Skeletal changes in multiparous, nulliparous and ovariectomized mice fed either a nutrient-sufficient or -deficient diet containing cadmium.

As a simulation of the etiological factors known for Itai-Itai disease, a syndrome characterized by osteomalacia and renal dysfunction in its Japanese victims, female mice were subjected to the individual and combined stresses of dietary cadmium, nutrient-deficient diet, multiparity and ovariectomy; the calcium-depleting effect of each factor was evaluated by determining Ca levels in femur and lumbar vertebrae. At age 68 days, female mice were given nutrient-sufficient (+) or -deficient (-), purified diets containing either 0.25 (environmental), 5, or 50 ppm Cd as CdCl2; the nutritional composition of (-) diet simulated that of food consumed by Japanese victims of Itai-Itai disease. At age 70 days, half of the females began a breeding regimen of six consecutive, 42-day rounds of pregnancy/lactation (PL mice); the remainder were maintained as virgin, non-pregnant controls (NP mice). Limited numbers of PL and NP mice were sacrificed at the end of each reproductive round. PL(+) mice taken at the end of round (R)-6 had successively borne litters in all six rounds, while PL(-) counterparts had nonsuccessively borne only three. At the conclusion of the 252-day reproductive period, remaining females entered the 392-day, post-reproductive phase of the experiment. At age 546 days (mid-R-12), PL females having successfully borne at least three litters were ovariectomized (OV) to mimic human menopause; at the same time, NP females were either ovariectomized or sham-operated (SO). After surgery, all females were maintained to age 714 days (mid-R-16), then sacrificed. During the post-reproductive period, food consumption by females of the same reproductive status was unaffected by elevated levels of Cd or nutrient-deficiencies in diet. However by R-16, Cd at 50 vs. 0.25 ppm had reduced body mass by 11% in both NP and PLOV females, femur and lumbar vertebral calcium content (TCa) by 20 and 25% in the respective groups, and femur and vertebral calcium/dry weight ratios (Ca/DW) by 12 and 11%. Alternative R-16 comparisons indicated that (-) diet also diminished skeletal Ca, but that the additional factors of (prior) multiparity and ovariectomy generated only small and non-significant effects. Comparison of skeletal status between the ends of the reproductive and post-reproductive periods indicated that (1) individual NP groups, regardless of Cd exposure, generally sustained small decreases in TCa and CaDW over time (consistent with aging), but PL groups without exception secured significant gains (consistent with cessation of multiparous activity), (2) skeletal integrity of PL groups was significantly more compromised by the combination of Itai etiological factors at the end of R-6 than R-16, and (3) among those factors, the most demineralizing over lifetime were chronic exposure to Cd followed by ingestion of (-) diet. Despite these findings, skeletal degeneration characteristic of the Itai-Itai syndrome was ultimately not duplicated in this mouse model suggesting that the full-blown disease required primary and profound skeletal demineralization secondarily supported and enhanced by renal dysfunction.

Kidney changes in multiparous, nulliparous and ovariectomized mice fed either a nutrient-sufficient or -deficient diet containing cadmium.

