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Influence of dietary iron deficiency on nickel, lead and cadmium intoxication
the Science of the Total E n v i r o n m e n t into the E m n m e n t
a n d In ~lsl~nshipw i t h Men
The Science of the Total Environment 148 (1994) 167-173
ELSEVIER
Influence of dietary iron deficiency on nickel, lead and cadmium intoxication S.K. Tandon*, S. Khandelwal, V.K. Jain, N. M a t h u r Industrial Toxicology Research Centre. Post Box 80. Lucknow-226001, India
Abstract The influence of dietary iron deficiency on acute nickel, lead or cadmium toxicity as reflected by the induction of hepatic, renal, and intestinal metallothionein (MT), disposition of the metals and alterations in hematological parameters, was investigated in young rats to ascertain whether the toxic effects of these metals modify under anemic conditions. The administration of Cd induced hepatic, renal and intestinal MT while that of Ni or Pb induced hepatic MT only. While dietary Fe deficiency did not affect MT induction by Cd, it enhanced the synthesis of renal and intestinal MT by Ni and Pb. The accumulation of Pb in liver and kidney and that of Cd in liver only, were enhanced by Fe deficiency; the tissue deposition of Ni remained unaffected by Fe deficiency. The induction of hepatic MT by Ni, Pb or Cd appears to be related to the concomitant rise in the hepatic Zn, Ca and Fe levels in normal rats. However, dietary Fe deficiency increased the hepatic Zn in response to Ni or Cd and the hepatic Ca in response to Pb administration. Key words: Dietary iron deficiency; Nickel; Cadmium; Metallothionein; Biochemical alterations; Metals; Rat
I. Introduction The toxic manifestations of heavy metals are governed among other things by dietary constituents particularly the essential trace elements (Schafer and Forth, 1985; Elsenhans et al., 1987). Thus, while dietary Fe deficiency increases absorption of Pb and Cd (Ragan, 1977; Hashmi et al., 1989a; Schafer et al., 1990) its excess decreases their absorption (Conrad and Barton, 1978; Schafer and Forth, 1985; Clark et al., 1988). However, Fe deficiency enhances susceptibility to the toxic effects of Ni without particularly affecting its uptake (Tandon and Mathur, 1986). The dietary * Corresponding author.
Fe status therefore seems to influence the disposition and effects of toxic metals following exposure. The dietary constituents, particularly the essential metals markedly influence the induction of metallothionein (MT) (Cousins, 1985; Bremner, 1987). Considering the fact that MT is induced in response to various stresses (Hidalgo et al., 1988; Bremner and Beattie, 1990), including administration of heavy metals such as Cd or Hg, the specific genes coding for this protein may be expressed as an acute-phase response of the body. Like synthesis of other acute-phase proteins, synthesis of MT under such circumstances is probably designed to impart stress resistance or tolerance to cells and to maintain homeostasis. The MT induction in the intestinal mucosa and brain in anemic rats
0048-9697/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved. SSDI 0048-9697(93)03996-F
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S.K. Tandon et al. / Sci. Total Environ. 148 (1994) 167-173
(Ribas et al., 1987) led the authors to propose the participation of MT in both the Fe absorption by the intestinal mucosa and the involvement of the brain in Fe homeostasis. An increase in MT-I concentration in blood cells and bone marrow, a reduction in MT level in the kidney and no change in hepatic MT as a consequence of Fe deficiency have all been reported (Robertson et al., 1989). In view of the current lack of knowledge of the biochemical mechanism of metal-metal interactions, the influence of dietary Fe deficiency on the acute toxicity of Ni, Pb or Cd as evaluated by tissue MT induction, metal disposition and certain related hematological alterations, was investigated in young rats.
2. Experimental 2.1. Animals and treatment
Forty-eight young male rats (70 ± 10 g) bred in the Industrial Toxicology Research Centre's colony were divided into two batches of 24. One batch was fed a Fe-sufficient and the other a Fe-deficient synthetic diet for 6 weeks (Table 1). The animals of the Fe-deficient group were subjected to orbital plexus puncturing twice a week for the first 2 weeks to ascertain if they were in a Fe-deficient state. After 6 weeks, the 24 animals in each batch were subdivided into four groups of six
Table 1 Chemical composition of diet Ingredients
g/kg
Casein Corn starch Sucrose Groundnut oil Mineral mixture (Modified AIN76 mineral mixture) Vitamin mixture (AIN-76 vitamin mixture) Choline chloride DL-methionine FeSO 4, 7 H20 in sucrose
220 370 300 50 45 10 1.5 2.0 1.5
Analytical grade FeSO 4.7H20 triturated in powered sucrose (1.5 g) to supply 35 mg or 5 mg Fe/kg diet for an Fe-sufficient or Fe-deficient diet, respectively
each. Three groups, each containing six animals from each batch were administered intraperitoneally a single dose of 120/~mol/kg Ni as NiCI2 6H20, 400 #mol/kg Pb as Pb (CH 3 COO)2 3H20 or 20 #mol/kg Cd as CdC12H20, dissolved in distilled water (2 ml/kg). The solution of lead acetate was prepared in boiled water and bubbled with nitrogen immediately prior to administration. The remaining group in each batch was given an equal volume of water and served as a control. The metals were given as equitoxic doses (i.e. MTD) previously determined and defined as the maximum dose which produced death in less than 10% of the exposed population (Eaton et al., 1980). The use of MTD provides information on the maximal effectiveness of the metal to induce MT at the time point selected. The animals were sacrificed 24 h after metal administration and blood drawn by cardiac puncture into heparinised vials. An aliquot of blood was centrifuged to separate the plasma. The liver and kidneys were removed and the small intestine (about 10 cm from the pylorus) excised, cut open and washed with saline. 2.2. Biochemical estimations
The liver and kidney were homogenised individually in 4 vols. and the intestine in 10 vols. of 0.01 M Tris-HC1 buffer (pH 7.4). The homogenates were centrifuged at 10 000 x g for 30 min and the heat stable fraction obtained by placing the post-mitochondrial supernatant in a boiling water bath for 5 min. The MT concentration was estimated in this fraction using the Cd/Hb affinity method (Eaton and Toal, 1982). Standard procedures were employed for the estimation of blood hemoglobin (Clegg and King, 1942); packed cell volume (Keele and Neil, 1966); plasmacholesterol (Zlatkis et al., 1953); total Fe and Fe-binding capacity (Peters et al., 1956). 2.3. Estimation o f metals
The weighed hepatic and renal samples were mineralized in nitric and perchloric acids (6:0.5). The residue was dissolved in 5% HNO 3 and the metals were determined using a flame atomic absorption spectrometer (Perkin-Elmer 5000). The readings were recorded for Cu (324.7 nm), Zn (213.9 nm), Ca (422.7 nm), Fe (248.3 nm), Ni
S.K. Tandon et al./ Sci. Total Environ. 148 (1994) 167-173
169
Table 2 Influence of dietary iron deficiency on m e t a l - i n d u c e d biochemical alterations in rat b l o o d Treatment
Normal Ni Pb Cd Fe-def. Fe-def. Fe-def. Fe-def.
