0 Phurtnucdugy & To.xico/ugjj 1996, 78, 289-295 Primd in Denmcrrk , A// righrs reserved Cupyrighr 0 ISSN 0901 -9928 Aluminium Accumulation in Some Tissues of Rats with Compromised Kidney Function Induced by Cadmium-Metallothionein Jenny Lid, Gunnar E Nordberg' and Wolfgang Frechz 'Department of Environmental Medicine, and 'Department of Analytical Chemistry. University of UmeA, S-901 87 Umei, Sweden (Received May 31. 1995; Accepted October 25, 1995) Abstract: Two experiments (I and 11) were performed to study aluminium accumulation in brain as well as in several other tissues in male Wistar rats. A single intraperitoneal injection of cadmium-metallothionein (CdMT, 0.1-0.4 mg Cd/kg b.wt.) was used to compromise kidney function 12 hr before the final aluminium injection in both experiments. In experiment 1, rats were maintained on diets deficient (O.Ol'%, w/w) in calcium (-Ca) or providing adequate (+Ca) dietary calcium (0.9'%)for 6 weeks. Among animals given a daily intraperitoneal dose of aluminium chloride (10.8 mg AVkg per day) on 6 consecutive days there was a tendency towards higher aluminium level in brains of rats with compromised kidney function from CdMT (in -Ca rats: the geometric mean [G]=288 versus 205 ng/g wet weight [w., wt.]. P=0.07, and in +Ca rats: G=242 versus 164, PC0.05) as compared to animals given no CdMT. The results from experiment I1 (all rats were given aluminium 5.6 mg AVkg 2 and 12 hr after CdMT injection) demonstrated a higher level of aluminium (C: 41 ngig w. wt.. P<0.05) in brains of rats with only slightly damaged kidney function (0.1 mg Cd/kg) than in those given no CdMT (G: 29 ng/g w. wt.). It was also observed that I) calcium deficiency had a statistically significant effect (P<O.O5) in increasing kidney retention of intraperitoneal aluminium (G: 327 pg/g w. wt.) as compared to rats with a normal calcium supply in the diet (G: 54 pg/g w. wt.); 2) when aluminium concentration in kidney was at and above 54 pg/g wet tissue, kidney damage was observed. The above results indicate that compromised kidney function including tubular damage induced by a low-dose of CdMT may play a crucial role in the accumulation of aluminium in brain and other tissues. Since tubular function decreases with age in human populations, these findings in rats may be of considerable importance if a similar phenomenon would occur in humans. Therefore, the possibility of increased aluminium retention in persons with low calcium and high aluminium intakes may need to be further investigated. It was originally believed that aluminium had no toxic effect in humans and that aluminium accumulation did not occur. Over the last decades, however, an increasing number of toxic effects of aluminium have been reported in the litera- ture. These include the first case of aluminium-related en- cephalopathy in a worker in 1962 (Mclaughlin et d.), and aluminium-related dialysis dementia in patients with renal failure (Alfrey et a/. 1972 & 1976). In recent years, although several studies in human found no difference in aluminium level in the brains of patients with Alzheimer's disease as compared to the controls (Mcdermott et a/. 1979; Traub et a/. 1981; Markesberry et al. 1981; Jacobs et a/. 1989), aluminium has been found in higher concentrations in the brains of some persons who died of Alzheimer's disease than in controls (Crapper et a/. 1973, 1975 & 1976; Trapp er rd. 1978; Per1 et al. 1980), and a selective accumulation of aluminium within the neurofibrillary tangle-bearing neurones was found in all 10 subjects with Alzheimer's dis- ease (Good ef a/. 1992). In animal experiments, local appli- cation of aluminium to the central nervous system of cats has given rise to learning and memory impairment similar Author for correspondence: Jenny Liu, Department of Environ- mental Medicine. University of Umei, S-901 87 UmeA, Sweden (fax 46-YO-779630). to those seen in patients with Alzheimer's disease (Crapp- er & Dalton 1973; Crapper 1973). Several epidemiological studies have found that an increased incidence of Alzhei- mer's disease in the areas surveyed was related to elevated aluminium concentrations in drinking water (Vogt 1986; Martyn et a/. 1989; Flaten 1990). Although no contusion can be drawn at present as to the role of aluminium in hu- man dementia disorders, Crapper e? a/. (1991) considered that the evidence from both clinical and epidemiological studies suggests a link between aluminium exposure and these disorders. Since aluminium-containing chemicals are widely used in medicines, food additives and cosmetics, and are also added to tap water in some areas as a flocculating agent during the water purification process, it is of importance to study factors that might increase, the retention of aluminium in tissues. However, few studies have been concerned with the potential interaction between factors such as calcium de- ficiency and kidney damage, and the accumulation of alu- minium in brain and other tissues. The interaction of alu- minium with nutritional factors might predispose some groups of people, considered to have normal renal function, to a chronic process of aluminium accumulation in the brain. It has been observed in primates that degenerative changes in brain tissue were induced by a diet containing
