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Effects of orally administered vanadium on the immune system and bone metabolism in experimental animals
The Science of the Total Ent~ronment, Supplement 1993 Elsevier Science Publishers B.V., Amsterdam
663
Effects of orally administered vanadium on the immune system and bone metabolism in experimental animals A. Mravcovfi, D. Jirovfi, H. Jan~i and J. L e n e r National Institute of Public Health Srobdrova 48, 100 42 Prague, Czechoslovakia
ABSTRACT Experiments were carried out to gain more information on the effects of long term exposure to low doses of vanadium administered to mice and rats in drinking water. The selective immunotoxic effects of vanadium were depression of phagocytosis, splenotoxicity, enlargement of spleen, elevation of peripheral blood leucocytes and T and B cell activation. Vanadium accumulates in hard tissues and influences the mineralisation of epiphyseal cartilage. This effect is obviously evident in young animals. Significant differences in vanadium concentration were found between young and adult animals. INTRODUCTION
The concentration of vanadium in the environment in Czechoslovakia is rising due to the combustion of fossil fuels and increasing use of vanadium in the metallurgical and chemical industry. A p a r t from exposure by inhalation there is a risk of vanadium intake via the gastrointestinal tract in the population. The obvious harmful effect of pollutants u p o n the immune system is an increase in various allergic disorders, infections, incidence of tumors, an increase in childhood and neonatal morbidities and a decline in life expectancy. W e wish to devise a test system that can be used for the screening of possible immunotoxicants. Basic assays have b e e n d e v e l o p e d which enable one to examine the influence of a suspected immunotoxicant on a specific aspect of the immune process [1]. Continuing our studies on the distribution of vanadium [2,3] current experiments were carried out to gain m o r e information on the effects of long term exposure to low doses of vanadium and its effect on the immune system. 1993 Elsevier Science Publishers B.V.
664
A. MRAVCOV,~ ET AL.
EXPERIMENTAL
Weanling and adult Wistar rats and mice ICR (males and females) were used. Rats: groups of 10 animals were given vanadium as V205 (Fluka A.G.) in deionized water (0, 1 and 100 mg W ' 1 - 1 ) for six months. Mice:
DISTRIBUTION OF 48V IN R A T S |
21 clays I - " " l ot(:l dP adult d'
TIBIA
INCISOR
#
21 d a y s old adutt 9
60"
II
30'
1
,'7
3
10
1
KIDNEY
3
10
SPLEEN
60-
30.
1 3 10 TIME AFTER APPLICATION {DAYSI
1
3
10
Fig. 1. Distribution of 4Sv in young and adult rats after subcutaneous application (300 ng V-kg -1, 111 kBq'kg-1); mean, SD, n = 6.
EFFEC'I~ O F O R A L L Y A D M I N I S T E R E D V A N A D I U M IN E X P E R I M E N T A L ANIMALS
665
Fig. 2. Autoradiogram of rat tibia after subcutaneous application of 4sv-labelled vanadate.
groups of 10 animals were given VeO 5 in 0.2 ml deionized water (0 or 6 mg V-kg -1) by gavage 5 days a week for 6 weeks. In distribution studies 48V-labelled sodium vanadate was used (300 ng V . k g -~ b.w., activity 111 kBq.kg-~). The results are expressed as %~ of applied dose .g-1. Autoradiography was done using emulsion ORWO AF4 [4]. Histopathology employed standard procedures [5]. For the immunotoxicological study adult male rats and mice were used. I m m u n e dysfunctions tested: immunopathology (spleen and thymus weight, spleen cellularity, number of peripheral blood leucocytes); nonspecific immunity (phagocytosis, natural
....
eS~.~" i ~ ' ~ k "
- ~ * ~ k ,
~i~
./~.. ~":.'~:?~.~
......
-,~ ~ . ~ , - , , , , . :,, : ' ' . ~ , ~ _ . . ~ , ~ , , - - , , , , ~ ,
Fig. 3. Growth zone of epiphyseal cartilage in control rat.
666
i
A. MRAVCOV.~ E T AI.
~
~
~ ~ "
I I ' I I I ~ _ I F " I L - . , I ~1~'
l l l ~ l i "
;Y
1 ~ . ~~
D:ll:
p ....
~,
.
.:,,:, .