diet
diet Ni Pb Cd
Blood H b (g/lO0 ml)
PCV (%)
Total cholesterol (mg/100 ml)
Total Fe (mequiv./100 ml)
Fe b i n d i n g capacity (mequiv./100 ml)
11.8 9.6 12.8 13.4 9.3 10.5 10.5 10.2
40.8 32.2 45.8 42.2 34.4 31.6 29.2 35.3
98.5 104.2 140.9 109.6 114.2 92.9 145.3 106.7
229.3 235.4 170.1 191.5 177.3 181.3 140.2 167.4
447.8 490.7 516.7 466.6 637.9 556.5 592.6 637.7
± ± ± ± ± ± ± ±
0.5 2.6 1.6 1.6 c 0.6 1.8 1.9 1.1
± ± ± ± ± ± ± ±
3.4 6.4 5.5 1.8 3.8 3.7 4.4 4.7
± ± ± ± ± ± ± ±
6.0 2.0 5.8 a 12.1 15.9 c 5.2Y 5.6 x 9.4
± ± ± ± ± ± ± ±
9.2 19.4 26.3 a 34.7 19.5 b 11.1 14.9Y 20.3
± a± q± ± ± ±
37.3 56.1 24.8 a 64.0 15.0 a 18.0Y 23.8 z 16.6
Each figure is m e a n ± S.D. (6). a p < 0.001, b p < 0.01, c p < 0.05 vs. n o r m a l diet. xp < 0.001, YP < 0.01, zp < 0.05 vs. Fe-deficient diet.
(232.0 nm), Pb (283.3 nm) and Cd (228.8 nm) against suitable standards, processed identically.
2.4. Statistical analysis A 2 x 2 randomised block factorial design was used to analyse various parameters using six replicates in each group (normal diet, normal diet + Ni, Fe-deficient diet and Fe-deficient diet + Ni). Prior to this analysis, the normality assumption for the data and homogeneity of variance between the treatment groups were ascertained. The means of the treatment groups from the controls were compared separately using Dunett's test (Zar, 1984). The same analysis was performed for Pb and Cd.
uninfluenced by dietary Fe deficiency. Ni and Pb were successful in inducing MT in liver only, but failed to induce the same in Fe-deficient rats. However, these metals could restore the Fe deficiency and led to a lowering in renal and intestinal MT levels (Table 3). Dietary Fe deficiency enhanced the accumulation of Pb in both liver and kidney and that of Cd in liver only. However, it had no effect on the tissue deposition of Ni (Fig. 1).
Table 3 Influence of dietary iron deficiency on the i n d u c t i o n o f tissue metallothionein by metals in rats
3. Results
Treatment
Liver
Dietary Fe deficiency decreased plasma Fe level but increased plasma cholesterol and Fe-binding capacity. The administration of Pb but not Ni or Cd caused similar effects in animals fed normal diets. The influence of Pb was more prominent in animals maintained on Fe-deficient diets (Table 2). Dietary Fe deficiency caused a significant decrease in renal and intestinal MT levels but the hepatic MT remained unaffected. Cd was highly effective in inducing MT synthesis in all the three tissues examined, an effect which remained
N o r m a l diet Ni Pb Cd Fe-def. diet Fe-def. Ni Fe-def. Pb Fe-def. Cd
4.5 14.4 6.5 46.2 5.4 5.8 4.7 63.0
± ± ± ± ± ± ± ±
Kidney
1.7 4.3 a 1.7 b 6.3 a 1.0 1.3 0.7 11.0 x
12.8 13.5 14.4 31.0 5.6 15.1 13.2 32.0
± ± ± + ± ± ± ±
Intestine
1.5 1.7 0.8 4.6 a 1.3 a 2.0 x 1.8 x 4.0 x
5.4 4.8 4.6 18.4 2.7 4.4 4.6 13.9
All concentrations is ttg/g, fresh tissue. Each mean q- S.D. (6). a p < 0.001, b p < 0.01, VS. n o r m a l diet. xp < 0.001, YP < 0.01, vs. Fe-deficient diet.
a+ + + ± ± ± ±
0.7 1.0 0.8 3.1 a 0.7 a 0.7 y 0.7Y 2.1 x
figure
is
170
S.K. Tandon et al. ~SoL Total Environ. 148 (1994) 167-173
diet however, decreased Zn and Fe contents of liver. The administration of Ni or Pb caused an increase in hepatic Zn or hepatic Ca contents, respectively while that of Cd produced an increase in hepatic Zn as well as increasing Fe levels in animals fed a Fe-deficient diet (Table 4). The administration of Cd enhanced renal Zn, Cu and Ca and the dietary Fe deficiency maintained such an influence of Cd. However, the injection of Pb decreased the renal level of Cu in normal animals which remained so under dietary Fe deficiency• Interestingly, the administration of Pb significantly increased the lowered renal level of Fe in dietary Fe-deficient animals (Table 5).
