., 1999;2000).HEX has the highest specific activity among the exoglycosidases of rat submandibulary gland (Fig. 2) (Kamada et al., 1989), similarly as in human gastric mucous membrane (Zwierz et al., 1981).A decrease of the specific activity of HEX during maturation of rats (Fig. 3) is in agreement with report of Przybyło et al. (2004).It has to be checked, if the decrease of HEX activity after chronical exposure of rats to cadmium increases the N-acetylhexosamine content in salivary glycoconjugates, both remaining in tissue and excreted in saliva: high (MG1) and low molecular mass (MG2) salivary mucins, lysozyme, lactoferrin, secretory immunoglobulins, acid phosphatase and b-glucuronidase (Zalewska et al., 1999).The changes in the structure and function of submandibulary gland may explain periodontopathies detected among cadmium smelter workers and the cigarette smokers (Ziętek, 1999).

Figure 1 .
Figure 1.Accumulation of cadmium in submandibulary glands of rats chronically exposed to cadmium.

Figure
Figure 2. The supernatant from submandibulary glands was preincubated with Cd for 20 min at 37 o C, than the reaction was started by addition of substrates.Further procedure as described in Materials and Methods.Other details see in description to Fig. 1.

Figure 3 .
Figure 3. Specific activity of N-acetyl-b-glucosaminidase (HEX) present in supernatant obtained from submandibulary gland homogenates of rats chronically exposed to cadmium.