TOXICOLOGY AND APPLIED PHARMACOLOGY 116, 155- 160 ( 1992)

Human and Experimental Studies on Renal Eicosanoid Response to Long-Term Cadmium Exposure

ALVARO CARDENAS,* ISABEL RAMIS,? GINA HOTTER,? JOAN ROSELL~,~ EMILIO GELP~,? HARRY ROELS,* ALFRED BERNARD,* AND ROBERT LAUWERYS*

*Industrial Toxicology and Occupational Medicine Unit, School of Medicine, Catholic University of Louvain. 30.54 Clos Chapelle-au-champs,

B-1200 Brussels, Belgium; and tDepartamento de Neuroquimica, Seccidn Eicosanoides, Centro de Investigacidn y Desarrollo (CSIC). Jorge Girona Salgado 18-26, 08034 Barcelona, Spain

Received February 10, 1992; accepted June 8, 1992

Human and Experimental Studies on Renal Eicosanoid Re- sponse to Long-Term Cadmium Exposure. CARDENAS, A., RAMIS, I., HOTTER, G., ROSELL~, J., GELPI, E., ROELS, H., BERNARD, A., AND LAUWERYS, R. (1992). Toxicol. Appl. Phar- macol. 116, 155-160.

In order to assess the effects of long-term exposure to cadmium (Cd) on the renal metabolism of eicosanoids, the urinary excre- tion of 6-keto-prostaglandin F,, (6-keto-PGF,,), prostaglandin Ez (PGE& prostaglandin FZu (PGF,,), and thromboxane Bz (TXB*) was determined in 37 workers exposed to Cd and in female Sprague-Dawley rats given 100 ppm Cd in drinking water for 10 months. Urinary output of sodium and calcium was also determined. The Cd-exposed workers showed an increased uri- nary concentration of 6-keto-PGF,,, PGEl, sodium, and cal- cium. The rise of 6-keto-PGF,, was related to Cd levels in blood and weakly correlated with urinary sodium. Calcium in urine was not related to the concentration of the metal in blood and urine. A slight elevation in urinary TXBz was also observed in workers with blood Cd higher than 5 fig/liter. After 10 months of exposure to Cd, female Sprague-Dawley rats presented an enhanced urinary excretion of albumin, transferrin, P2-micro- glulin, sodium, and PGEz in urine. The latter was significantly correlated with albuminuria and transferrinuria. In conclusion the results show that chronic exposure to Cd induces changes in the urinary excretion of some eicosanoids. The possible re- lation of these changes to Cd-induced kidney dysfunction are discussed. Q 1992 Academic PRSS, IX.

The main target organs of cadmium (Cd) are lung, bone, and kidney, the latter being considered the critical organ following long-term exposure (Friberg, 1950; Kazantzis et al., 1963; Kjellstrom 1986a,b). The early renal disturbances observed in man or animal chronically exposed to Cd consist of an increased urinary excretion of low- or high-molecular- weight plasma proteins. This proteinuria may be accompa- nied by an increased excretion of enzymes, amino acids, tubular antigens, glucose, calcium, or phosphorus (Lauwerys et al., 1974; Bernard et al., 1979, 1981, 1992; Cardenas et

al., 1991; Friberg, 1950; Kazantzis et al., 1963; Scott et al., 1976; 1978; Greenberg et al., 1986).

In the kidney, prostaglandins may influence several func- tional parameters such as renal hemodynamics and tubular transport of water and electrolytes (e.g., sodium, calcium) and modulate the action of other hormones affecting renal function (Ganick, 199 1). It has therefore been suggested that alteration in the renal synthesis of eicosanoids could be im- plicated in the development of some renal diseases and hy- pertension (Stork et al., 1986; Zoja et al., 1989; Garrick, 199 1). Furthermore in various pathological conditions which may have an adverse repercussion on renal function, the integrity of prostaglandin synthesis seems important for the maintenance of renal blood flow and/or glomerular filtration rate (GFR) (Dunn, 1984; Stork et al., 1986; Patron0 and Dunn, 1987; Olson and Heptinstall, 1988).