[ ' ~ Normal+ Metal Ir~ Fe Def.+ Metal NICKEL
2.0'
.•,
_l.,
-I0,0
.L
".'
1.0-
•
.
•
•
°..
-S.O
.°, •••
~.
_.
--:
-- ".'- "r ","
15't0
I
L
LEAD
15.01
i
IX
r10.0
•
/
r b~_.
I:::1
5.0
~
"I |
I I
. . . . . .
15.0" 10"0• ~
CADMIUM
L4.0
.'-h ::
5.0 .
_°
--%
LIVER A
B
I ""1"•
I
A
"I " [
|
B
KIDNEY
Fig. 1. Influenceof dietary iron deficiencyon the levels of nickel, lead or cadmium in rat liver and kidney. Each bar is mean ± S.D. (6) aP < 0.001, bp < 0.01, cp < 0.05 vs normal diet alone represented by continuous horizontal line ( ), xp < 0.001 vs. Fe-deficient diet alone represented by broken horizontal line ( - - - ) .
The administration of all three metals (Ni, Pb or Cd) increased the hepatic Zn, Ca and Fe levels of rats maintained on normal diets• The Fe-deficient
4. Discussion
The hematopoietic alterations under Fe deficiency are generally akin to Pb or Cd exposure (Hashmi et al., 1989b; Schafer et al., 1990; Baynes and Bothwell, 1990) but in the present study only Pb seems to support this observation and Ni appears to reduce the influence of Fe deficiency. The stimulating effect of the metals on the hepatic synthesis of cholesterol is believed to result in the increased blood cholesterol, phospholipid, or triglycerides in experimental animals (Yoshikawa et al., 1974; Mathur and Tandon, 1979; Hashmi et al., 1989b). This proposition has been supported by Pb only irrespective of the dietary status of the animals• The higher retention of Cd or Pb in the liver and kidney of rats maintained on a Fedeficient diet may be attributed to the greater availability of binding sites for these metals under Fe deficiency (Ragan, 1977; Schafer et al., 1990)• The results however, indicate that the body status of Fe has no influence on the retention of Ni in the intestinal mucosa. The fact that dietary Fe deficiency decreased the native levels of renal and intestinal MT but not hepatic MT indicates the selective influence of dietary Fe status on MT in different tissues• However, the enhanced MT synthesis by Cd in all the three organs examined irrespective of body Fe status shows that this is due to the relative ability of various metals to induce MT rather than the selective response of the tissue towards such an induction. On the other hand, Ni and Pb could
171
S.K. Tandon et al. / Sci. Total Environ. 148 (1994) 167-173
Table 4 Influence of dietary iron deficiency on metal-induced alterations in essential elements in rat liver Treatment
Zn
Cu
Ca
Fe
Normal diet Ni
32.5 ± 4.4 41.4 ± 3.4 b
3.4 ± 1.1 4.2 ± 0.4
17.1 ± 1.5 20.7 ± 2.5 b
182.6 ± 25.8 246.9 ± 22.3 a
Pb
41.1 ± 4.6 b
3.6 ± 0.4
22.2 ± 2.9 c
239.6 ± 28.8 b
Cd Fe-def. diet
45.1 ± 3.2 a 25.7 ± 2.1 b
4.0 ± 0.5 4.4 ± 0.8
21.1 ± 2.6 c 18.0 ± 2.0
207.0 4- 34.0 84.6 ± 15.3 a
Fe-def. Ni Fe-def. Pb Fe-def. Cd
33.8 ± 2.3 y 26.1 ± 4.1 52.5 ± 3.4 x
3.9 ± 0.4 3.5 ± 0.3 4.2 ± 0.6
19.0 ± 0.6 35.8 ± 4.9 x 19.7 ± 2.5
71.8 ± 9.8 63.4 ± 9.7 119.3 ± 4.6 z
All concentrations in #g/g, fresh tissue. Each figure is mean ± S.D. (6). a p < 0.001, bp < 0.01, c p < 0.05 vs. normal diet. xp < 0.001, YP < 0.01, z p < 0.05 vs. Fe-deficient diet.
