Toxicology, 63 (1990) 53--62 Elsevier Scientific Publishers Ireland Ltd.
Biological monitoring for occupational cadmium exposure: the urinary metallothionein Z . A . Shaikh a, K.J. Ellis b, K.S. S u b r a m a n i a n c a n d A. G r e e n b e r g d ~Department of Pharmacology and Toxicology, University of Rhode Island, Kingston, RI 02881, ~Children's Nutrition Research Center, Baylor College of Medicine, Houston, TX 77096, CEnvironmental Health Center, Health and Welfare Canada, Tunney's Pasture Ottawa, Ontario (Canada) and aDepartment of Medicine, University of Pittsburgh, Pittsburgh, PA 15261 (U.S.A.)
(Received June 1st, 1989; accepted March 21st, 1990)
Summary The relationship between urinary metallothionein and kidney and liver cadmium levels was examined in 68 active and retired smelter workers. Metallothionein was analyzed by a radioimmunoassay and liver and kidney cadmium levels were determined by in vivo neutron activation. Four workers suffered from severe renal dysfunction and excreted high amounts of total protein and/3:microglobulin and greater than l mg metallothionein/g creatinine. In the remaining 64 workers the urinary metallothionein levels correlated significantly with the cadmium levels in both liver and kidney. Similarly, in these individuals urinary metallothionein was significantly related to cadmium in blood and urine. These results demonstrate that urinary metallothionein is a sensitive biological indicator of cadmium exposure and body burden, before the onset of severe renal dysfunction. Key words: Cadmium; Occupational exposure; Urinalysis; Metallothionein; Biological monitoring; In
vivo neutron activation analysis; Radioimmunoassay
Introduction E x p o s u r e to c a d m i u m (Cd) induces the synthesis o f m e t a l l o t h i o n e i n (MT), a 6600 D a p r o t e i n , in liver, k i d n e y a n d o t h e r tissues. This p r o t e i n binds the metal a n d is r e s p o n s i b l e for the a c c u m u l a t i o n o f c a d m i u m in v a r i o u s tissues [1]. O u r p r e v i o u s studies in c a d m i u m - t r e a t e d a n i m a l s have s h o w n that a l t h o u g h M T is a c y t o p l a s m i c p r o t e i n , it is released into the c i r c u l a t i o n in small quantities [2]. T h e circulating C d - M T c o m p l e x , d u e to its low m o l e c u l a r weight, is efficiently filtered by the kidneys. A l t h o u g h , m o s t o f the filtered M T is r e a b s o r b e d by the p r o x i m a l tubules, a small f r a c t i o n is excreted in urine [2,3] in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r [4]. M e a s u r e m e n t o f u r i n a r y M T in C d - e x p o s e d i n d i v i d u a l s m a y , therefore, o f f e r a p r o m i s i n g a p p r o a c h for the biological m o n i t o r i n g o f c u m u l a t i v e C d body burden. U r i n a r y a n d b l o o d C d , two c o m m o n l y used indices o f b o d y b u r d e n , are often u n r e l i a b l e d u e to the p o s s i b i l i t y o f c o n t a m i n a t i o n d u r i n g s a m p l i n g . F u r t h e r m o r e ,
Correspondence to: Z.A. Shaikh. 0300-483X/90/$03.50 ~3 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
53
blood and urinary Cd, at high exposure, reflect current exposure rather than the body burden [5]. Direct measurement of liver and kidney Cd burdens, can be performed by in vivo neutron activation analysis. Using this technique Ellis et al. [6--10] determined the tissue Cd burden of a group of smelter workers. In a limited number of these workers, Tohyama et al. [11] examined urinary MT concentration and found that the levels of this protein correlated significantly with the renal and hepatic Cd. The present investigation was undertaken to document the relationship between urinary MT and tissue Cd burden in a larger group of smelter workers who were either actively employed or retired at the time of the study. Methods
Occupationally exposed subjects The subjects included in this study were 68 males workers, ranging in age from 22 to 92 years, who had been employed at a cadmium smelter in the United States. All subjects were volunteers and participated in the study after an informed consent was obtained. Forty-one of the workers were actively employed at the time of the study; the remaining 27 subjects were retired workers. Descriptions of the cadmium smelter, the working conditions and the degree of exposure of workers to Cd have been previously described [6--10].
Urinanalysis Spot and 8- or 24-h urine samples were collected from each individual and stored between - 2 0 and - 8 0 ° C until analysis. Generally, the retired workers followed a 24-h rather than 8-h urine collection schedule. MT was analyzed by a modification of the radioimmunoassay method described earlier [2]. The modification consisted of using Pansorbin (Calbiochem, La Jolla, CA) to separate the bound antigen. Urinary creatinine was determined using a colorimetric assay kit (Sigma Chemical Company, St. Louis, MO), urinary protein was analyzed by the method of Bradford [12], and urinary fl2-microglobulin (fl2-MG) was quantitated by radioimmunoassay (Pharmacia, Uppsala, Sweden). Blood urea nitrogen and serum creatinine were assayed by an autoanalyzer.