Transcript
0 Phurtnucdugy & To.xico/ugjj 1996, 78, 289-295 P r i m d in Denmcrrk , A/ / righrs reserved
Cupyrighr 0
I S S N 0901 -9928
Aluminium Accumulation in Some Tissues of Rats with Compromised Kidney Function Induced by
Cadmium-Metallothionein Jenny Lid, Gunnar E Nordberg' and Wolfgang Frechz
'Department of Environmental Medicine, and 'Department of Analytical Chemistry. University of UmeA, S-901 87 Umei, Sweden
(Received May 31. 1995; Accepted October 25, 1995)
Abstract: Two experiments (I and 11) were performed to study aluminium accumulation in brain as well as in several other tissues in male Wistar rats. A single intraperitoneal injection of cadmium-metallothionein (CdMT, 0.1-0.4 mg Cd/kg b.wt.) was used to compromise kidney function 12 hr before the final aluminium injection in both experiments. In experiment 1, rats were maintained on diets deficient (O.Ol'%, w/w) in calcium (-Ca) or providing adequate (+Ca) dietary calcium (0.9'%) for 6 weeks. Among animals given a daily intraperitoneal dose of aluminium chloride (10.8 mg AVkg per day) on 6 consecutive days there was a tendency towards higher aluminium level in brains of rats with compromised kidney function from CdMT (in -Ca rats: the geometric mean [G]=288 versus 205 ng/g wet weight [w., wt.]. P=0.07, and in +Ca rats: G=242 versus 164, PC0.05) as compared to animals given no CdMT. The results from experiment I1 (all rats were given aluminium 5.6 mg AVkg 2 and 12 hr after CdMT injection) demonstrated a higher level of aluminium (C: 41 ngig w. wt.. P<0.05) in brains of rats with only slightly damaged kidney function (0.1 mg Cd/kg) than in those given no CdMT (G: 29 ng/g w. wt.). I t was also observed that I ) calcium deficiency had a statistically significant effect (P<O.O5) in increasing kidney retention of intraperitoneal aluminium (G: 327 pg/g w. wt.) as compared to rats with a normal calcium supply in the diet (G: 54 pg/g w. wt.); 2) when aluminium concentration in kidney was at and above 54 pg/g wet tissue, kidney damage was observed. The above results indicate that compromised kidney function including tubular damage induced by a low-dose of CdMT may play a crucial role in the accumulation of aluminium in brain and other tissues. Since tubular function decreases with age in human populations, these findings in rats may be of considerable importance if a similar phenomenon would occur in humans. Therefore, the possibility of increased aluminium retention in persons with low calcium and high aluminium intakes may need to be further investigated.
It was originally believed that aluminium had no toxic effect in humans and that aluminium accumulation did not occur. Over the last decades, however, an increasing number of toxic effects of aluminium have been reported in the litera- ture. These include the first case of aluminium-related en- cephalopathy in a worker in 1962 (Mclaughlin et d.), and aluminium-related dialysis dementia in patients with renal failure (Alfrey et a/. 1972 & 1976). In recent years, although several studies in human found no difference in aluminium level in the brains of patients with Alzheimer's disease as compared to the controls (Mcdermott et a/. 1979; Traub et a/. 1981; Markesberry et al. 1981; Jacobs et a/. 1989), aluminium has been found in higher concentrations in the brains of some persons who died of Alzheimer's disease than in controls (Crapper et a/. 1973, 1975 & 1976; Trapp er r d . 1978; Per1 et al. 1980), and a selective accumulation of aluminium within the neurofibrillary tangle-bearing neurones was found in all 10 subjects with Alzheimer's dis- ease (Good ef a/. 1992). In animal experiments, local appli- cation of aluminium to the central nervous system of cats has given rise to learning and memory impairment similar
Author for correspondence: Jenny Liu, Department of Environ- mental Medicine. University of Umei, S-901 87 UmeA, Sweden (fax 46-YO-779630).