cance observed (control versus experimental groups) in kidney and intestinal MT, in response to Ni or Pb administration in Fe-deficient animals, is actually due to the decrease in MT levels attributable to the body Fe-deficiency. In other words, Ni or Pb might have compensated for the decreasing influence of Fe defciency alone on the tissue MT induction. The increase in hepatic Zn in response to Ni, Pb or Cd administration in normal diet-fed animals and in response to Cd in Fe-deficient diet-animals and the increase in renal Zn and Cu in response to Cd injection both in normal diet as well as in Fedeficient diet-animals could be attributed to the
biosynthesise the hepatic MT only and to a far lesser extent than Cd. While Cd is a well known strong tissue MT inducer (Waalkes and Klaassen, 1985), Ni (Khandelwal et al., 1990) and Pb (Maitani et al., 1986) have occasionally been shown to be hepatic and/or renal MT inducers. Interestingly, while Ni or Pb were able to increase the biosynthesis of hepatic MT in animals fed a normal diet, they failed to do so in animals fed a Fe-deficient diet and the administration of these two metals significantly raised the renal and intestinal MT levels which were lowered under Fedeficiency. It appears that Ni and Pb are capable of inducing MT in liver only and that the signifi-
Table 5 Influence of dietary iron deficiency on metal-induced alterations in essential elements in rat kidney Treatment
Zn
Cu
Normal diet
34.6 ± 3.5 35.5 ± 3.5
7.1 ± 0.4 6.9 ± 1.6
Cd
35.8 ± 3.6 49.6 ± 5.8 c
5.5 ± 1.0 b 8.6 ± 1.2 c
Fe-def. diet Fe-def. Ni
37.2 ± 4.0 34.8 ± 3.3
7.3 ± 1.2 6.2 ± 1.1
7.6 ± 1.3 7.1 ± 1.0
Fe-def. Pb Fe-def. Cd
30.1 ± 2.5 z 47.6 ± 2.9Y
5.4 ± 0.6 x 9.0 ± 1.6 z
6.1 ± 0.6 9.1 + 1.1
Ni
Pb
Ca 7.5 ± 2.4 7.8 ± 1.5 6.9 + 2.0 10.9 ± 2.2 b
All concentrations in tzg/g, fresh tissue. Each figure is mean + S.D. (6) a p < 0.001, b p < 0.01, c p < 0.05 vs. normal diet. xp < 0.001, YP < 0.01, zp < 0.05 vs. Fe-deficient diet.
Fe 120.2 + 5.1 89.2 + 14.7 a 118.9 122.6 51.4 75.1 87.0
± ± ± ± ±
5.4 13.2 15.1 a 9.4 7.2 x
50.2 ± 10.6
172
induction of tissue MT by these toxic metals, capable of strongly binding additional Zn and Cu. The increase in hepatic Ca and Fe levels following Ni, Pb or Cd administration particularly in normal diet-animals may be the result of their increased uptake or retention upon heavy metal exposure. The marked increase in hepatic Ca level in response to Pb administration in Fe-deficient animals indicates that Ca uptake must accompany Pb accumulation. However, the extent of Cd-induced hepatic and renal MT and Pb- or Ni-induced hepatic MT appear related to the intracellular Ca levels in the present study. A simultaneous rise in Ca and MT following CdCI 2 administration has also been observed earlier (Yamane et al., 1990). 5. Conclusion Effects of dietary Fe deficiency in response to Ni, Pb or Cd were variable and metal-metal interactions may be influenced by their nature. 6. Acknowledgement Thanks are due to Ms N.S. Hashmi for some biochemical estimations. 7. References Baynes, R.D. and T.H. Bothwell, 1990. Iron deficiency. Annu. Rev. Nutr., 10: 133. Bremner, I., 1987. Nutritional and physiological significance of metallothionein+ In: H.R. Kagi and Y. Kojima, (Eds.), "Metallothionein II Birkhauser", Basel, Switzerland, p. 81. Bremner, I. and J.H. Beattie, 1990. Metallothionein and the trace minerals. Annu. Rev. Nutr., 10: 63. Clark, M., Royal, J. and R. Seeler, 1988. Interaction of iron deficiency and lead and the hematological findings in children with severe lead poisoning. Pediatrics, 81: 247. Clegg, J.W. and E.J. King, 1942. Estimation of hemoglobin by the alkaline hematin method. Br. Med. J., 2: 329. Conrad, M.E. and J.C. Barton Jr., 1978. Factors affecting the absorption and excretion of lead in rats. Gastroenterology, 74: 731. Cousins, R.J., 1985. Absorption, transport and hepatic metabolism of copper and zinc: Special reference to metallothionein and ceruloplasmin. Physiol. Rev., 65: 238. Eaton, D.L. and B.F. Toal, 1982. Evaluation of Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues. Toxicol. Appl. Pharrnacol., 66: 134.
S.K. Tandon et al./Sci. Total Environ. 148 (1994) 167-173
Eaton, D.L., N.H. Stacey, K.L. Wong and C.D. Klaassen, 1980. Dose response effect of various metal ions on rat liver metallothionein, glutathione, heme oxygenase and cytochrome P-450. Toxicol. Appl. Pharmacol., 55: 393. Elsenhans, B., G. Schmolke, K. Kolb, J. Stokes and W. Forth, 1987. Metal-metal interactions among dietary toxic and essential trace metals in the rat. Ecotoxicol. Environ. Safety, 14: 275. Hashmi, N.S., D.N. Kachru, S. Khandelwal, and S.K. Tandon, 1989a. Interrelationship between iron deficiency and lead intoxication (II). Biol. Trace Elem. Res., 22: 299. Hashmi, N.S., D.N. Kachru, and S.K. Tandon, 1989b. Interrelationship between iron deficiency and lead intoxication (I). Biol. Trace Elem. Res., 22: 287. Hidalgo, J., L. Company, M. Borras, J.S. Garvey and A. Armario, 1988. Metallothionein response to stress in rats: role in free radical scavenging. Am. J. Physiol., 255: 518. Keele, C.A. and E. Neil, 1966. Samsons Wright's "Applied Physiology". 2nd ed. Oxford Medical publication, ELBS and Oxford University Press, London, p. 12. Khandelwal, S., S.J.S. Flora and S.K. Tandon, 1990. Nickelselenium interaction. Time dependent biochemical alterations and metal decorporation in rats. Chem.-Biol. Interact., 75: 341. Maitani, T., A. Watahiki, and K.T. Suzuki, 1986. Induction of metallothionein after lead administration by three injection routes in mice. Toxicol. Appl. Pharmacol., 83:211. Mathur, A.K. and S.K. Tandon, 1979. Some biochemical alterations in early nickel toxicity. Chemosphere, 8: 983. Peters, T., T.J. Giovanniello, L. Apt, and J.F. Ross, 1956. A new method for the determination of serum iron II. J. Lab. Clin. Med., 48: 280. Ragan, H.A., 1977. Effects of iron deficiency on absorption and distribution of lead and cadmium in rats. J. Lab. Clin. Med., 90: 700. Ribas, B., M.A. Brenes, F.J. De pascual, J. Del Rio and M.I. Sanchez-Reus, 1987. Participation of metallothionein and cerebral structures in iron homeostasis of anemic rats. In: P. Bratter and P. Schramel (Eds.), Trace Element-Analytical Chemistry in Medicine and Biology, Vol. 4, Walter de Gruyter & Co., Berlin, p. 317. Robertson, A., J.N. Morrison, A.M. Wood and I. Bremner, 1989. Effects of iron deficiency on metallothionein-I: concentrations in blood and tissue of rats. J. Nutr., 119: 439. Schafer, S.G. and W. Forth, 1985. The interaction between cadmium and iron: A review of the literature. Trace Elem. Med., 2: 158. Schafer, S.G., U. Schwegler and K. Schumann, 1990. Retention of cadmium in cadmium-naive normal and iron-deficient rats as well as in cadmium-induced iron-deficient animals. Ecotoxicol. Environ. Safety, 20: 71. Tandon, S.K. and A.K. Mathur, 1986. Role of iron deficiency in suscptibility to nickel toxicity. J. Environ. Biol., 7: 201. Waalkes, M.P. and C.D. Klaassen, 1985. Concentration of metallothionein in major organs of rats after administration of various metals. Fundam. Appl. Toxicol., 5: 473.