Cadmium analysis Blood and urine Cd were determined by atomic absorption spectrophotometry [13]. The Cd concentration in the liver and the Cd content in the left kidney of each subject were measured using the in vivo neutron activation technique previously described [6].
Data analysis All urinary data were normalized with respect to creatinine to minimize variation due to dilution. Statistical evaluation was performed on log-transformed data [14]. Parallelism between the two slopes was checked by Student's t-test. Paired t-test analysis was used to test the significance of the difference between MT levels in the spot and timed urine samples from each subject.
54
Results
Urinary MT levels in spot and timed urine samples obtained from each subject were compared (Fig. 1). MT levels in samples collected over time were markedly lower than the spot samples, especially at the lower end of the concentration range. The samples collected for 8 h (active workers) were no better than those collected by the retired workers over the 24-h time period. Because of these observations, only the spot urine data were used for all subsequent statistical analyses. The MT was significantly related to total urinary protein, as well as/]2-MG, in both active and retired workers (Figs. 2 and 3). On a group basis, the retired workers excreted more total protein, ~2-MG and MT than the active workers. However, there was no apparent difference between the active and retired workers with respect to the relationship of total protein or /]2-MG with urinary MT. Since both urinary protein and/I2-MG are used as indices of renal function in Cd-exposed populations, the relationship between the two was examined and is shown in Fig 4. On the basis of normal or abnormal levels of total protein and ~2-MG, the population was divided into two main groups; those excreting less than or equal to 250 mg protein or 500 ~g ~2-MG/g creatinine (normal renal function) and those excreting more than these amounts (renal dysfunction). All but two individuals in the latter group were retired workers and were older than the other group. Among the renal dysfunction group, four individuals were identified as severe cases based on very high total urinary protein and fJ2-MG excretion. Two of these individuals had elevated blood urea nitrogen and all four had
._= C N 0
•
10 =
•c~ w
a
.,-"
~
v 0 C
T
-
t
1
..........................
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10 ~
10 =
10'
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Timed Urine (POMT/o CreaUnine) Fig. 1. Comparison of MT levels in spot urine and urine collected over 8--24 h. The graph includes both active (r-l) and retired ( I ) workers. The linear regression line (--) is plotted using the following equation: log Y = 0.506 log X + 1.375 (N = 68; r = 0.84; P < 0.001). The dashed line represents the hypothetical regression line if the MT concentration in the two urine specimens from each individual was the same.
55
A
,~,
l°'F
tD
°
" i .
,o. F
e
'=
] 10'
. . . . . . . 10 =
10'
Urine Protein Img/g Creatinine) Fig. 2. The relationship between total protein and MT in spot urine samples. The graph includes both active (r-I) and retired (11) workers. The linear regression line is plotted using the following equation: log Y = 0.835 log X + 0.704 (N = 68; r = 0.76; P < 0.001).
elevated serum creatinine concentration (Table I). These individuals were excluded from all subsequent correlation analyses. Age o f the individuals was related significantly to the duration of their exposures. Age also showed a positive correlation with all parameters analyzed (data not shown). The effect o f age on these parameters, therefore, could not be separated from the effect o f cadmium exposure. Figure 5 depicts the relationship between urinary MT and Cd content in the left kidney. All subjects, including those with mild to moderate renal dysfuncA
Q)
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10 4
0
o
a
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o
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.:=) 10'
1 .......................................... 10'
10 =
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Urine p,MG (POLO Creatinine) Fig. 3. The relationship between /J2-MG and MT in spot urine samples. The graph includes both active (I-I) and retired (11) workers. The linear regression line is plotted using the following equation: log Y = 0.295 l o g X + 1.819(N = 6 8 ; r = 0 . 6 6 ; P < 0.001).
56
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Fig. 4. The relationship between total protein and 32-MG levels in spot urine samples of active and retired workers. The group is subdivided according to normal (O) and abnormal ( e , & ) levels of protein and ~2-MG in urine. Normal upper limit for protein was considered to be 250 mg/g creatinine and that for/J2-MG to be 500 ~g/g creatinine. Four individuals (&) had excessively high levels of urinary protein, fl2-MG, MT, blood urea nitrogen and serum creatinine (Table l). The linear regression line is plotted using the following equation: log Y = 1.802 log X + 1.585 ( N = 68; r = 0.74; P
Biological monitoring for occupational cadmium exposure: the urinary metallothionein Z . A . Shaikh a, K.J. Ellis b, K.S. S u b r a m a n i a n c a n d A. G r e e n b e r g d ~Department of Pharmacology and Toxicology, University of Rhode Island, Kingston, RI 02881, ~Children's Nutrition Research Center, Baylor College of Medicine, Houston, TX 77096, CEnvironmental Health Center, Health and Welfare Canada, Tunney's Pasture Ottawa, Ontario (Canada) and aDepartment of Medicine, University of Pittsburgh, Pittsburgh, PA 15261 (U.S.A.)