Several studies suggest that Cd may interfere with arachi- donic acid metabolism. It has been observed that in rabbits treatment with Cd induces an increase of the serum levels of thromboxane B2 (TXB2) and stimulates the production of 6-keto-prostaglandin F,, (6-keto-PGF,,) by aortic walls in vitro (Caprino et al., 1982). Cd also stimulates prosta- glandin E2 (PGE2) formation and bone reabsorption in os- teoblast-like cells and in fetal mouse calvaria in vitro (Suzuki et al., 1989a,b).

The aim of the present study was to assess, in humans and in rats, whether chronic Cd exposure interferes with the renal synthesis of some eicosanoids by measuring their uri- nary excretion. We also examined whether the changes in eicosanoid excretion were related to natriuria, calciuria, and proteinuria.

MATERIAL AND METHODS

Human study. The population was composed of 50 male workers ex- posed to Cd for at least 1 year in nonferrous smelters (exposed group) and 50 matched subjects from the same plants who had never been occupationally exposed to this metal (control group).

The criteria used for the selection of the workers were the following: (a) no kidney disease or disease likely to impair the renal function (e.g., diabetes,

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156 CARDENAS ET AL

gout); (b) no regular consumption of drugs with potential nephrotoxicity or likely to influence cyclooxygenase metabolism (e.g., analgesics, lithium); (c) concentration of mercury in urine and lead in blood below 5 fig/g creatinine and 350 &liter. respectively; and (d) concentration of Cd in urine below 2 pg/g creatinine in controls and above this level in exposed workers. After application of these selection criteria the number of subjects remaining in the control and exposed groups were 43 and 37, respectively. The workers had been exposed to Cd on average for I 1.3 years (range, 1.1-36.4). In the control group the age ranged from 23.0 to 57.9 years with a mean of 44.9 and in the exposed group it ranged from 23.2 to 59.3 years with a mean of 43.2.

A sample of venous blood (10 ml) on sodium EDTA (10 ~1, 10%) was taken from each worker for heavy metal and zinc-protoporphyrin (ZPP) determinations. A spot-urine was also collected from each participant: 10 ml was immediately transferred to a tube containing 10 nl of lysine acetyl salicylate (1% in water) and kept at -40°C until eicosanoid analysis and 5 ml was stored at -20°C for sodium and calcium determination. Syringes, tubes, and urine containers were previously checked for lack of heavy metal contamination.

Experimental study. Ten female Sprague-Dawley rats ( 100- 150 g, 8 weeks old) were given 100 ppm Cd (CdCIz) in deionized drinking water for up to IO months. The control group was composed of IO rats receiving only deionized drinking water. The animals were housed in an air-conditioned room with a conventional 12-hr light/dark cycle. Food (A.03 pellets UAR, Epinay-sur-Orge, France) containing 0.13 ppm Cd and 110 ppm Zn was given an libitum.

At the end of the treatment the animals were placed in individual me- tabolism cages equipped with urine/feces separators and their urine samples were collected during 24 hr in a tube kept in dry ice and containing 20 ~1 of sodium azide (10%) and 10 ~1 of lysine acetyl salicylate. Urine samples were stored at -40°C until eicosanoid and protein analysis. A separate 24- hr urine collection was performed in a tube without preservative for sodium and calcium determination.

Biological analyses. Cd levels in blood (CdB) and in urine (CdU) and lead concentration in blood were measured by electrothermal atomic ab- sorption spectrometry as described previously (Roels et al., 199 1). Mercury in urine was analysed by an automated “cold vapour” atomic absorption technique (Magos and Clarkson, 1972). The concentration of ZPP in eryth- rocytes was measured using a hematofluorimeter (AVIV Associates, Lake- wood, NJ). Creatinine in urine was determined according to Jaffe’s picrate method using a Technicon RAIOOO automate (Tarrytown, USA). Sodium and calcium levels in urine were analyzed by flame atomic absorption meth- ods (Perkin-Elmer, I97 I).

The urinary concentration of 6-keto-PGF,, (the stable metabolite ofpros- tacyclin), PGEZ. prostaglandin Fza (PGF,,), and TXB, (the stable metabolite ofthromboxane AZ) were determined by radioimmunoassay after extraction with Sep-Pak Cl8 cartridges and purification by high-performance liquid chromatography as described previously (Gelpi et al., 1989). Human urine samples with abnormally high values of PGEz (>900 rig/g creatinine) were suspected of seminal contamination and were discarded (four cases in the control group and two in the exposed group).