to those seen in patients with Alzheimer's disease (Crapp- er & Dalton 1973; Crapper 1973). Several epidemiological studies have found that an increased incidence of Alzhei- mer's disease in the areas surveyed was related to elevated aluminium concentrations in drinking water (Vogt 1986; Martyn et a/. 1989; Flaten 1990). Although no contusion can be drawn at present as to the role of aluminium in hu- man dementia disorders, Crapper e? a/. (1991) considered that the evidence from both clinical and epidemiological studies suggests a link between aluminium exposure and these disorders.
Since aluminium-containing chemicals are widely used in medicines, food additives and cosmetics, and are also added to tap water in some areas as a flocculating agent during the water purification process, it is of importance to study factors that might increase, the retention of aluminium in tissues. However, few studies have been concerned with the potential interaction between factors such as calcium de- ficiency and kidney damage, and the accumulation of alu- minium in brain and other tissues. The interaction of alu- minium with nutritional factors might predispose some groups of people, considered to have normal renal function, to a chronic process of aluminium accumulation in the brain. It has been observed in primates that degenerative changes in brain tissue were induced by a diet containing
290 JENNY LIU ET AL.
high amounts of aluminium and low concentrations of cal- cium and magnesium (Yano et al. 1989).
As mentioned, aluminium is well recognized as causative agent in dialysis-dementia, and the importance of severe re- nal disease in increasing aluminium retention is also well established, both in humans and in experimental animals (Berlyne et al. 1970 & 1972; Alfrey et al. 1976; Platts et al. 1977; Parkinson et al. 1979). However, a possible role of minor renal damage of the tubular type in increasing the aluminium retention has not been investigated. If such kid- ney damage would have an effect on aluminium retention this could be of considerable practical interest, since it is well known that kidney function, and in particular the tubu- lar function decreases in the older age groups. It is well known that both in humans and in animals long-term ex- posure to cadmium may give rise to kidney damage mainly of tubular type (Dalhamn & Friberg 1957; Kjellstrom 1986). By injection of cadmium-metallothionein (CdMT) in experimental animals, similar tubular damage can be in- duced (Nordberg et al. 1975a; Cherian et al. 1976; Squibb et al. 1984). In the present experiments, this model employing varying doses of CdMT to compromise the kidney function was used to study whether such compromised kidney func- tion would influence the retention of aluminium (also given by injection) in the same animals.
Materials and Methods Animals and experimental design. The experimental design is shown in table I . Experiment 1 was designed to observe the effects of cal- cium deficiency and kidney damage on the retention of aluminium in tissues. Thirty-two male Wistar rats (ALAB, Stockholm) weighing 396 ? 29 g used in experiment I were divided into 8 groups 1 to 4 being provided with a pelleted diet R-542, (calcium-deficient), and groups 5 to 8 with R-543 (normal calcium content). A daily alumin- ium injection (AIC13, 10.8 mg Alikg per day) was administered
Table 1. Experimental design. Four rats in each group in experiment I and six in each group in experiment 11. 0.15% of magnesium as MgC12 in water was provided for all groups in both experiments.
a A daily intraperitoneal aluminium chloride (in mg Al/kg body weight) injection was repeated on 6 consecutive days at the last week. A single intraperitoneal CdMT (in mg Cdikg) injection was given 12 hr before the final aluminium injection. %, w/w, fresh weight. Aluminium chloride injections were given intraperitoneally 2 and 12 hr after CdMT injection.
intraperitoneally to rats in groups 3, 4, 7 and 8 and repeated on 6 consecutive days during the last week of this experiment. Rats in groups 2, 4, 6, and 8 were given a single intraperitoneal injection of CdMT (0.4 mg Cd/kg) 12 hr prior to the final aluminium injection.