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Yamane, Y., M. Fukuchi, C. Li and T. Koizumi, 1990. Protective effect of sodium molybdate against the acute toxicity of cadmium chloride. Toxicology, 60: 235. Yoshikawa, H., M. Ohsawa, and M. Kaneta, 1974. Clinicochemical studies on subacute cadmium poisoning in rabbits. Ind. Health, 12: 127.
173 Zar, J.H., 1984. Biostatistical Analysis, 2nd Ed. p. 194, Prentice-Hall, Englewood Cliffs, NJ. Zlatkis, A., B. Zak and A.J. Boyle, 1953. A new method for the direct determination of serum cholesterol J. Lab. Clin. Med., 41: 486.
a n d In ~lsl~nshipw i t h Men
The Science of the Total Environment 148 (1994) 167-173
ELSEVIER
Influence of dietary iron deficiency on nickel, lead and cadmium intoxication S.K. Tandon*, S. Khandelwal, V.K. Jain, N. M a t h u r Industrial Toxicology Research Centre. Post Box 80. Lucknow-226001, India
Abstract The influence of dietary iron deficiency on acute nickel, lead or cadmium toxicity as reflected by the induction of hepatic, renal, and intestinal metallothionein (MT), disposition of the metals and alterations in hematological parameters, was investigated in young rats to ascertain whether the toxic effects of these metals modify under anemic conditions. The administration of Cd induced hepatic, renal and intestinal MT while that of Ni or Pb induced hepatic MT only. While dietary Fe deficiency did not affect MT induction by Cd, it enhanced the synthesis of renal and intestinal MT by Ni and Pb. The accumulation of Pb in liver and kidney and that of Cd in liver only, were enhanced by Fe deficiency; the tissue deposition of Ni remained unaffected by Fe deficiency. The induction of hepatic MT by Ni, Pb or Cd appears to be related to the concomitant rise in the hepatic Zn, Ca and Fe levels in normal rats. However, dietary Fe deficiency increased the hepatic Zn in response to Ni or Cd and the hepatic Ca in response to Pb administration. Key words: Dietary iron deficiency; Nickel; Cadmium; Metallothionein; Biochemical alterations; Metals; Rat
I. Introduction The toxic manifestations of heavy metals are governed among other things by dietary constituents particularly the essential trace elements (Schafer and Forth, 1985; Elsenhans et al., 1987). Thus, while dietary Fe deficiency increases absorption of Pb and Cd (Ragan, 1977; Hashmi et al., 1989a; Schafer et al., 1990) its excess decreases their absorption (Conrad and Barton, 1978; Schafer and Forth, 1985; Clark et al., 1988). However, Fe deficiency enhances susceptibility to the toxic effects of Ni without particularly affecting its uptake (Tandon and Mathur, 1986). The dietary * Corresponding author.
Fe status therefore seems to influence the disposition and effects of toxic metals following exposure. The dietary constituents, particularly the essential metals markedly influence the induction of metallothionein (MT) (Cousins, 1985; Bremner, 1987). Considering the fact that MT is induced in response to various stresses (Hidalgo et al., 1988; Bremner and Beattie, 1990), including administration of heavy metals such as Cd or Hg, the specific genes coding for this protein may be expressed as an acute-phase response of the body. Like synthesis of other acute-phase proteins, synthesis of MT under such circumstances is probably designed to impart stress resistance or tolerance to cells and to maintain homeostasis. The MT induction in the intestinal mucosa and brain in anemic rats
0048-9697/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved. SSDI 0048-9697(93)03996-F
168
S.K. Tandon et al. / Sci. Total Environ. 148 (1994) 167-173
(Ribas et al., 1987) led the authors to propose the participation of MT in both the Fe absorption by the intestinal mucosa and the involvement of the brain in Fe homeostasis. An increase in MT-I concentration in blood cells and bone marrow, a reduction in MT level in the kidney and no change in hepatic MT as a consequence of Fe deficiency have all been reported (Robertson et al., 1989). In view of the current lack of knowledge of the biochemical mechanism of metal-metal interactions, the influence of dietary Fe deficiency on the acute toxicity of Ni, Pb or Cd as evaluated by tissue MT induction, metal disposition and certain related hematological alterations, was investigated in young rats.