The urinary concentrations of albumin, transfenin, &microglobulin (@2- m), and retinal binding protein (RBP) were determined by an automated immunoassay based on latex particle agglutination (Bernard and Lauwerys, 1983). The antibodies against human proteins were obtained from Dakopatts (Glostrup, Denmark), those against rat albumin and transferrin were from USB (Cleveland, Ohio, USA), while those against 02-m were produced in our laboratory as described previously (Viau e/ a/., 1986). The activity of N-acetyl+D-glucosaminidase (NAG) in rat urine was measured fluori- metrically by the method of Tucker et al. (1975).

Statistical analyses. When parameters were not normally distributed statistical analysis was performed on log-transformed data. The differences between groups were assessed by Student’s t test or Duncan’s test. Associations between variables were evaluated by Pearson correlation coefficient.

RESULTS

Human study. The biological parameters reflecting the internal doses of the metals are summarized in Table 1. The concentration of Cd in urine exceeded 10 pg/g creatinine in seven exposed workers and that of Cd in blood was higher than 10 fig/liter in six exposed workers. The concentrations of Cd in blood and urine from cadmium workers were well correlated (r = 0.49, y1 = 37, p < 0.001). No differences were observed between exposed and control groups with re- gard to urinary mercury or blood levels of lead and ZPP (Table 1).

By comparison with the control group the workers exposed to Cd showed a significantly higher mean urinary concen- tration of albumin, transferrin, fl2-m, and NAG activity (Table 2). The urinary levels of 6-keto-PGF,,, PGE2, sodium, and calcium were also increased in the Cd-exposed workers (Table 3). With the exception of urinary calcium all these effects were significantly correlated, although weakly, with the levels of Cd in urine and blood, at least when both control and Cd workers were combined (v between 0.3 and 0.5, p < 0.01). In the exposed group, only the urinary excretion of albumin, transferrin, and NAG activity was significantly correlated with the level of urinary Cd and with duration of exposure (Y between 0.36 and 0.57. p < 0.05) while urinary 6-keto-PGF,, was the sole marker significantly associated with the level of Cd in blood (Y = 0.34, p < 0.05).

In the total population, 6-keto-PGF,, in urine was weakly but significantly associated with urinary sodium, calcium, transferrin, NAG, and 02-m (Y between 0.25 and 0.3 1, p < 0.05). In the exposed group 6-keto-PGF,, was slightly cor- related with NAG, 62-m, and sodium (Y between 0.28 and 0.38, p < 0.1) but not with urinary calcium (Y = 0.03) or transferrin. Nevertheless, all these associations must be in-

TABLE 1 Biological Indicators of Exposure to Cd, Pb, and Hg in

Control and Cd-Exposed Workers

Control workers Cd-exposed workers (n = 43) (If = 37) P”

Cd in blood * t rglliter) 0.8 (0.3-2.8) 5.5 (1.6-14.6) <o.ooo 1

Cd in urineb (pg/g creatinine) 0.7 (0.1-1.9) 5.4 (2.1-16.4) <o.ooo 1

Hg in urineh (@g/g creatinine) I .9 (0.8-4.8) 2.0 (0.7-4.0) NS

Pb in blood’ t rg/liW 126 (48-314) 154 (50-327) NS

ZPP in blood‘ (fig/g Hb) 1.2 (0.4-4.7) I .3 (0.7-2.2) NS

n Student’s r test. b Geometric means and ranges. ’ Arithmetic means and ranges.

RENAL EICOSANOID RESPONSE TO CADMIUM 157

TABLE 2 Urinary Concentration of Some Proteins and NAG Activity in

Control and Cd-Exposed Workers

Control workers Cd-exposed workers (n = 43) (n = 37) P”

Albumin (m&g creatinine) 5.7 (2.4-25.5) 8.0 (2.6-154) <0.05

Transferrin ( rg/g creatinine) 184 (50-1710) 345 (65-8070) <o.o 1

/32-m (j&g creatinine) 47.7 (4.7-250) 89.9 (11.7-7250) <o.o I

RBP ( pg/g creatinine) 57.3 (17.1-378) 66.8 (21.6-2550) NS

NAG (UI/g creatinine) 0.89 (0.18-1.71) 1.19 (0.46-4.48) <O.Ol

200 2

2 100 1 ‘Z 3 .E

G 0 0 .- i? 400 -

Note. Values are geometric means and ranges. ’ Student’s t test.

terpreted with caution because only spot urine samples were collected. It cannot be excluded that the urinary levels of these markers are influenced by a common determinant (for example glomerular flow rate) and their association may not necessarily be causal. Furthermore, the dietary intake of so- dium and calcium was not monitored.