In second experiment (experiment 11), which was performed in order to confirm the tendency of an increased aluminium accumu- lation in relation to CdMT-induced kidney damage observed in ex- periment I, two doses of CdMT were used to allow also an assess- ment of minor renal tubular damage on aluminium retention and lower doses of aluminium were administered to avoid possible kid- ney damage by aluminium. Seventeen male Wistar rats (148* 12.1 g) from Msllegaard Breeding center Itd., Denmark, were divided into 3 groups (6 rats in groups 1 and 2, and 5 in group 3). Food pellets (R-543) were provided to all groups during the 6 weeks. Rats in group 2 were administered a single intraperitoneal injection of CdMT at 0.1 mg cadmium, and rats in group 3 were administered 0.4 mg cadmiumikg body weight on the final day of week 6. All animals were given 2 intraperitoneal injections of aluminium chlor- ide (5.6 mg Alikglper dose) 2 and 12 hr after CdMT injection.
All animals used in these two experiments had free access to pel- lets and water during the whole period of the experiments, and the untreated rats were injected with the same solution as that used to dissolve the aluminium or CdMT. Rats were placed individually in metabolic cages immediately after the CdMT injection and were euthanatized by an intraperitoneal injection of pentothal sodium (50-100 mgikg) 47 (experiment I) or 48 hr (experiment 11) after the CdMT administration. Brain, kidneys, liver, spleen, and serum were collected in a dust-free environment (laminar-flow sterile bench, class 100) using stainless instruments washed frequently in deion- ized water during the operation. Samples were stored at -70" until analysis. These two experiments were approved by the ethics com- mittee for animal experiments in Umeil.
Food and water. The pellets R-542 and R-543 were obtained from EWOS, Sweden. According to the specification, they consisted of 18% casein (EDTA washed), 2% egg white powder, 13.5% glucose, 13.5%) sugar H, 37.58% (35.58% for R-543) corn starch, 3% cellu- lose, 4% salt 918, 1% VF rats and mice special, 7% soybean oil. 0.42% sodium salt to provide phosphate, and 2% calcium carbonate (only for R-543). The concentrations of zinc, copper and mag- nesium were 5.5, 4.5, and 39 p.p.m. (62 p.p.m. for R-543), respec- tively. The calcium concentrations in the pellets are 0.01% (wiw) for R-542 and 0.9Ya for R-543. Because of the lower supply of mag- nesium provided by both of these diets compared to a normal pel- leted diet, deionized water containing 0.1 5% Mg as MgClz was pro- vided for all rats in these two experiments.
Chemicals. Aluminium chloride (AICI2.6H2O) was obtained from Deventer, Netherlands (lot no. 437342); magnesium chloride (MgCI2.6H2O) and citric acid (C6H807.H20) were provided by MERCK, Germany; pentothal sodium was from ABBOTT, Italy. CdMT (Cd:Zn=2.5:1 by weight) was prepared from liver tissue of male Wistar rats as described by Nordberg et al. (1975b).
The solution for aluminium injection was prepared in 0.9%) so- dium chloride, buffered with citric acid solution (0.19 g C6H807/ ml) and 2.5 M sodium hydroxide to a pH of 6.0.
Analysis. All laboratory utensils used for aluminium analysis and storage of samples were rinsed overnight in nitric acid, washed with deionized water, and checked for possible aluminium contami- nation. The nitric acid used for aluminium determination (including washing) was purified by using a sub-boiling still (Hans Kurner Analysentechnik, Rosenheim, Germany).
Aluminium. A Perkin-Elmer Model 3030 atomic absorption spectro- photometer and HGA-500 furnace with AS-40 autosampler were used for the determination of aluminium in brain, kidney, liver, spleen, and serum. Pyrolytically coated graphite tubes and pyro- lytical platforms were used with 20 pI sample aliquots (for the de-
29 1 KIDNEY FUNCTION A N D ALUMINIUM ACCUMULATION
1000 -
316 -
100 - 31.6 -
10 - 3.16 -
1 -
0.32 -
ng/g wet wt.