2. Experimental 2.1. Animals and treatment
Forty-eight young male rats (70 ± 10 g) bred in the Industrial Toxicology Research Centre's colony were divided into two batches of 24. One batch was fed a Fe-sufficient and the other a Fe-deficient synthetic diet for 6 weeks (Table 1). The animals of the Fe-deficient group were subjected to orbital plexus puncturing twice a week for the first 2 weeks to ascertain if they were in a Fe-deficient state. After 6 weeks, the 24 animals in each batch were subdivided into four groups of six
Table 1 Chemical composition of diet Ingredients
g/kg
Casein Corn starch Sucrose Groundnut oil Mineral mixture (Modified AIN76 mineral mixture) Vitamin mixture (AIN-76 vitamin mixture) Choline chloride DL-methionine FeSO 4, 7 H20 in sucrose
220 370 300 50 45 10 1.5 2.0 1.5
Analytical grade FeSO 4.7H20 triturated in powered sucrose (1.5 g) to supply 35 mg or 5 mg Fe/kg diet for an Fe-sufficient or Fe-deficient diet, respectively
each. Three groups, each containing six animals from each batch were administered intraperitoneally a single dose of 120/~mol/kg Ni as NiCI2 6H20, 400 #mol/kg Pb as Pb (CH 3 COO)2 3H20 or 20 #mol/kg Cd as CdC12H20, dissolved in distilled water (2 ml/kg). The solution of lead acetate was prepared in boiled water and bubbled with nitrogen immediately prior to administration. The remaining group in each batch was given an equal volume of water and served as a control. The metals were given as equitoxic doses (i.e. MTD) previously determined and defined as the maximum dose which produced death in less than 10% of the exposed population (Eaton et al., 1980). The use of MTD provides information on the maximal effectiveness of the metal to induce MT at the time point selected. The animals were sacrificed 24 h after metal administration and blood drawn by cardiac puncture into heparinised vials. An aliquot of blood was centrifuged to separate the plasma. The liver and kidneys were removed and the small intestine (about 10 cm from the pylorus) excised, cut open and washed with saline. 2.2. Biochemical estimations
The liver and kidney were homogenised individually in 4 vols. and the intestine in 10 vols. of 0.01 M Tris-HC1 buffer (pH 7.4). The homogenates were centrifuged at 10 000 x g for 30 min and the heat stable fraction obtained by placing the post-mitochondrial supernatant in a boiling water bath for 5 min. The MT concentration was estimated in this fraction using the Cd/Hb affinity method (Eaton and Toal, 1982). Standard procedures were employed for the estimation of blood hemoglobin (Clegg and King, 1942); packed cell volume (Keele and Neil, 1966); plasmacholesterol (Zlatkis et al., 1953); total Fe and Fe-binding capacity (Peters et al., 1956). 2.3. Estimation o f metals
The weighed hepatic and renal samples were mineralized in nitric and perchloric acids (6:0.5). The residue was dissolved in 5% HNO 3 and the metals were determined using a flame atomic absorption spectrometer (Perkin-Elmer 5000). The readings were recorded for Cu (324.7 nm), Zn (213.9 nm), Ca (422.7 nm), Fe (248.3 nm), Ni
S.K. Tandon et al./ Sci. Total Environ. 148 (1994) 167-173
169
Table 2 Influence of dietary iron deficiency on m e t a l - i n d u c e d biochemical alterations in rat b l o o d Treatment
Normal Ni Pb Cd Fe-def. Fe-def. Fe-def. Fe-def.
diet
diet Ni Pb Cd
Blood H b (g/lO0 ml)
PCV (%)
Total cholesterol (mg/100 ml)
Total Fe (mequiv./100 ml)
Fe b i n d i n g capacity (mequiv./100 ml)
11.8 9.6 12.8 13.4 9.3 10.5 10.5 10.2
40.8 32.2 45.8 42.2 34.4 31.6 29.2 35.3
98.5 104.2 140.9 109.6 114.2 92.9 145.3 106.7
229.3 235.4 170.1 191.5 177.3 181.3 140.2 167.4
447.8 490.7 516.7 466.6 637.9 556.5 592.6 637.7
± ± ± ± ± ± ± ±
0.5 2.6 1.6 1.6 c 0.6 1.8 1.9 1.1
± ± ± ± ± ± ± ±
3.4 6.4 5.5 1.8 3.8 3.7 4.4 4.7
± ± ± ± ± ± ± ±
6.0 2.0 5.8 a 12.1 15.9 c 5.2Y 5.6 x 9.4
± ± ± ± ± ± ± ±
9.2 19.4 26.3 a 34.7 19.5 b 11.1 14.9Y 20.3
± a± q± ± ± ±
37.3 56.1 24.8 a 64.0 15.0 a 18.0Y 23.8 z 16.6
Each figure is m e a n ± S.D. (6). a p < 0.001, b p < 0.01, c p < 0.05 vs. n o r m a l diet. xp < 0.001, YP < 0.01, zp < 0.05 vs. Fe-deficient diet.
(232.0 nm), Pb (283.3 nm) and Cd (228.8 nm) against suitable standards, processed identically.
2.4. Statistical analysis A 2 x 2 randomised block factorial design was used to analyse various parameters using six replicates in each group (normal diet, normal diet + Ni, Fe-deficient diet and Fe-deficient diet + Ni). Prior to this analysis, the normality assumption for the data and homogeneity of variance between the treatment groups were ascertained. The means of the treatment groups from the controls were compared separately using Dunett's test (Zar, 1984). The same analysis was performed for Pb and Cd.
uninfluenced by dietary Fe deficiency. Ni and Pb were successful in inducing MT in liver only, but failed to induce the same in Fe-deficient rats. However, these metals could restore the Fe deficiency and led to a lowering in renal and intestinal MT levels (Table 3). Dietary Fe deficiency enhanced the accumulation of Pb in both liver and kidney and that of Cd in liver only. However, it had no effect on the tissue deposition of Ni (Fig. 1).