.,
',~
-~.:~¢~ ~ , ' / S ~ . _ ~
ri,--: ~
!l~JIb
,iv'-
.lt.ll
"° - -,..;ll~,-
Fig. 4. G r o w t h z o n e o f e p i p h y s e a l cartilage in v a n a d i u m - t r e a t e d rat.
killer cell activity - - NK cells); humoral-mediated immunity (plaqueforming cells to T-dependent antigen - - SRBC); cell-mediated immunity (lymphocyte blastogenesis to mitogens - - PHA, Con A, PWM); conventional procedures [1,3]. Experimental results are expressed as percentage of results in controls (100%). Differences were evaluated using an unpaired t-test. RESULTS
It is apparent that vanadium accumulates in the skeleton (Fig. 1). Significantly higher concentrations of vanadium were found in young compared to adult animals. Autoradiography of tibia shows the accumulation of vanadium in epiphyseal cartilage and subperiosteous zone of the diaphysis (Fig. 2). Histological examination revealed the influence of vanadium on the ossification of epiphyseal cartilage (Figs. 3 and 4). Immunotoxicological study proved enlargement of spleen in a group of rats given a toxic dose (100 mg. 1-1) of vanadium (Fig. 6). A similar phenomenon was found in mice, although a cellularity of spleen was depressed. The thymus weight was not affected. The number of peripheral blood leucocytes was significantly (P < 0.05) elevated (Fig. 5). These changes may be related to the splenotoxicity of vanadium. A depression of phagocytosis was found in mice and in a dose-dependent manner in rats (Figs. 5 and 6). Mice exposed to low dose of vanadium exhibited higher mitogen responsiveness and a very high stimulation of B cells in PFC assay. No changes in NK cell activity were found for mice (Fig. 5). These results document the activation of T and B cell immune responsiveness.
667
E F F E C T S O F O R A L L Y A D M I N I S T E R E D V A N A D I U M IN E X P E R I M E N T A L ANIMALS
I DOS£:
6 ag V.kg~
200
T T
T
controls
100
"FW
$v
$C
L
PH
NK
PFC
pHA
Con A
Fig. 5. Vanadium immunotoxicityevaluation in mice. TW, thymus weight; SW, spleen weight; SC, spleen cellularity;1, leucocytes(peripheralblood);PH, phagocytosis;NK, natural killers; PFC, plaque-formingcells; PHA, phytohemagglutininresponsiveness;Con A, concanavalin responsiveness. CONCLUSIONS Vanadium deposition in the skeleton is greatest in areas of recent bone formation and mineralisation. This may be due to the insulin-like effect of vanadium since insulin-like factors are considered potent mitogens of the bone. A selective immunotoxicity of vanadium in low doses which differs from general toxicity following high dose exposure is suggested. Immunotoxic effects are depression of phagocytosis, splenotoxicity, enlargement of spleen and T and B cell activation. The changes are accompanied by elevation of peripheral blood leucocytes. Our results strongly support other findings [6,7]. Depression of phagocytosis may have an important role in host resistance to infections. The T and B cell
668
A. MRAVCOVA ET AL.
T
Fts. 6
I
,|
X ~ESt
S
loo
-a
v.Cz
2OO
S'~
~
PHA
Con A
Fig. 6. Vanadium immunotoxicity evaluation in rats. SW, spleen weight; PH, phagocytosis; PHA, phytohemagglutinin responsiveness; Con A, concanavalin A responsiveness; PWM, pokeweed mitogen responsiveness.
activation and particularly the high Con A response of T suppressor cells suggest the possibility of vanadium-related hypersensitivity REFERENCES 1 M.I. Luster, A.E. Munson, P.T. Thomas et al., Development of a testing battery to asses chemical-induced immunotoxicity. Fundam. Appl. Toxicol., 10 (1988) 2-19. 2 A. Mravcovfi, A. Babicloj and J. Lener, On the vanadium metabolism in rats. Physiol. Bohemoslov., 37 (1988) 549. 3 D. Jirov~, A. Mravcovfi and J. Lener, Effect of vanadium on some reactions of cell-mediated immunity. Physiol. Bohemoslov., 39 (1990) 550.
E F F E C T S OF O R A L L Y A D M I N I S T E R E D V A N A D I U M IN E X P E R I M E N T A L ANIMALS
669
4 F. My~k, A. Mravcov~t, M. Krej~i et al., Distribution of 48V labelled vanadate in bones of experimental animals. Nukleonika, in press. 5 A. Mravcov~ and L. Puzanov~, Toxicological parameters of vanadium exposure. Procc. 6th international trace element symposium, 1 (1989) 93-98. 6 M.D. Cohen and C.J. Wei, Effect of ammonium metavanadate on the murine immune response. J. Toxicol. Environ. Health, 19 (1986) 279. 7 A.W. Musk and J.G. Tus, Asthma caused by occupational exposure to vanadium compounds. Med. J. Aust., 1/4 (1982) 183-184.
