urinary excretion of copper bound to low molecular

International Journal of Occupational Medicine and Environmental Health, Vol. 7, N o 4, 3 4 5 -3 5 3 , 1994

URINARY EXCRETION OF COPPER BOUND TO LOW MOLECULAR WEIGHT PROTEINS IN THE POPULATION EXPOSED TO CADMIUM IN COMMUNITY AND OCCUPATIONAL ENVIRONMENTS

TERESA W RO ŃSKA-NO FER1. TADEUSZ H AŁATEK1. JU ST Y N A W IŚNIEW KSA-KNYPL1 and G RAŻY NA RAŹNIEW SKA2

‘Department o f Biochemistry 2Bepartment of Biological Monitoring The N ofer Institute o f Occupational Medicine Lodz. Poland

Key words: Cadmium exposure, Communal/occupational exposure, Copper-binding protein, /f2-micro- globulin, Retinol-binding protein, Urine, Human

A bstract People living in Cd-polluted areas excrete increased amounts of copper with urine. A substantial quantity o f this is eliminated with metallothionein the concentration of which in urine increases in people exposed to cadmium. Therefore, the measurement of metallothionein in urine is applied as a marker of renal function in people exposed to cadmium in addition to other low m olecular weight proteins, /J2-microglobulins (j52M G) and retinol binding proteins (RBP). In this study copper bound to metallothionein-like proteins of low molecular weight (CuBP) — a newly proposed marker of cadmium nephrotoxicity, as well as /J2M G and RBP, were evaluated in those exposed to cadmium in the community and in occupational environments.

The results obtained indicated that people exposed to cadmium in both polluted environments excreted greater amount of CuBP in urine than people not exposed to cadmium. In groups excreting cadmium in urine in amounts ranging from 1 to 10 ng/1 the urinary level of CuBP was closely associated with the levels of /?2M G and RBP. A considerable increase in the excretion of urinary CuBP began when Cd concentration in urine exceeded 4 ng/1. The amount of CuBP excreted was higher in people with renal disfunction than in those with a normal renal function.

It is suggested that urinary excretion of CuBP may be considered as a specific marker of renal function in people exposed to cadmium.

IN TR O D U CTIO N

It has been widely documented that workers employed in factories using cadmium (Cd) and people living in Cd-polluted areas show a high prevalence of renal tubular dysfunction characterized by proteinuria (3, 5 — 10, 13, 15 — 16,

Address reprint reguests to Prof. T. Wrońska-Nofer, Department of Biochemistry, The Nofer Insitute of Occupational Medicine, P.O. Box 199, 90-950 Lodz, Poland.

346 T. Wrońska-Nofer et al.

21 — 24, 31 — 32). The increase in urinary excretion of low-molecular weight proteins such as retinol-binding protein (RBP) or /?2microglobulin (/J2MG) is regarded as a sign of tubular renal dysfunction (3, 10, 15). Recently aj-microglobulin (oijMG) has been considered as another indicator of early renal damage (11, 30). There is also evidence that changes in the activity of some enzymes, e.g. urinary N-acetyl- /?-D-glucosaminidase (NAG) is sensitive and reliable measure of nephrotoxicity (3, 10, 20).

An elevated level of ^ M G and RBP as well as the activity o f NAG, is not a specific sign associated with the nephrotoxic effect of cadmium. The level of low-molecular weight proteins increases in the case of therapeutic treatm ent with some aminoglycoside drugs like gentamicin (6, 17), tubulo-interstitial diseases or renal hypertension.

Numerous studies indicate that the level of urinary M T increases in persons environmentally or occupationally exposed to cadmium as well as in experimentally exposed rats (5, 23 — 24, 18, 26 — 29). Furthermore, an elevated excretion of MT is well correlated with a urinary level of /?2M G and RBP (5,24). The excretion of MT is directly involved in the enhanced elimination of copper in the urine of people living in Cd-polluted areas (14, 18). The level of urinary copper correlates significantly with the level of metallothionein and [i2M G or RBP, indicators of renal tubular dysfunction (14, 17, 19).