Table 3 Influence of dietary iron deficiency on the i n d u c t i o n o f tissue metallothionein by metals in rats
3. Results
Treatment
Liver
Dietary Fe deficiency decreased plasma Fe level but increased plasma cholesterol and Fe-binding capacity. The administration of Pb but not Ni or Cd caused similar effects in animals fed normal diets. The influence of Pb was more prominent in animals maintained on Fe-deficient diets (Table 2). Dietary Fe deficiency caused a significant decrease in renal and intestinal MT levels but the hepatic MT remained unaffected. Cd was highly effective in inducing MT synthesis in all the three tissues examined, an effect which remained
N o r m a l diet Ni Pb Cd Fe-def. diet Fe-def. Ni Fe-def. Pb Fe-def. Cd
4.5 14.4 6.5 46.2 5.4 5.8 4.7 63.0
± ± ± ± ± ± ± ±
Kidney
1.7 4.3 a 1.7 b 6.3 a 1.0 1.3 0.7 11.0 x
12.8 13.5 14.4 31.0 5.6 15.1 13.2 32.0
± ± ± + ± ± ± ±
Intestine
1.5 1.7 0.8 4.6 a 1.3 a 2.0 x 1.8 x 4.0 x
5.4 4.8 4.6 18.4 2.7 4.4 4.6 13.9
All concentrations is ttg/g, fresh tissue. Each mean q- S.D. (6). a p < 0.001, b p < 0.01, VS. n o r m a l diet. xp < 0.001, YP < 0.01, vs. Fe-deficient diet.
a+ + + ± ± ± ±
0.7 1.0 0.8 3.1 a 0.7 a 0.7 y 0.7Y 2.1 x
figure
is
170
S.K. Tandon et al. ~SoL Total Environ. 148 (1994) 167-173
diet however, decreased Zn and Fe contents of liver. The administration of Ni or Pb caused an increase in hepatic Zn or hepatic Ca contents, respectively while that of Cd produced an increase in hepatic Zn as well as increasing Fe levels in animals fed a Fe-deficient diet (Table 4). The administration of Cd enhanced renal Zn, Cu and Ca and the dietary Fe deficiency maintained such an influence of Cd. However, the injection of Pb decreased the renal level of Cu in normal animals which remained so under dietary Fe deficiency• Interestingly, the administration of Pb significantly increased the lowered renal level of Fe in dietary Fe-deficient animals (Table 5).
[ ' ~ Normal+ Metal Ir~ Fe Def.+ Metal NICKEL
2.0'
.•,
_l.,
-I0,0
.L
".'
1.0-
•
.
•
•
°..
-S.O
.°, •••
~.
_.
--:
-- ".'- "r ","
15't0
I
L
LEAD
15.01
i
IX
r10.0
•
/
r b~_.
I:::1
5.0
~
"I |
I I
. . . . . .
15.0" 10"0• ~
CADMIUM
L4.0
.'-h ::
5.0 .
_°
--%
LIVER A
B
I ""1"•
I
A
"I " [
|
B
KIDNEY
Fig. 1. Influenceof dietary iron deficiencyon the levels of nickel, lead or cadmium in rat liver and kidney. Each bar is mean ± S.D. (6) aP < 0.001, bp < 0.01, cp < 0.05 vs normal diet alone represented by continuous horizontal line ( ), xp < 0.001 vs. Fe-deficient diet alone represented by broken horizontal line ( - - - ) .
The administration of all three metals (Ni, Pb or Cd) increased the hepatic Zn, Ca and Fe levels of rats maintained on normal diets• The Fe-deficient
4. Discussion
The hematopoietic alterations under Fe deficiency are generally akin to Pb or Cd exposure (Hashmi et al., 1989b; Schafer et al., 1990; Baynes and Bothwell, 1990) but in the present study only Pb seems to support this observation and Ni appears to reduce the influence of Fe deficiency. The stimulating effect of the metals on the hepatic synthesis of cholesterol is believed to result in the increased blood cholesterol, phospholipid, or triglycerides in experimental animals (Yoshikawa et al., 1974; Mathur and Tandon, 1979; Hashmi et al., 1989b). This proposition has been supported by Pb only irrespective of the dietary status of the animals• The higher retention of Cd or Pb in the liver and kidney of rats maintained on a Fedeficient diet may be attributed to the greater availability of binding sites for these metals under Fe deficiency (Ragan, 1977; Schafer et al., 1990)• The results however, indicate that the body status of Fe has no influence on the retention of Ni in the intestinal mucosa. The fact that dietary Fe deficiency decreased the native levels of renal and intestinal MT but not hepatic MT indicates the selective influence of dietary Fe status on MT in different tissues• However, the enhanced MT synthesis by Cd in all the three organs examined irrespective of body Fe status shows that this is due to the relative ability of various metals to induce MT rather than the selective response of the tissue towards such an induction. On the other hand, Ni and Pb could
171
S.K. Tandon et al. / Sci. Total Environ. 148 (1994) 167-173
Table 4 Influence of dietary iron deficiency on metal-induced alterations in essential elements in rat liver Treatment
Zn
Cu
Ca
Fe
Normal diet Ni
32.5 ± 4.4 41.4 ± 3.4 b
3.4 ± 1.1 4.2 ± 0.4
17.1 ± 1.5 20.7 ± 2.5 b
182.6 ± 25.8 246.9 ± 22.3 a
Pb
41.1 ± 4.6 b
3.6 ± 0.4
22.2 ± 2.9 c
239.6 ± 28.8 b
Cd Fe-def. diet
45.1 ± 3.2 a 25.7 ± 2.1 b
4.0 ± 0.5 4.4 ± 0.8
21.1 ± 2.6 c 18.0 ± 2.0
207.0 4- 34.0 84.6 ± 15.3 a
Fe-def. Ni Fe-def. Pb Fe-def. Cd
33.8 ± 2.3 y 26.1 ± 4.1 52.5 ± 3.4 x
3.9 ± 0.4 3.5 ± 0.3 4.2 ± 0.6
19.0 ± 0.6 35.8 ± 4.9 x 19.7 ± 2.5
71.8 ± 9.8 63.4 ± 9.7 119.3 ± 4.6 z
All concentrations in #g/g, fresh tissue. Each figure is mean ± S.D. (6). a p < 0.001, bp < 0.01, c p < 0.05 vs. normal diet. xp < 0.001, YP < 0.01, z p < 0.05 vs. Fe-deficient diet.