663
Effects of orally administered vanadium on the immune system and bone metabolism in experimental animals A. Mravcovfi, D. Jirovfi, H. Jan~i and J. L e n e r National Institute of Public Health Srobdrova 48, 100 42 Prague, Czechoslovakia
ABSTRACT Experiments were carried out to gain more information on the effects of long term exposure to low doses of vanadium administered to mice and rats in drinking water. The selective immunotoxic effects of vanadium were depression of phagocytosis, splenotoxicity, enlargement of spleen, elevation of peripheral blood leucocytes and T and B cell activation. Vanadium accumulates in hard tissues and influences the mineralisation of epiphyseal cartilage. This effect is obviously evident in young animals. Significant differences in vanadium concentration were found between young and adult animals. INTRODUCTION
The concentration of vanadium in the environment in Czechoslovakia is rising due to the combustion of fossil fuels and increasing use of vanadium in the metallurgical and chemical industry. A p a r t from exposure by inhalation there is a risk of vanadium intake via the gastrointestinal tract in the population. The obvious harmful effect of pollutants u p o n the immune system is an increase in various allergic disorders, infections, incidence of tumors, an increase in childhood and neonatal morbidities and a decline in life expectancy. W e wish to devise a test system that can be used for the screening of possible immunotoxicants. Basic assays have b e e n d e v e l o p e d which enable one to examine the influence of a suspected immunotoxicant on a specific aspect of the immune process [1]. Continuing our studies on the distribution of vanadium [2,3] current experiments were carried out to gain m o r e information on the effects of long term exposure to low doses of vanadium and its effect on the immune system. 1993 Elsevier Science Publishers B.V.
664
A. MRAVCOV,~ ET AL.
EXPERIMENTAL
Weanling and adult Wistar rats and mice ICR (males and females) were used. Rats: groups of 10 animals were given vanadium as V205 (Fluka A.G.) in deionized water (0, 1 and 100 mg W ' 1 - 1 ) for six months. Mice:
DISTRIBUTION OF 48V IN R A T S |
21 clays I - " " l ot(:l dP adult d'
TIBIA
INCISOR
#
21 d a y s old adutt 9
60"
II
30'
1
,'7
3
10
1
KIDNEY
3
10
SPLEEN
60-
30.
1 3 10 TIME AFTER APPLICATION {DAYSI
1
3
10
Fig. 1. Distribution of 4Sv in young and adult rats after subcutaneous application (300 ng V-kg -1, 111 kBq'kg-1); mean, SD, n = 6.
EFFEC'I~ O F O R A L L Y A D M I N I S T E R E D V A N A D I U M IN E X P E R I M E N T A L ANIMALS
665
Fig. 2. Autoradiogram of rat tibia after subcutaneous application of 4sv-labelled vanadate.
groups of 10 animals were given VeO 5 in 0.2 ml deionized water (0 or 6 mg V-kg -1) by gavage 5 days a week for 6 weeks. In distribution studies 48V-labelled sodium vanadate was used (300 ng V . k g -~ b.w., activity 111 kBq.kg-~). The results are expressed as %~ of applied dose .g-1. Autoradiography was done using emulsion ORWO AF4 [4]. Histopathology employed standard procedures [5]. For the immunotoxicological study adult male rats and mice were used. I m m u n e dysfunctions tested: immunopathology (spleen and thymus weight, spleen cellularity, number of peripheral blood leucocytes); nonspecific immunity (phagocytosis, natural
....
eS~.~" i ~ ' ~ k "
- ~ * ~ k ,
~i~
./~.. ~":.'~:?~.~
......
-,~ ~ . ~ , - , , , , . :,, : ' ' . ~ , ~ _ . . ~ , ~ , , - - , , , , ~ ,
Fig. 3. Growth zone of epiphyseal cartilage in control rat.
666
i
A. MRAVCOV.~ E T AI.
~
~
~ ~ "
I I ' I I I ~ _ I F " I L - . , I ~1~'
l l l ~ l i "
;Y
1 ~ . ~~
D:ll:
p ....
~,
.
.:,,:, .