This study was undertaken in order to evaluate the urinary excretion of copper bound to low-molecular weight proteins (CuBP) in populations exposed to cadmium in communal (low exposure) and occupational (high exposure) environments. The assessment of copper in the metal-protein complex similar to metallothionein would allow a more specifically assessment of the impact of cadmium on renal function. This in turn would justify the adoption of urinary CuPB determination as a biological m arker of cadmium nephrotoxicity.

MATERIALS AND M ETHODS

Studied population

Group I — reference, unexposed population, inhabitans of rural areas (small villages) in the north-eastern part of Poland (n = 144 persons):

G roup II — population exposed to cadmium in a community area, located in ctose vicinity to a nonferrous mill with a cadmium production division (n = 123 persons):

G roup III — population exposed to cadmium in the occupational environment in an alkaline battery plant (n = 57 workers).

Biochemical assays

Low-molecular weight proteins from daily urine specimens were isolated by organic solvent fractionation. The fraction of high-molecular weight proteins from the urine was precipitated with a cold mixture of 1.05 and 0.8 ethanol and chloroform (in a respective ratio to urine) (12). The precipitate was spun down at 10000 g for 10 min. Low-molecular weight proteins from the supernatants were precipitated with 5 volumes of acetone while chilled overnight at — 20°C: the protein precipitate was sedimented at 10000 g for 10 min.

Urinary copper binding proteins in cadmium exposure 347

The determination of cadmium in urine (CdU), after nitric acid digestion was car­ried out by the method of Stoppler and Brandt (25) in two variants, depending on the cadmium concentrations: (a) directly in urine diluted with water (1:1) when concentra­tions were higher than 10 \ig Cd/1 and (b) by MIBK extraction of the cadmium com­plex with diethyldithiocarbazone when cadmium concentrations were below 10 jj.g/1.

Copper in the low-molecular weight proteins fraction was determined by AAS after dissolving the protein in 0.8 M H N 0 3. AAS-ET Pye-Unicam Type SP9 with a P L —9095 graphite furnace was used for cadmium and copper determination.

External quality control for the cadmium determination of cadmium was performed within a Quality Central Programme for Toxic Metals and Organic Compounds in Urine organized by the Institute of Occupational Health in Helsinki (expected values: 0.22 and 6.27 Cd/1: found: <0.9 and 5,71 |ig Cd/l), IUPAC Cooperative Interlaboratory Survey (expected values: 0.3; 6.6; 1.1; 25.5; 6.4 and 1.03 jxg Cd/1, found: < 0.6; 6.2; 1.5; 23.8; 5.6 and 0.93 |ig Cd/1) and Coronel Laboratory for Occupational and Environmental Health, University of Amsterdam (results acceptable, with a score of 100% in runs 1 and 2 and a score of 50% in run 3). Internal quality control for cadmium and copper determination was performed using Lanonorm Metals 1 — 3 from the Behring Institute and the precision of the obtained results was within a one-two standard deviation of the reference values.

The contents of RPB and jS2M G in urine were measured by means of latex immunoassay (LIA) (2) subjected to external quality control within a program supervised by the Coronel Laboratory at the University of Amsterdam.

All the data was evaluated statistically after logarithmical transformation. The relationship between the continuous variables was examined by linear analysis.

RESULTS

All the mean values of the evaluated parameters characterizing the exposure to Cd (the level of Cd in urine) and renal function (urinary /?2MG, RBP) in the unexposed (control) group were at the normal level. The mean concentration of cadmium in the urine of control groups was 1 p.g/1. The subjects exposed to cadmium in the community environment in relation to the nonexposed persons excreted greater amounts of CuBP (50%) /?2M G and RBP (15%) with urine. The most substantial elimination of CuBP as well as j82MG, RBP and Cd was observed in people exposed in the workplace (plant producing alkaline batteries) (Table 1).

Fig. 1 shows the urinary excretion of CuBP and RBP in six groups of people with a growing cadmium concentration in urine (from 1 to 10 jj.g/1). A considerable increase in the CuBP level in urine began when the Cd concentration exceeded4 jxg/1. When the concentration of Cd in urine reached 10 (j.g/1, the amount of excreted CuBP was twice as high as in the unexposed group. Unlike CuBP, a substantial increase in the RBP level in urine was observed only when the concentration of urinary cadmium reached the value of 8 — 10 jj.g/1.