cance observed (control versus experimental groups) in kidney and intestinal MT, in response to Ni or Pb administration in Fe-deficient animals, is actually due to the decrease in MT levels attributable to the body Fe-deficiency. In other words, Ni or Pb might have compensated for the decreasing influence of Fe defciency alone on the tissue MT induction. The increase in hepatic Zn in response to Ni, Pb or Cd administration in normal diet-fed animals and in response to Cd in Fe-deficient diet-animals and the increase in renal Zn and Cu in response to Cd injection both in normal diet as well as in Fedeficient diet-animals could be attributed to the
biosynthesise the hepatic MT only and to a far lesser extent than Cd. While Cd is a well known strong tissue MT inducer (Waalkes and Klaassen, 1985), Ni (Khandelwal et al., 1990) and Pb (Maitani et al., 1986) have occasionally been shown to be hepatic and/or renal MT inducers. Interestingly, while Ni or Pb were able to increase the biosynthesis of hepatic MT in animals fed a normal diet, they failed to do so in animals fed a Fe-deficient diet and the administration of these two metals significantly raised the renal and intestinal MT levels which were lowered under Fedeficiency. It appears that Ni and Pb are capable of inducing MT in liver only and that the signifi-
Table 5 Influence of dietary iron deficiency on metal-induced alterations in essential elements in rat kidney Treatment
Zn
Cu
Normal diet
34.6 ± 3.5 35.5 ± 3.5
7.1 ± 0.4 6.9 ± 1.6
Cd
35.8 ± 3.6 49.6 ± 5.8 c
5.5 ± 1.0 b 8.6 ± 1.2 c
Fe-def. diet Fe-def. Ni
37.2 ± 4.0 34.8 ± 3.3
7.3 ± 1.2 6.2 ± 1.1
7.6 ± 1.3 7.1 ± 1.0
Fe-def. Pb Fe-def. Cd
30.1 ± 2.5 z 47.6 ± 2.9Y
5.4 ± 0.6 x 9.0 ± 1.6 z
6.1 ± 0.6 9.1 + 1.1
Ni
Pb
Ca 7.5 ± 2.4 7.8 ± 1.5 6.9 + 2.0 10.9 ± 2.2 b
All concentrations in tzg/g, fresh tissue. Each figure is mean + S.D. (6) a p < 0.001, b p < 0.01, c p < 0.05 vs. normal diet. xp < 0.001, YP < 0.01, zp < 0.05 vs. Fe-deficient diet.
Fe 120.2 + 5.1 89.2 + 14.7 a 118.9 122.6 51.4 75.1 87.0
± ± ± ± ±
5.4 13.2 15.1 a 9.4 7.2 x
50.2 ± 10.6
172
induction of tissue MT by these toxic metals, capable of strongly binding additional Zn and Cu. The increase in hepatic Ca and Fe levels following Ni, Pb or Cd administration particularly in normal diet-animals may be the result of their increased uptake or retention upon heavy metal exposure. The marked increase in hepatic Ca level in response to Pb administration in Fe-deficient animals indicates that Ca uptake must accompany Pb accumulation. However, the extent of Cd-induced hepatic and renal MT and Pb- or Ni-induced hepatic MT appear related to the intracellular Ca levels in the present study. A simultaneous rise in Ca and MT following CdCI 2 administration has also been observed earlier (Yamane et al., 1990). 5. Conclusion Effects of dietary Fe deficiency in response to Ni, Pb or Cd were variable and metal-metal interactions may be influenced by their nature. 6. Acknowledgement Thanks are due to Ms N.S. Hashmi for some biochemical estimations. 7. References Baynes, R.D. and T.H. Bothwell, 1990. Iron deficiency. Annu. Rev. Nutr., 10: 133. Bremner, I., 1987. Nutritional and physiological significance of metallothionein+ In: H.R. Kagi and Y. Kojima, (Eds.), "Metallothionein II Birkhauser", Basel, Switzerland, p. 81. Bremner, I. and J.H. Beattie, 1990. Metallothionein and the trace minerals. Annu. Rev. Nutr., 10: 63. Clark, M., Royal, J. and R. Seeler, 1988. Interaction of iron deficiency and lead and the hematological findings in children with severe lead poisoning. Pediatrics, 81: 247. Clegg, J.W. and E.J. King, 1942. Estimation of hemoglobin by the alkaline hematin method. Br. Med. J., 2: 329. Conrad, M.E. and J.C. Barton Jr., 1978. Factors affecting the absorption and excretion of lead in rats. Gastroenterology, 74: 731. Cousins, R.J., 1985. Absorption, transport and hepatic metabolism of copper and zinc: Special reference to metallothionein and ceruloplasmin. Physiol. Rev., 65: 238. Eaton, D.L. and B.F. Toal, 1982. Evaluation of Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues. Toxicol. Appl. Pharrnacol., 66: 134.
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