.,
',~
-~.:~¢~ ~ , ' / S ~ . _ ~
ri,--: ~
!l~JIb
,iv'-
.lt.ll
"° - -,..;ll~,-
Fig. 4. G r o w t h z o n e o f e p i p h y s e a l cartilage in v a n a d i u m - t r e a t e d rat.
killer cell activity - - NK cells); humoral-mediated immunity (plaqueforming cells to T-dependent antigen - - SRBC); cell-mediated immunity (lymphocyte blastogenesis to mitogens - - PHA, Con A, PWM); conventional procedures [1,3]. Experimental results are expressed as percentage of results in controls (100%). Differences were evaluated using an unpaired t-test. RESULTS
It is apparent that vanadium accumulates in the skeleton (Fig. 1). Significantly higher concentrations of vanadium were found in young compared to adult animals. Autoradiography of tibia shows the accumulation of vanadium in epiphyseal cartilage and subperiosteous zone of the diaphysis (Fig. 2). Histological examination revealed the influence of vanadium on the ossification of epiphyseal cartilage (Figs. 3 and 4). Immunotoxicological study proved enlargement of spleen in a group of rats given a toxic dose (100 mg. 1-1) of vanadium (Fig. 6). A similar phenomenon was found in mice, although a cellularity of spleen was depressed. The thymus weight was not affected. The number of peripheral blood leucocytes was significantly (P < 0.05) elevated (Fig. 5). These changes may be related to the splenotoxicity of vanadium. A depression of phagocytosis was found in mice and in a dose-dependent manner in rats (Figs. 5 and 6). Mice exposed to low dose of vanadium exhibited higher mitogen responsiveness and a very high stimulation of B cells in PFC assay. No changes in NK cell activity were found for mice (Fig. 5). These results document the activation of T and B cell immune responsiveness.
667
E F F E C T S O F O R A L L Y A D M I N I S T E R E D V A N A D I U M IN E X P E R I M E N T A L ANIMALS
I DOS£:
6 ag V.kg~
200
T T
T
controls
100
"FW
$v
$C
L
PH
NK
PFC
pHA
Con A
Fig. 5. Vanadium immunotoxicityevaluation in mice. TW, thymus weight; SW, spleen weight; SC, spleen cellularity;1, leucocytes(peripheralblood);PH, phagocytosis;NK, natural killers; PFC, plaque-formingcells; PHA, phytohemagglutininresponsiveness;Con A, concanavalin responsiveness. CONCLUSIONS Vanadium deposition in the skeleton is greatest in areas of recent bone formation and mineralisation. This may be due to the insulin-like effect of vanadium since insulin-like factors are considered potent mitogens of the bone. A selective immunotoxicity of vanadium in low doses which differs from general toxicity following high dose exposure is suggested. Immunotoxic effects are depression of phagocytosis, splenotoxicity, enlargement of spleen and T and B cell activation. The changes are accompanied by elevation of peripheral blood leucocytes. Our results strongly support other findings [6,7]. Depression of phagocytosis may have an important role in host resistance to infections. The T and B cell
668
A. MRAVCOVA ET AL.
T
Fts. 6
I
,|
X ~ESt
S
loo
-a
v.Cz
2OO
S'~
~
PHA
Con A
Fig. 6. Vanadium immunotoxicity evaluation in rats. SW, spleen weight; PH, phagocytosis; PHA, phytohemagglutinin responsiveness; Con A, concanavalin A responsiveness; PWM, pokeweed mitogen responsiveness.
activation and particularly the high Con A response of T suppressor cells suggest the possibility of vanadium-related hypersensitivity REFERENCES 1 M.I. Luster, A.E. Munson, P.T. Thomas et al., Development of a testing battery to asses chemical-induced immunotoxicity. Fundam. Appl. Toxicol., 10 (1988) 2-19. 2 A. Mravcovfi, A. Babicloj and J. Lener, On the vanadium metabolism in rats. Physiol. Bohemoslov., 37 (1988) 549. 3 D. Jirov~, A. Mravcovfi and J. Lener, Effect of vanadium on some reactions of cell-mediated immunity. Physiol. Bohemoslov., 39 (1990) 550.
E F F E C T S OF O R A L L Y A D M I N I S T E R E D V A N A D I U M IN E X P E R I M E N T A L ANIMALS
669
4 F. My~k, A. Mravcov~t, M. Krej~i et al., Distribution of 48V labelled vanadate in bones of experimental animals. Nukleonika, in press. 5 A. Mravcov~ and L. Puzanov~, Toxicological parameters of vanadium exposure. Procc. 6th international trace element symposium, 1 (1989) 93-98. 6 M.D. Cohen and C.J. Wei, Effect of ammonium metavanadate on the murine immune response. J. Toxicol. Environ. Health, 19 (1986) 279. 7 A.W. Musk and J.G. Tus, Asthma caused by occupational exposure to vanadium compounds. Med. J. Aust., 1/4 (1982) 183-184.