As a simulation of the etiological factors known for Itai-Itai disease, a syndrome characterized by renal dysfunction and osteomalacia in its Japanese victims, female mice were subjected to the individual and combined stresses of dietary Cd, nutrient-deficient diet, multiparity and ovariectomy. Renal function as affected by the etiological factors was periodically evaluated by determination of protein, amino acid, glucose and Cd concentrations in urine; periodic changes in skeletal Ca status were assessed relative to current renal function. Renal metabolism of Cd, Zn and Cu was also examined. At age 68 days, female mice were given nutrient-sufficient (+) or -deficient (-), purified diets containing either 0.25 (environmental), 5, or 50 ppm Cd as CdCl(2); the nutritional composition of (-) diet simulated that of food consumed by Japanese victims of Itai-Itai disease. At age 70 days, half of the females began a breeding regimen of six consecutive, 42-day rounds of pregnancy/lactation (PL mice); the remainder were maintained as virgin, non-pregnant controls (NP mice). Limited numbers of PL and NP mice were sacrificed at the end of each reproductive round. PL( + ) mice taken in a given round had successively borne litters in that round and all preceding ones. PL(-) females taken at the end of round (R)-1, -2 and -3 had successively borne litters through those rounds; those taken at the end of R-5 or R-6 had nonsuccessively borne litters in four of five or three of six rounds, respectively. At the conclusion of the 252-day reproductive period, remaining females entered the 392-day, post-reproductive phase of the experiment. At age 546 days (mid-R-12), PL females having successfully borne at least three litters were ovariectomized (OV) to mimic human menopause; at the same time, NP females were either ovariectomized or sham-operated (SO). After surgery, all females were maintained to age 714 days (mid-R-16), then sacrificed. Spot urine samples were taken from individual mice at the end of most reproductive rounds (R-2-->6), prior to surgery (mid-R-10), and prior to final sacrifice (late-R-15); samples were also collected via metabolism cages at the end of R-10. Food consumption, monitored on a weekly basis over the first nine rounds, was generally not significantly affected by dietary Cd level or nutrient deficiencies in females of the same reproductive status; consumption was increased about 2.5-fold in PL versus NP groups during the reproductive period and about 1.4-fold during the post-reproductive period. At each of the three dietary Cd levels and after all reproductive rounds, mean renal Cd concentrations were 1.2- to 5.6-fold higher in PL than NP mice. After six reproductive rounds, renal Cd concentrations in PL(+) and (-) groups exposed to 50 ppm Cd had reached 155 and 179 microg Cd/g kidney, respectively. Although these levels fell within a concentration range (145-200 microg Cd/g) where cadmium-induced renal dysfunction could be anticipated, no significant, Cd-dependent changes in mean urinary amino acid or protein concentrations were found. Moreover, among the same population, a 12% incidence of elevated urinary Cd (> or = 250 ng/ml) was noted, however none of the affected individuals exhibited depressed total calcium content (TCa) or calcium:dry weight ratios (Ca:DW) for femur. Such results suggested that the Cd-induced, skeletal demineralization observed in mice during the reproductive period (Bhattacharyya et al., Toxicology 1988a; 50: 193-204; Whelton et al., Toxicology 1994: 91: 235-251) likely occurred in the general absence of cadmium-induced renal dysfunction. By the end of the post-reproductive period, the incidence of elevated urinary Cd increased to 26% among ovariectomized females: of these, 89% with urinary Cd > or = 345 ng/ml exhibited decreases in TCa and/or Ca:DW values for femur or lumbar vertebrae that exceeded one S.D. of their group mean. Such results suggested that skeletal demineralization observed at

Hepatic levels of cadmium, zinc and copper in multiparous, nulliparous and ovariectomized mice fed either a nutrient-sufficient or -deficient diet containing cadmium.

As a simulation of etiological factors known for Itai-Itai disease, female mice were subjected to the individual and combined stresses of dietary cadmium, nutrient-deficient diet, multiparity and ovariectomy. From age 68 days, female mice were maintained on either nutrient-sufficient (+) or -deficient (-), purified diets containing either 0.25 (environmental), 5, or 50 ppm Cd as CdCl(2); the nutritional composition of (-) diet simulated that of food consumed by Japanese women who contracted Itai-Itai disease. At age 70 days, half of the mice began a breeding regimen of six consecutive, 42-day rounds of pregnancy/lactation (PL mice); the remainder were maintained as virgin, non-pregnant controls (NP mice). Limited numbers of PL and NP mice were sacrificed at the end of each reproductive round. PL(+) mice taken in a given round had successively borne litters in that round and all preceding ones. PL(-) females taken at the end of round (R)-1, -2 and -3 had successively borne litters through those rounds; those taken at the end of R-5 or -6 had nonsuccessively borne litters in four of five or three of six rounds, respectively. At the end of the 252-day reproductive period, remaining females entered the 392-day, post-reproductive phase of the experiment. At age 546 days (mid-R-12), PL females having successfully borne at least three litters were ovariectomized (OV) to mimic human menopause, while NP females were either ovariectomized or sham-operated (SO). After surgery, all females were maintained to age 714 days (mid-R-16), then sacrificed. Food consumption, monitored on a weekly basis over the first nine rounds, was in general not significantly affected by dietary Cd level or nutrient deficiencies for females of the same reproductive status; consumption was increased about 2.5-fold in PL versus NP groups during the reproductive period and about 1.4-fold during the post-reproductive period. Over the reproductive period, small increases in liver concentrations of Zn and Cu were observed (ca. 3.1- and 2.5-fold, respectively) with far larger increases for Cd (ca. 22200-fold). Threshold hepatic Cd concentrations below which the concentrations of Zn and Cu were relatively constant and independent of Cd concentration were identified; they were 2.7 microg Cd/g liver for Zn and 3.3 microg Cd/g liver for Cu for females consuming (+) diet, and 4.9 microg Cd/g liver for Zn and 4.5 microg Cd/g liver for Cu for females consuming (-) diet. Regardless of Cd exposure level, round-by-round hepatic concentrations of Cd were generally 2- to 6-fold higher in PL than NP mice, while Zn or Cu levels were generally only 1.1- to 2.5-fold higher. For each reproductive round, hepatic concentrations of Cd in NP females were consistently about 10-fold greater in mice exposed to 50 than 5 ppm dietary Cd: corresponding Zn levels were essentially equivalent. For PL females. Cd levels were about 7-fold greater in 50 than 5 ppm Cd-exposed groups, however Zn concentrations were about 45% decreased. The pattern of Cd, Zn and Cu sequestration established during the reproductive period clearly differed from that of the post-reproductive period. Between R-6 and -16, hepatic concentrations of Cd, Zn and Cu appreciably decreased (14-69%) in 5 ppm Cd-exposed NPOV and PLOV females regardless of diet-type consumed. At the 50 ppm Cd level, Cd and Zn concencentrations dramatically rose with increases in Cd (37-129%) exceeding those of Zn (12-21%).