The amount of excreted CuBP was related to the amount of Cd in urine in the whole range of urinary cadmium concentration examined (Fig. 2). It was shown (Fig. 3.) that urinary excretion of CuBP is closely associated to the urinary level of P2M G and RBP indices of renal dysfunction (the correlation between urinary /?2MG, RBP and CuBP was calculated in groups of subjects whose urinary cadmium con- centraion did not exceed 15 jj.g/1).

348 T. W rośska-Nofer et al.

Table 1. Biological markers o f nephrotoxic effects o f cadmium in population exposed to cadmium in community and occupational environments

GroupNumber of

subjectsAge

(years)*CdU

RBP(ng/i)b

P2m g CuBP

Non-exposed 144 59 + 8 1.1 ± 1 .95 79 + 2.80 41 ± 3 .8 5 9.3 ± 3 .32

Cd-exposedin community environment

123 61 ± 9 2.4 + 2.03° 90 ±4 .22 45 ± 4 .2 6 14.2 ± 3.12c

Cd-exposedin occupational environment

57 34 + 7 25.8 + 5.41^ 247 ± 3.42cd 1 1 4 ± 4 .8 5 cd 28.6 ± 2.03cd

CdU — cadmium in urine RBP — retinol binding protein 0 2MG — 02-microglobulinRBP and /?2M G were adopted as a marker of renal function statusCuBP — copper bound to low molecular weight protein: a postulated marker of cadmium nephrotoxic effects 8 — arithmetic mean + SD: b — geometric mean ± S D ;c,a - significantly different at p<0.05 vs non-exposed group and Cd-exposed in community environment groups, respectively.

Fig. 1. Urinary excretion of CuBP and RBP as dependent on urinary cadmium concentration in population analyzed, divided into six subgroups according to CdU level. CuBP (striped bars) and RBP (dotted bars) represent values for a number of subjects given over the bars. Significant difference of CuBP and RBP values vs values for the lowest CdU concentration was marked by x and y, respectively. Details as in Table. 1.

In order to find out whether urinary CuBP reflects the status of renal function, the amount of excreted CuBP was calculated for the two separated subgroups: those with symptoms of renal dysfunction and those with normal renal function (Table 2). Persons with renal dysfunction were selected both from the population exposed to cadmium in the community (low exposure) and in the occupational (high exposure)

Urinary copper binding proteins in cadmium exposure 349

OJ

o_‘ 18mDo

Fig. 2. Relationship between urinary concentration of CuBP and cadmium among the analysed subjects divided into subgroups according to CdU level. Results presented experimental data (asterisks) and calculated according to equation for linear plot (squares). Details in Table 1.

Table 2. Urinary level of CuBP marker as dependent on renal function status in people exposed to cadmium in community and occupational environments

In order to discriminate the subjects with impaired renal function a threshold limit value of excreted proteins was applied: 02M G > 200 ng/1 and RBP > 250 jxg/1.Results are the arithmetic mean with the range in round brackets.The number of subjects are given in square brackets.O ther details as in Table 1.

environments. In order to separate the subgroups vs renal function status the urinary level of RBP and /?2M G was adopted as a discriminating factor. The adopted discriminating “critical value” of RBP and MG, identifying people with symptoms of renal dysfunction amounted > 250 and 200 (j.g/1, respectively. As shown in Table 2 the subjects with renal dysfunction, selected from the population exposed to low and high concentrations of cadmium, excreted considerably more CuBP in urine than people with normal renal function.

Excretion

Group

of CuBP vs renal function CuBP (ng/1)

normal renal function impaired renal function

Cd-exposed 10.7 43.1in community environment (1 -6 3 .2 ) (97) (1 5 -8 6 .5 ) (13)

Cd-exposed 17.7 43.3in occupational environment (2 -4 0 ) (25) (1 1 .3 -1 8 2 ) (18)

350 T. Wrońska-Nofer et al.

Fig. 3. Relationship between urinary concentration of CuBP and /i2MG or RBP among inhabitants of cadmium polluted area. Details as in Table 1 and Fig. 2.