The exposed workers were divided into two subgroups as a function of Cd level in blood or urine and duration of exposure (Fig. 1). The urinary output of 6-keto-PGF1, was higher in the group with the highest internal dose of Cd. The subgroup of workers with blood Cd higher than 5 pg/liter also presented an enhanced excretion of TXB,. Calciuria, natriuria, and urinary PGEz excretion were not statistically different between both subgroups. PGEl showed a tendency to rise with the duration of exposure.

,T A

TABLE 3 Urinary Concentration of Some Eicosanoids, Sodium, and

Calcium in Control and Cd-Exposed Workers

200 2

100 1

0 0

Control workers Cd-exposed workers (n = 43) (n = 37) Pa

&keto- PGE, PGF,,z TXB, Na+ ca”

mFlci

6-keto-PGF,, (rig/g creatinine) 113 (34-231)

PGE2 (rig/g creatinine) 137 (56-314)

PGFz. (rig/g creatinine) 245 (85-1229)

TXB2 (rig/g creatinine) 44.4 (8.2-141)

Na+ (mmol/g creatinine) 67 (19-210)

Ca’+ (mmol/g creatinine) 1.68 (0.10-5.27)

175 (53-320) <o.ooo I

245 (78-609) <o.ooo 1

238 (21-949) NS

50.0 (15.3-111) NS

97 (44-194) <o.oo I

2.67 (0.39-8.02) <O.Ol

FIG. 1. Urinary concentration of some eicosanoids, sodium, and calcium in male workers as a function of Cd in blood (CdB, expressed in *g/liter), Cd in urine (CdU, expressed in pg/g creatinine), or duration of exposure (Y exp, expressed in years). Values are geometric means f SEM. Values with the same letter did not differ significantly (Duncan’s test).

Experimental study. After 10 months of Cd administra- tion ( 100 ppm in drinking water), female rats had glomerular damage as reflected by a markedly increased albuminuria and transferrinuria (Table 4). Cd treatment also induced an elevation of kidney weight, which was significantly correlated with albuminuria and transferrinuria (r = 0.45, p < 0.05). The urinary excretion of ,L?2-m was only slightly enhanced (p = 9.05). Urinary NAG activity was not affected by the Note. Values are geometric means and ranges.

’ Student’s t test. cadmium treatment (Table 4).

r4

r 4 IJ Yexp=O (n=43)

158 CARDENAS ET AL.

Effect of Long-Term Administration of Cadmium to Female Rats (100 ppm in Drinking Water for 10 Months) on Body and Kidney Weight and on Urinary Excretion of Albumin, Trans- ferrin, /32-Microglobulin (/32-m), and NAG Activity

TABLE 4 TABLE 5 Effect of Long-Term Administration of Cadmium to Female

Rats (100 ppm in Drinking Water for 10 Months) on Urinary Excretion of Some Eicosanoids, Sodium, and Calcium

Control rats Cd-treated rats (n = 10) (n = 10) P’

Body weightb (g) 321 f 14 314 + 12 NS Kidney weightb (g) 2.08 + 0.08 2.36 f 0.07 <0.05 Albumin’

(424 hr) 356 (150-I 110) 1580 (430-5840) <o.oo 1 Transferrin’

(~gt24 hr) 9.98 (3.08-50.4) 57.1 (17.3-210) <o.oo I P2-m’ (&g/24 hr) 13.0 (7.44-29.0) 22.4 (8.58-58.1) 0.05 NAG’ (mUI/ hr) 94.5 (59.5-145) 102 (64.1-134) NS

Control rats Cd-treated rats (n = 10) (n = 10) Pa

6-keto-PGF,, (%I24 hr)

PGEz (ng/24 hr) PGF,, (ng/24 hr) TXBz (ng/24 hr) Na+ (pmo1/24 hr) Car+ (rmo1/24 hr)

11.4 (6.78-17.7) 14.0 (7.82-18.4) NS 6.84 (3.68-16.7) 12.60 (6.45-20.3) <o.o 1

32.0 (14.8-72.6) 33.0 (18.1-59.7) NS 1.64 (0.55-2.68) 1.90 (0.72-7.53) NS 627 (451-1033) 796 (577-l 158) co.05 5.13 (2.02-26.6) 6.14 (2.74-21.7) NS

Note. Values are geometric means and ranges. ’ Student’s t test.