0
0
Aluminium in brain
0 0 0
0
%O 0 "Oe 0%
0 0 00
0
0
0.1 -1, I I I I I I I I
1 2 3 4 * 5 6 7 8 *
Group Fig. I . Brain aluminium from experiment I. There are 4 data points in each group. Aluminium chloride (10.8 mg Al/kg per day) was administered intraperitoneally to groups 3, 4, 7 and 8 on 6 consecu- tive days on the last week of the experiment. Groups I , 2. 5, and 6 are non-aluminium treated controls. Groups 1 4 were maintained on a calcium-deficient diet and groups 5-8 a calcium-adequate diet for 6 weeks. A single intraperitoneal CdMT injection was given 12 hr before the final dose of aluminium. Rats were sacrificed 47 hr after the CdMT injection. * One-side between groups 4 and 3 (P= 0.07) or groups 8 and 7 (P=0.04).
tailed analytical procedure, see Slanina et a/. 1984). The standard addition method was used to determine aluminium concentrations in samples, so that the matrix effect was matched for samples of different dilutions from different tissues. The accuracy of the ana- lytical procedure was tested using two different standard reference materials in which aluminium was determined under the same con- ditions as for the samples. Of these, the first was lyophilized serum (sample no. 1353) from Belgium (Dr. J. Versieck, University of Ghent. Department of Internal Medicine, Division of Gastroenter- ology, University Hospital, De Pintelaan 185, B-9000 Ghent, Bel- gium): a value of 28+1.6 p.p,b. dry weight (n=3) was found for this material (the certified value was 17.5-23.3 p.p.b.). The second was lyophilized human reference serum from Norway (Nycomed Phar- ma AS, Division Diagnostica, PO. Box 4284-Torshov, N-0401 Oslo 4, Norway). Values of 107+2 p.p.b. (n=3) were found in the recon- stituted serum (the certified value was 106z6 p.p.b).
Dissolufion of'sanzples. About 3 g of concentrated nitric acid per g fresh tissue was added to all tissue and serum samples prepared for the analysis of aluminium (in acid-washed quartz beakers). Samples were dissolved on a hot plate (temperature: 125-175") with dust- free. class 100 acid-proof ventilation until there was very little liquid left at the bottom of the beaker. These acid-digested samples were diluted with deionized water to 5 or 10 times the sample weight on a dust-free bench and stored at 04'. Aluminium was determined within one week after dissolution.
Crrarininr in serum and calcium in kidney. Creatinine in serum was determined as described by Hare (1950), and calcium in kidney was determined as described by Liu & Nordberg (1995). The standard materials, bovine liver 1577a, from the National Bureau of Stan- dards (U.S.A) was analyzed for calcium at the same time. A value of 121.6 p.p.m. was found for this material (the certified value for calcium was 12027 p.p.m).
Stofi.sfics or dattr anulysis. After doing a log transformation on the data. a Student's t-test was carried out to test for significance using
the statistical package Status I1 (Foresco AB, Sweden). The ac- cepted significance level for most of the comparison between groups was Pi =0.05. For the comparison of aluminium level in brain be- tween groups in experiment I, the significance level is indicated as one-side test P<=O.O7.
Results
This paper is concerned mainly with aluminium accumu- lation in brain and other selected tissues; the data on kidney damage will only be mentioned briefly since such data from experiment I has been reported elsewhere (Liu & Nordberg 1995).
Aluminium concentrution in brain. Fig. 1 and 2 show the concentrations of aluminium in brain in experiments I and 11, respectively. All the individual measurements were used to plot the figures. The results of both exeperiments proved that the administration of CdMT significantly increased the accumulation of aluminium in brain.
Results from experiment I (fig. 1) showed that in rats given a daily intraperitoneal dose of aluminium chloride (10. 8 mg Al/kg per day) on 6 consecutive days there was a tendency towards higher aluminium levels (P< =0.07, one- side) in brains of rats with compromised kidney function from CdMT, i.e. group 4: geometric mean (G)=288 ng/g wet weight (w. w.t) and geometric mean standard deviation (G.S.D.) 1.4 as compared to rats given no CdMT (group 3: G=205 and G.S.D. 1.2). In animals on a normal calcium intake, there was also a higher aluminium level (P<0.05) in brains of rats with compromised kidney function from CdMT (group 8: G=242 and G.S.D. 1.4) as compared to those given no CdMT (group 7: G= 164 and G.S.D. 1. I ) .
ng/g wet wt.