Skeletal changes in multiparous and nulliparous mice fed a nutrient-deficient diet containing cadmium.

Female mice were given nutrient-deficient, purified diets containing either 0.25 (environmental), 5, or 50 ppm Cd; the nutrient quality of each was patterned after deficiencies known to be present in food consumed by Japanese women who contracted Itai-Itai disease. One-half of the mice were bred for six consecutive, 42-day rounds of pregnancy/lactation (PL mice); remaining females were non-pregnant, virgin controls (NP mice). PL and NP mice were sacrificed at the end of rounds 1, 2, 3, 5, or 6. PL mice taken during the first three rounds were successively pregnant; those taken in later rounds experienced gestation/lactation either four (round 5) or three (round 6) non-successive times. No consistent round-by-round decreases in diet consumption or body weight occurred among NP mice during the 252 days of cadmium exposure, however a significant decrease in femur calcium content (11-17%) was observed in virgin groups exposed to 50 vs. 0.25 ppm Cd. Similar femur decalcification (14-20%) was observed in PL mice, however calcium loss at 50 ppm Cd paralleled decreases in food consumption (24%) and body weight (9-17%). Significant but smaller decreases in the calcium/dry weight (Ca/DW) ratio were found for NP and PL groups consuming 50 ppm dietary Cd. Over the 6-round experiment, exposure to cadmium was found to effect smaller decreases in both femur Ca content and Ca/DW ratio than either consumption of nutrient-deficient diet or multiparous experience. Demineralization results for PL mice provide evidence that the combination of chronic ingestion of cadmium in a nutrient-deficient diet and multiparous activity likely played a role in the etiology of Itai-Itai disease; results for NP mice additionally suggest that decalcification may have been initiated in human females at a time prior to the multiparous and menopausal stages of life.

Cadmium-109 metabolism in mice. IV. Diet versus maternal stores as a source of cadmium transfer to mouse fetuses and pups during gestation and lactation.

The transfer of 109Cd from dam to offspring during gestation and lactation was studied in uniparous mice. From 70 to 210 d of age and during the subsequent reproductive period, young adult female mice received drinking water containing tracer amounts of 109Cd (8 ppb total Cd) and nutrient-sufficient or -deficient solid diet containing stable Cd (5 ppm Cd). The nutrient quality of the deficient diet was patterned after that consumed by Japanese women who contracted itai-itai disease. To evaluate established maternal stores as a potential source of cadmium transfer to pups, some dams were switched to water with no 109Cd and diet with an environmental or control level of cadmium (0.25 ppm Cd) during the reproductive period. The resulting pups were analyzed for 109Cd at birth and at 7-d intervals throughout the lactation period. Pup 109Cd content at birth, representative of the amount transferred via the placenta during gestation, accounted for less than 1% of the total 109Cd transferred during the full reproductive period. During lactation, 109Cd levels in pups from dams with current 109Cd exposure approximately tripled with each 7-d interval; no significant differences occurred due to nutrient quality of the dams' diet. For 21-d-old pups, 98% of the 109Cd burden came from the diet of the dam, while only 2% came from her tissue stores, primarily the hepatic one. Such fractions represented a transfer per pup of about 0.01% of the oral 109Cd dose ingested by the dam during the reproductive period and about 0.05% of the 109Cd in her tissue stores. Overall, transfer per litter amounted to about 7% of the dietary 109Cd dose absorbed and retained by the dam during that interval and about 0.2% of the 109Cd from tissue stores. On lactation d 21, 90% of the total 109Cd in pups was sequestered in the gastrointestinal tract. Cadmium transfer was additionally examined in multiparous mice that began a repetitive breeding program at 70 d of age at the time of introduction to the same diet/water regimens already described. Overall, females consuming nutrient-sufficient diet experienced 5 consecutive 42-d rounds of gestation/lactation, while their deficient diet counterparts experienced 3 nonconsecutive rounds during an equivalent period. Transfer was examined during their last gestation/lactation experience. Throughout the lactation interval, 109Cd transfer to pups was about 30% increased for multiparous versus uniparous females; however, transfer again was not significantly affected by nutrient quality of the dams' diet.(ABSTRACT TRUNCATED AT 400 WORDS)

Cadmium-109 metabolism in mice. III. Organ retention in mice ovariectomized after experiencing multiple rounds of gestation and lactation.