’ Student’s t test. b Arithmetic means f SEM. ’ Geometric means and ranges.

In rats, the only significant effect of the prolonged admin- istration of Cd on eicosanoid excretion was an increased PGE2 (Table 5). The urinary output of 6-keto-PGFi,, TXB2, and PGF,, was not significantly different from that in control rats, although output of 6-keto-PGF,, and TXB2 was slightly enhanced in the Cd-treated group. The exposed group also showed a higher natriuria than the control but the urinary excretion of calcium was not significantly different between both groups (Table 5). During both urine collections, the mean urine flow of the Cd-treated rats was not significantly different from that of the control group (means * SEM: 17.9 f 2.2 vs 15.4 + 1.9 ml/24 hr during the first urine collec- tion and 16.4 + 2.6 vs 13.6 f 1.5 ml/24 hr during the second one).

study suggests that Cd can interfere with arachidonic me- tabolism at the glomerular level (increased excretion of 6- keto-PGF,, in man and possibly PGE2 in rat) as well as in

1 cJooo1

.

0

log y = 1.66 + 1.26 log x

r=0.64 p -C 0.005

The urinary excretion of PGE2, 6-keto-PGF,, , NAG, Na+, and Ca2+ was significantly (p < 0.0 1) correlated with diuresis (r = 0.60, 0.60, 0.64, 0.78, and 0.55, respectively). There was no significant association between albuminuria, trans- ferrinuria, or PZmicroglobinuria and diuresis. Of special in- terest, however was the finding that urinary PGE2 correlated well with the albuminuria and the transferrinuria but not with the P2-microglobulin excretion (Fig. 2).

I .

DISCUSSION

loo-

10.

This study shows that both in humans and in rats, chronic Cd exposure is associated with an increased urinary excretion of some eicosanoids, which probably results from changes in the renal metabolism of arachidonic acid. Indeed, it is generally accepted that the concentration of eicosanoids in urine is a good indicator of the amount synthesized in the kidney (Catella et al., 1986; Patron0 and Dunn, 1987).

14 2.5

r=O.69 p < 0.001

I I 7 5 10 20

Unnary excretion of FGE (ng/Z4h)

In view of the distribution of prostaglandin synthesis in the kidney (Bonvalet et al., 1987; Garrick, 199 l), the present

FIG. 2. Correlations between albuminuria or transfertinuria and urinary excretion of PGE2 in female Sprague-Dawley rats. Dark diamond, Cd-treated rats (100 ppm in drinking water for 10 months) (n = 10); open diamond, control rats (n = 10).

RENAL EICOSANOID RESPONSE TO CADMIUM 159

the medulla (increased excretion of PGE2 in man and rat). The vasculature might also be involved as suggested by ex- perimental data (Caprino et al., 1982) and by the increased excretion of 6-keto-PGF1, in workers exposed to Cd. The slight differences between the results observed in man and in rat probably result from differences in the renal metab- olism of arachidonic acid between both species (Bonvalet et al., 1987; Garrick, 1991).