(Received June 1st, 1989; accepted March 21st, 1990)
Summary The relationship between urinary metallothionein and kidney and liver cadmium levels was examined in 68 active and retired smelter workers. Metallothionein was analyzed by a radioimmunoassay and liver and kidney cadmium levels were determined by in vivo neutron activation. Four workers suffered from severe renal dysfunction and excreted high amounts of total protein and/3:microglobulin and greater than l mg metallothionein/g creatinine. In the remaining 64 workers the urinary metallothionein levels correlated significantly with the cadmium levels in both liver and kidney. Similarly, in these individuals urinary metallothionein was significantly related to cadmium in blood and urine. These results demonstrate that urinary metallothionein is a sensitive biological indicator of cadmium exposure and body burden, before the onset of severe renal dysfunction. Key words: Cadmium; Occupational exposure; Urinalysis; Metallothionein; Biological monitoring; In
vivo neutron activation analysis; Radioimmunoassay
Introduction E x p o s u r e to c a d m i u m (Cd) induces the synthesis o f m e t a l l o t h i o n e i n (MT), a 6600 D a p r o t e i n , in liver, k i d n e y a n d o t h e r tissues. This p r o t e i n binds the metal a n d is r e s p o n s i b l e for the a c c u m u l a t i o n o f c a d m i u m in v a r i o u s tissues [1]. O u r p r e v i o u s studies in c a d m i u m - t r e a t e d a n i m a l s have s h o w n that a l t h o u g h M T is a c y t o p l a s m i c p r o t e i n , it is released into the c i r c u l a t i o n in small quantities [2]. T h e circulating C d - M T c o m p l e x , d u e to its low m o l e c u l a r weight, is efficiently filtered by the kidneys. A l t h o u g h , m o s t o f the filtered M T is r e a b s o r b e d by the p r o x i m a l tubules, a small f r a c t i o n is excreted in urine [2,3] in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r [4]. M e a s u r e m e n t o f u r i n a r y M T in C d - e x p o s e d i n d i v i d u a l s m a y , therefore, o f f e r a p r o m i s i n g a p p r o a c h for the biological m o n i t o r i n g o f c u m u l a t i v e C d body burden. U r i n a r y a n d b l o o d C d , two c o m m o n l y used indices o f b o d y b u r d e n , are often u n r e l i a b l e d u e to the p o s s i b i l i t y o f c o n t a m i n a t i o n d u r i n g s a m p l i n g . F u r t h e r m o r e ,
Correspondence to: Z.A. Shaikh. 0300-483X/90/$03.50 ~3 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
53
blood and urinary Cd, at high exposure, reflect current exposure rather than the body burden [5]. Direct measurement of liver and kidney Cd burdens, can be performed by in vivo neutron activation analysis. Using this technique Ellis et al. [6--10] determined the tissue Cd burden of a group of smelter workers. In a limited number of these workers, Tohyama et al. [11] examined urinary MT concentration and found that the levels of this protein correlated significantly with the renal and hepatic Cd. The present investigation was undertaken to document the relationship between urinary MT and tissue Cd burden in a larger group of smelter workers who were either actively employed or retired at the time of the study. Methods
Occupationally exposed subjects The subjects included in this study were 68 males workers, ranging in age from 22 to 92 years, who had been employed at a cadmium smelter in the United States. All subjects were volunteers and participated in the study after an informed consent was obtained. Forty-one of the workers were actively employed at the time of the study; the remaining 27 subjects were retired workers. Descriptions of the cadmium smelter, the working conditions and the degree of exposure of workers to Cd have been previously described [6--10].
Urinanalysis Spot and 8- or 24-h urine samples were collected from each individual and stored between - 2 0 and - 8 0 ° C until analysis. Generally, the retired workers followed a 24-h rather than 8-h urine collection schedule. MT was analyzed by a modification of the radioimmunoassay method described earlier [2]. The modification consisted of using Pansorbin (Calbiochem, La Jolla, CA) to separate the bound antigen. Urinary creatinine was determined using a colorimetric assay kit (Sigma Chemical Company, St. Louis, MO), urinary protein was analyzed by the method of Bradford [12], and urinary fl2-microglobulin (fl2-MG) was quantitated by radioimmunoassay (Pharmacia, Uppsala, Sweden). Blood urea nitrogen and serum creatinine were assayed by an autoanalyzer.