Aluminium In brain
joo0 1 100 316 4 0
0
lo I 3.16 I I I I
1 (0.0Cd) 2 (0.1 Cd)* 3 (0.4Cd)n
Group
Fig. 2. Brain aluminium from experiment 11. There are 6 data points in each group (5 in group 3). Aluminium chloride (5.6 mg AUkg per dose) was administered intraperitoneally to all groups 2 and 12 hr after CdMT injection (mg Cd/kg) on the final day of the experi- ment. Group 1 is a non-CdMT treated control. All rats were main- tained on a calcium-adequate diet for 6 weeks. Rats were sacrificed 48 hr after CdMT injection. * P<0.05 as compared to group 1 and 0 P<0.05 as compared to either group I or 2.
292 JENNY LIU ET AL.
Table 2. Aluminium concentrations" in kidney, spleen, serum and liver in experiment I. Each group consisted of 4 rats. For detailed experimental design. see table 1. Values are oresented as geometric mean (G) based on wet weight and geometric standard deviation (G. S. D.).
ngig wet weight for groups 1, 2, 5 and and pgig wet weight for groups 3, 4, 7 and 8. * P<0.05 and ** P<O.OI as compared with groups indicated by the superscript numbers.
Results from experiment I1 (fig. 2) also demonstrated a higher level of aluminium (G: 41 ng/g w.wt, P<0.05) in brains of rats with only slightly damaged kidney function (0.1 mg Cd/kg, group 2) than in those given no CdMT (G: 29 nglg w.wt., group 1).
No statistically significant effect of a low-calcium diet on the accumulation of aluminium in brain tissue was observed in experiment I, although the concentration of brain alu- minium in group 7 or 8 (given a diet providing an adequate amount of calcium) was lower than that in group 3 or 4, respectively.
Aluminium concentrations in tissues other than brain. The concentrations of aluminium in kidney, liver, spleen and serum found in experiment I are shown in table 2. These data clearly demonstrate (groups 3 ,4 ,7 and 8) that, of these organs, the kidneys retained the highest level of aluminium, on a per gram of wet tissue basis. It is also clear that, when the diet was low in calcium, a larger amount of aluminium (328 pg/g w.wt., group 3) accumulated in the normally func- tioning kidneys (no CdMT administration), namely 6 times that in rats maintained on a calcium-adequate diet (54 pg/ g w.wt., group 7). When rats were maintained on a low- calcium diet and injected intraperitoneally with CdMT 12 hr before the final injection of aluminium, a lower alumin- ium concentration in kidney was observed than in animals not given CdMT (group 4 versus group 3); in those on a
calcium-adequate diet, however, aluminium accumulation in kidney was not significantly affected by the injection of CdMT (group 8 versus 7). Significantly higher concen- tration of aluminium was seen in serum in groups 4 and 8 as compared to the non-CdMT-treated controls (groups 3 and 7). Although the similar finding was observed in the spleen, the comparison between groups 7 and 8 was not statistically significant. It is evident that the accumulation of aluminium in liver was independent of the calcium con- centration in the diet by this experimental design.
Table 3 presents the data from experiment 11, in which all animals were administered aluminium. As can been seen from table 3, the intraperitoneal injection of CdMT, corre- sponding to either 0.1 or 0.4 mg cadmium/kg. had a statisti- cally significant effect in increasing aluminium accumu- lation in all the organs analysed except the spleen.
Although it is well known that bone avidly accumulates aluminium (Goyer 1991), it was not determined in bone in the present study because the major concern was to examine the relationship between calcium deficiency, kidney damage and aluminium accumulation in some selected tissues.
Creatinine in serum and calcium in kidney. In order to show the extent of the kidney damage induced by the injection of CdMT, creatinine levels in serum and concentrations of calcium in kidney are summarized in table 4. The intraper- itoneal injection of CdMT at a level of 0.4 mg cadmiudkg
Table 3. Aluminium concentrations" in kidnev. serum. liver and soleen in exoeriment 11.