DISCUSSION

Exposure to Cd results in an elevated urinary excretion of both copper and metallothionein (MT). According to Mitane et al. (18) more than 60% of urinary Cu is bound to M T and the enhanced excretion of Cu was due to the increased elimination of this metal-protein complex. When M T undergoes an oxidation process, Cu may be redistributed into the fraction of high and low molecular weight proteins. In this study, the amount of copper excreted with the fraction of low molecular weight proteins (10000 — 30000) was evaluated in the urine of people exposed to cadmium at low level (community environment) and high level

Urinary copper binding proteins in cadmium exposure 351

(occupational environment). It was indicated that people exposed to Cd in comparison to those unexposed excreted a higher concentration of P2M G and RBP (the protein indices of renal tubular dysfunction) as well as a greater amounts of CuBP in urine.

The relationship between the amount of CuBP and CdU excreted by the subjects whose level of urinary cadmium ranged from 1 to 10 jxg/1 should be stressed. W ith a very low concentration of Cd in urine (1—4 jj.g/1) there was no increase in the urinary RBP excretion (see Fig. 1). The results obtained confirmed the Jakubowski’s (8) earlier data, indicating that there was, in people living in Cd-polluted areas, no relationship between urinary cadmium concentration in a range of 0.65 — 7.85 jxg/1 and the excretion of J?2M G and RBP. The findings were also consistent with the data given by Kawada et al. (10), where no correlation was found between urinary cadmium at a level of below 10 jj.g/g creatinine and the /?2M G level.

The gradual increase in CuBP excretion corresponds to an increment in the level of CdU, reaching a statistically significant increase when CdU concentration amounted to 4 (j.g/1 (cf. Fig. 1). The enhancement of RBP level in urine did not occur when the level of urinary cadmium was below 5 |xg/l. A significant increase of RBP in urine was found when the urine cadmium concentration was 8 (ag/1. Comparing the excretion of CuBP and RBP one can conclude that an enhanced CuBP excretion can be detected earlier than RBP, the recognized indicator of renal tubular dysfunction. Thus, the CuBP indicator approximates towards more sensitive tests of renal dysfunction — the excretion of M T and NAG with urine as found by Kawada et al. (10) at the level of Cd-U in the range 0.2 —9.7 jag/1.

The excreted am ount of CuBP was much more pronounced in subjects with renal dysfunction than in those with normal renal function. In both subgroups with renal dysfunction, regardless of the magnitude of exposure to cadmium concen­trations (low in the community and high in the occupational environment) the amount of CuBP excreted with urine was at the same very high level (Table 2). One can conclude that the increased CuBP excretion is due to renal dysfunction. Nevertheless, the magnitude of Cd exposure affected the excretion of CuBP with urine in the group of people considered as having normal renal function (RBP < 250 jj.g/1 and /i2M G < 200 jig/1). In this group, the level of CuBP was elevated in the urine of people exposed to higher concentration of Cd. It seems that the assessment of the effect of Cd on renal function by marker measurement of CuBP may fulfill the requirements for the evaluation of the early nephrotoxic effects of Cd in the general population where the urinary level of cadmium amounted to 10 ng/1. According to Abdulla and Chmielnicka (1) an increased elimination of copper with urine is a good m arker of renal dysfunction and c ccurs when the concentration of Cd in the kidney reaches 50 |xg/g of tissue in humans and 10 — 20 ^g Cd/g in animals. The results of recent investigations on the effect of Cd on renal function in the general population in Belgium led to the conclusion that the risk of kidney impairment can be observed when urinary excretion of Cd exceeded 2 — 3 (j.g Cd/day (4), which was found in 10% of the population. Such an excretion rate corresponds to an accumulation of 50 |ag Cd/g kidney cortex.

ACKNOW LEDGEM ENTS

This study was partly supported by program PB/0647/93 and IM P 4.3.

352 T. Wrońska-Nofer et al.

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Received for publication: September 30, 1994Approved for publication: October 13, 1994