The mechanism of this action of Cd is not known; it could result from a direct effect of Cd on eicosanoid metabolism and/or from a compensatory reaction to the toxic effects of the metal on the kidney. The studies of Suzuki and co-work- ers ( 1989a,b) and Caprino et al. ( 1982) argue in favor of the first hypothesis. The Japanese group showed that Cd stim- ulates in vitro PGE;! production in osteoblast-like cells (Su- zuki et al., 1989b) and in fetal mouse calvaria (Suzuki et al., 1989a). Caprino et al. (1982) also observed an increase in the synthesis of TXB2 in platelets and of PG12 in aortic walls in vitro after acute administration of Cd to rabbits. However, arguments can also be offered in favor of the second hy- pothesis. An increased synthesis of the vasodilatatory pros- taglandins PGE2 and PG12 has been observed in different models of glomerular injury, including diabetes and renal ablation (Shambelan et al., 1985; Rahman et al., 1987; Stahl et al., 1987; Klarh and Purkeson, 1989). It has been postu- lated that the increase of vasodilatatory prostaglandins may represent a compensatory reaction to the glomerular injury, which plays a role in the maintenance of renal blood flow and GFR (Stork et al., 1986; Patron0 and Dunn, 1987; Olson and Heptinstall, 1988; Klarh and Purkeson, 1989).

Rats chronically exposed to Cd via drinking water develop a selective high-molecular-weight proteinuria of glomerular origin which precedes by several months the rise of &-mi- croglobulin excretion (Bernard et al., 198 1, 1992; C&-denas et al., 1991). In this model of Cd nephropathy urinary &- microglobulin is thus a less sensitive indicator of renal injury than albumin or transferrin. Further evidence that prolonged administration of Cd via the oral route induces a glomerular injury in rats is provided by studies showing a glomerular swelling in these animals (Kawamura et al., 1978; Aughey et al., 1984). The increase in kidney weight (Table 4) and urinary PGE2 excretion (Table 5) and the finding of an as- sociation between urinary PGE2 and albuminuria or trans- ferrinuria in rats (but not &-microglobulinuria) (Fig. 2) fit well with the hypothesis of a compensatory reaction to the nephrotoxic effects of Cd. Although we have previously shown that Cd-induced albuminuria is associated with a bio- chemical alteration (loss of sialic acid residues) of the glo- merular capillary wall (Bernard et al., 1992; CBrdenas et al., 1991) and that these changes are not associated with a sig- nificant reduction ofthe GFR (Bernard et al., 1992), it cannot be excluded that the increased glomerular synthesis of PGE2 can be partly responsible for the increased high-molecular- weight proteinuria. By causing mesangial relaxation and va-

sodilatation of glomerular capillaries, PGE2 increases the single glomerular filtration rate (Olson and Heptinstall, 1988) and hence the filtration of plasma proteins. This mechanism might thus explain the association between urinary PGE2 and high-molecular-weight proteinuria. It might also provide an attractive explanation to the potentiation of the glomer- ular proteinuria caused by diabetes and the concomitant ex- posure to Cd in rat (Bernard et al., 199 I), since both Cd and diabetes may increase prostaglandin synthesis in the glo- meruli (Shambelan et al., 1985).

Natriuria was found to be increased in both humans and rats exposed to cadmium (Tables 3 and 5) which is in agree- ment with our previous observations in Cd-exposed workers (Roels et al., 1990). This effect might be related to the in- creased production of PGE2 which has natriuretic properties probably through its action on the distal tubule (Raymond and Lifschitz, 1986; Bonvalet et al., 1987).

As already reported by other authors (Kazantzis et al., 1963; Scott et al., 1976, 1978) an increased urinary excretion of calcium was found in Cd-exposed workers, which is likely to represent the main factor responsible for the higher prev- alence of renal stones frequently reported in these workers (Friberg, 1950; Scott et al., 1976). Increased calciuria was not observed in Cd-treated rats (Table 5). However, with the same experimental model Kawamura et al. (1978) showed an increased fractional excretion of calcium despite a de- crease in the urinary excretion of calcium. More recently, it has been postulated that Cd stimulates bone resorption and that this stimulation is mediated by PGE2 (Suzuki et al., 1989a). However, the results reported here do not permit the suggestion of a link between changes in eicosanoid and calcium metabolism.

In conclusion this study provides evidence that chronic exposure to Cd causes changes in the renal metabolism of eicosanoids in both human and rat. Further studies are needed to assess the mechanism of these renal metabolic changes and their relation to Cd-induced kidney dysfunction.

ACKNOWLEDGMENTS

A.C. was supported by a sectorial grant from the Commission of the European Communities. This study was financially supported by the Com- mission of the European Communities and the Belgian State (Prime Min- ister’s Service-Science Policy Office). A. Bernard is Maitre de recherches du Fonds National Belge de la Recherche Scientifique.

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