Treatmentsb Kidney Serum Liver Spleen
Group Al CdMT Ca G G.S.D. G G.S.D. G G.S.D. G G.S.D. 1 + - + 6.7 I .2 0.2 1.2 7.0 1.3 17.8 1.4 2 + + + 9.1 ' * 1.2 0.3'** 1.2 11.5'** 1.1 15.8 I .2 3 + + + 14.5'**.2** 1.4 0.7'* 3.1 12.2'* 1.5 16.6 I .4
* pg/g wet tissue Intraperitoneal aluminium chloride injections (5.6 mg Alikg per dose) were given 2 and 12 hr after CdMT injection. A single intraperitoneal CdMT injection (0.1 mg Cdikg for group 2 and 0.4 for group 3) was given on the last day of the experiment. Calcium content in the diet was 0.9% (wiw, fresh weight). G and G.S.D.: Geometric mean and geometric standard deviation.
* P<0.05 and ** P<=O.OI by Student's t-test on log transformed data.
KIDNEY FUNCTION AND ALUMINIUM ACCUMULATION 293
Table 4. Concentrations of calcium in kidney and creatinine in serum.
Calcium in kidnev" (G and G.S.D.) Creatinine in serumh (MeankS.D.1
data for calcium). * P = or c0.05 and ** P= or <0.01 as compared with the group indicated by the superscript numbers by Student's t-test (log transformed
had a significant effect in increasing both creatinine levels in serum and calcium concentrations in kidney. Although the calcium level was higher in group 2 (0.1 Cd/kg) than in group 1 (no cadmium given), the creatinine level in serum was close to that in the control group. A slight increase in serum creatinine was also found in rats injected only with aluminium in experiment I (groups 3 and 7 ) reported in detail by Liu & Nordberg (1995), while no increase was observed for creatinine in serum in rats of group 1 in experi- ment 11 (with two lower intraperitoneal doses of aluminium injections).
Discussion
Whether aluminium accumulates, or can accumulate, in brain is of the greatest importance, because it can exert toxic effects on that organ. It has been stated that, with normal kidney function and a Western diet, the aluminium in the brain of a healthy person will not reach the toxic level of 1.5 to 5 pg/g w.wt. (the average normal concentration in human adults is about 0.5 pg/g w.wt.) as reported in the literature and summarized by Ganrot (l986), until that person is 100 years old. While this may be the case for most of a population, it may not be true for people with decreased renal function, those with a medicinal intake of aluminium, or those exposed to an unbalanced diet for most of their lives.
The findings of the present study clearly demonstrate el- evated aluminium concentrations in both brain and other tissues of rats either given 6 intraperitoneal doses of alu- minium or combined with a single intraperitoneal dose of CdMT. Significantly higher aluminium concentration in brain was found in rats with compromised kidney function induced by the injection of CdMT than in those given no CdMT. It was observed for the first time that even minor kidney damage induced by the injection of a small amount of CdMT (0.1 mg Cd/kg) gave rise to a significantly higher brain aluminium level than that observed in rats given no CdMT. As mentioned in the introduction, the renal effect of CdMT is mainly of a tubular type. It was demonstrated in the present study that even less severe damage of this
type may give rise to an increase in aluminium retention. This phenomenon may be of practical health concern for population groups which permanently dwell in a cadmium contaminated area, or older people whose tubular function is progressively declining.
The present study demonstrates that significantly elev- ated tissue aluminium concentrations occur in animals with calcium deficiency (Similar findings were previously re- ported by Provan & Yokel 1990). A diet deficient in calcium will help to accumulate more aluminium in tissues even if the kidney function is close to normal. It was observed that the calcium-deficiency-related aluminium accumulation was more pronounced in the kidney than in other tissues. A high accumulation of aluminium in kidney was seen in rats maintained on a low-calcium diet, while aluminium concen- trations in the liver, spleen and serum did not seem to be significantly affected by a diet of this type. The underlying mechanism is unknown. However, a hypothesis has been advanced which may explain the relationship between alu- minium accumulation and the calcium deficiency. Pro- longed calcium deficiency results in secondary hyperpara- thyroidism, which in turn increases the calcium concen- tration in body fluids as more calcium is withdrawn from the diet, bone and kidneys (MacManus et ul. 1982). The hormone, PTH, excreted by the parathyroid, has been re- ported to increase aluminium deposition in rats (Mayor C t
ul. 1977). The kidneys will increase tubular calcium reab- sorption in responding to the regulation by PTH, as in cal- cium deficiency and, in the process, increase aluminium re- absorption (Provan & Yokel 1990). In the present study concentrations of aluminium in the kidney were higher in rats fed a low-calcium diet than in those on a calcium-ade- quate diet.