Cadmium analysis Blood and urine Cd were determined by atomic absorption spectrophotometry [13]. The Cd concentration in the liver and the Cd content in the left kidney of each subject were measured using the in vivo neutron activation technique previously described [6].
Data analysis All urinary data were normalized with respect to creatinine to minimize variation due to dilution. Statistical evaluation was performed on log-transformed data [14]. Parallelism between the two slopes was checked by Student's t-test. Paired t-test analysis was used to test the significance of the difference between MT levels in the spot and timed urine samples from each subject.
54
Results
Urinary MT levels in spot and timed urine samples obtained from each subject were compared (Fig. 1). MT levels in samples collected over time were markedly lower than the spot samples, especially at the lower end of the concentration range. The samples collected for 8 h (active workers) were no better than those collected by the retired workers over the 24-h time period. Because of these observations, only the spot urine data were used for all subsequent statistical analyses. The MT was significantly related to total urinary protein, as well as/]2-MG, in both active and retired workers (Figs. 2 and 3). On a group basis, the retired workers excreted more total protein, ~2-MG and MT than the active workers. However, there was no apparent difference between the active and retired workers with respect to the relationship of total protein or /]2-MG with urinary MT. Since both urinary protein and/I2-MG are used as indices of renal function in Cd-exposed populations, the relationship between the two was examined and is shown in Fig 4. On the basis of normal or abnormal levels of total protein and ~2-MG, the population was divided into two main groups; those excreting less than or equal to 250 mg protein or 500 ~g ~2-MG/g creatinine (normal renal function) and those excreting more than these amounts (renal dysfunction). All but two individuals in the latter group were retired workers and were older than the other group. Among the renal dysfunction group, four individuals were identified as severe cases based on very high total urinary protein and fJ2-MG excretion. Two of these individuals had elevated blood urea nitrogen and all four had
._= C N 0
•
10 =
•c~ w
a
.,-"
~
v 0 C
T
-
t
1
..........................
Q,
10 ~
10 =
10'
10 +
Timed Urine (POMT/o CreaUnine) Fig. 1. Comparison of MT levels in spot urine and urine collected over 8--24 h. The graph includes both active (r-l) and retired ( I ) workers. The linear regression line (--) is plotted using the following equation: log Y = 0.506 log X + 1.375 (N = 68; r = 0.84; P < 0.001). The dashed line represents the hypothetical regression line if the MT concentration in the two urine specimens from each individual was the same.
55
A
,~,
l°'F
tD
°
" i .
,o. F
e
'=
] 10'
. . . . . . . 10 =
10'
Urine Protein Img/g Creatinine) Fig. 2. The relationship between total protein and MT in spot urine samples. The graph includes both active (r-I) and retired (11) workers. The linear regression line is plotted using the following equation: log Y = 0.835 log X + 0.704 (N = 68; r = 0.76; P < 0.001).
elevated serum creatinine concentration (Table I). These individuals were excluded from all subsequent correlation analyses. Age o f the individuals was related significantly to the duration of their exposures. Age also showed a positive correlation with all parameters analyzed (data not shown). The effect o f age on these parameters, therefore, could not be separated from the effect o f cadmium exposure. Figure 5 depicts the relationship between urinary MT and Cd content in the left kidney. All subjects, including those with mild to moderate renal dysfuncA
Q)
._c .-
10 4
0
o
a
10' '
l r
O "1
I:E
•
o
~°~E
~
•
i
10 =
0 C
.:=) 10'
1 .......................................... 10'
10 =
10 =
1 10'
10 s
Urine p,MG (POLO Creatinine) Fig. 3. The relationship between /J2-MG and MT in spot urine samples. The graph includes both active (I-I) and retired (11) workers. The linear regression line is plotted using the following equation: log Y = 0.295 l o g X + 1.819(N = 6 8 ; r = 0 . 6 6 ; P < 0.001).
56
,II
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j,.~j.40 • r
",-
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"
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t
10 =
Urine
Protein
10 =
(mglg
Creatinine)
Fig. 4. The relationship between total protein and 32-MG levels in spot urine samples of active and retired workers. The group is subdivided according to normal (O) and abnormal ( e , & ) levels of protein and ~2-MG in urine. Normal upper limit for protein was considered to be 250 mg/g creatinine and that for/J2-MG to be 500 ~g/g creatinine. Four individuals (&) had excessively high levels of urinary protein, fl2-MG, MT, blood urea nitrogen and serum creatinine (Table l). The linear regression line is plotted using the following equation: log Y = 1.802 log X + 1.585 ( N = 68; r = 0.74; P