A low-calcium diet increased aluminium accumulation in rat brain in studies by Provan & Yokel (1990) who did not advance an explanation for this phenomenon. The results from the present study may indicate that it might have been an effect secondary to aluminium toxicity to the kidney. Our results did not show a clear effect of the low dietary calcium on brain aluminium retention. This may in part be
294 JENNY LIU ET AL.
due to the smaller amount of aluminium adminstered in the present study so that the adverse effect on kidney function was smaller. It has been reported by Liu & Nordberg (1995) that, when administered to rats (experiment I), aluminium is nephrotoxic and that the damage to kidney was increased when the rats were fed a calcium-deficient diet. The data reported in the present study show that the toxic effect of aluminium on the kidney may be concentration-related. This is indicated by the fact that no toxic effects were seen in the animals in experiment I1 when the aluminium con- centration in kidney is 6.7 pg aluminium/g wet weight or lower; while at or above 54 pg aluminiudg wet weight, slightly damaged kidney function was observed in animals in experiment I. In the study by Provan & Yokel (1990), aluminium chloride was administered to rats at the same dose as that adopted in our study (10.8 mg Allkg per day) by the intraperitoneal route 5 days a week for 4 consecutive weeks. It is quite possible that more severe kidney damage was caused by the more extensive aluminium exposure and accumulation (1 01 3 pg/g dry weight in rats on calcium-de- ficient diet) in these rats although the kidney effects were not recorded. The probable kidney damage may well have contributed to the increased aluminium concentration in the brain of those rats, particularly in those given a low- calcium diet.
In experiment I, a significantly higher level of aluminium in serum was observed in rats injected with CdMT as com- pared to the non-CdMT-treated controls. It was also no- ticed, at the same time, that the aluminium concentration in kidney was decreased but that in spleen was increased. It seems quite possible that the extensive tubular damage in- duced by CdMT (Liu & Nordberg 1995) was responsible for the decreased aluminium concentration in that organ. There was a concomitant increase in aluminium in spleen and plasma in these rats. Spleen and plasma probably repre- sent a common compartment for aluminium distribution and the concentration in this compartment is probably de- pendent on the extent of renal elimination of aluminium.
It has been reported that aluminium accumulation in- creases with age in the human brain (Mcdermott et al. 1979; Markesberry et al. 1981), and that once aluminium has entered the brain it may not be eliminated (Ganrot 1986). Although kidney is quite efficient in eliminating absorbed aluminium, it remains possible that continous human ex- posure to various sources of aluminium, sometimes involv- ing large doses, might pose a problem from the point of view of human health.
Sunzmary. It was observed in this study that: (1) slight kid- ney damage induced by injection of a small amount of CdMT significantly increased aluminium accumulation in brain tissue; (2) food deficient in calcium is largely respon- sible for the increased aluminium accumulation in rats with normal renal function. These observations may therefore indicate the need for greater attention to be given to popu- lation groups with low calcium and high aluminium intakes, particularly if they are also exposed to cadmium.
Acknowledgements We would like to extend our thanks to Mrs. K. Olsson
and Dr. D. Baxter, Department of Analytical Chemistry, Umei University, for their help in determining the alumin- ium concentrations in samples. We would also like to thank Professor S. Sjoberg, Department of Inorganic Chemistry, Umei University, and Dr. M. Sjostrbm, Department of Or- ganic Chemistry, Umei University, and Dr. T. Jin and Mrs. S. Sandberg, Department of Environmental Medicine, Umei University, for their valuable advice and technical assistance. This work was supported in part by the Swedish National Environmental Protection Agency.
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