Organ retention of 109Cd was studied in multiparous female mice 6 wk after ovariectomy or sham-control surgery. Females previously had experienced from three to five rounds of gestation/lactation during a maximum of 5 successive, 42-d reproductive periods. Throughout the experiment, mice were provided with tracer amounts of 109Cd in drinking water as well as stable Cd appropriate for the itai-itai experience in solid diets otherwise sufficient or deficient in nutrient quality. For sufficient-diet females, organ 109Cd content and concentration values were somewhat lower in the ovariectomized group compared to the sham control group. For deficient-diet females, the opposite trend occurred. When ovariectomized groups were compared with round 5 breeder groups of the same dietary experience, in almost all instances organ 109Cd content and concentration values were lower in the ovariectomized animals. Exceptions to this trend occurred in hepatic and especially renal tissues, where 109Cd concentrations were higher in the deficient-diet, ovariectomized group. Finally, when the deficient-diet, ovariectomized group was compared with its sufficient-diet counterpart, significantly higher 109Cd content values were found for liver, kidneys, and whole body (minus GI tract) in the former group. Conversely, when the same tissue content values were compared between the sham-control groups, differences without exception proved to be nonsignificant.

Cadmium-109 metabolism in mice. I. Organ retention in mice fed a nutritionally sufficient diet during successive rounds of gestation and lactation.

Organ retention of 109Cd was studied in multiparous and virgin female mice provided tracer amounts of 109Cd in drinking water and stable Cd appropriate for the itai-itai experience in an otherwise nutrient replete solid diet. Breeder females maximally experienced 6 consecutive, 42-d rounds of gestation/lactation. On a round-by-round basis, breeder organ 109Cd content and concentration values were compared with those from their time-matched virgin controls. By the end of round 5, the 109Cd contents of some organs appeared to have plateaued in consecutive breeders. Comparing breeder with control values at that point, the following increases were observed: whole body (minus gastrointestinal tract), 4.7-fold; mammary tissue, 14.1-fold; liver, 5.9-fold; and kidney, 3.8-fold. For 109Cd concentrations, analogous increases were mammary tissue, 15.3-fold; liver, 4.0-fold; and kidney, 2.4-fold. Through the six rounds, a temporal shift in fractional 109Cd distribution was noted for breeder tissues where transfer occurred from those of the mammaries, remaining carcass, and liver to the kidneys. In spite of this shift, at the end of round 6 109Cd content in hepatic tissue still exceeded that in renal tissue; however, 109Cd concentration was 3.3-fold greater in the kidneys. For virgin female mice over the same period, a relatively smaller shift was observed from remaining carcass to kidneys. Unlike breeders, 109Cd content was identical in hepatic and renal tissues, while 109Cd concentration was 4.6-fold greater in the kidneys. With respect to renal 109Cd increases, the larger portion of these shifts had occurred by the end of round 2 for virgin mice and by the end of round 6 for breeder mice. Comparison of content and concentration measures for a single, time-matched, virgin male group with those from a virgin female group at the end of round 6 revealed distinguishable differences only for the mammary tissues; by either measure these were about threefold higher in the female one.

Cadmium-109 metabolism in mice. II. Organ retention in mice fed a nutritionally deficient diet during successive rounds of gestation and lactation.

Organ retention of 109Cd was studied in multiparous and virgin female mice provided trace amounts of 109Cd in drinking water and stable Cd as well as certain nutrient minerals, vitamins, and fat all apportioned in solid diet in amounts appropriate for the itai-itai experience. Breeder females maximally experienced 4 nonconsecutive rounds of gestation/lactation in a total of 5 such 42-d periods. On a round-by-round basis, breeder organ 109Cd content and concentration values were compared with those from their time-matched virgin controls. By the end of round 5, most organ 109Cd content values in breeders were still increasing. Relative to control values at that point, the following increases were observed: whole body (minus gastrointestinal tract), 4.7-fold; mammary tissue, 12.5-fold; liver, 4.7-fold; and kidney, 4.8-fold. Analogous increases in 109Cd concentration values were mammary tissue, 9.8-fold; liver, 2.8-fold; and kidney, 2.9-fold. Through the five rounds, a temporal shift in fractional Cd distribution was noted for breeder tissues where transfer occurred from those of the mammaries and remaining carcass to kidneys. Although by the end of the period 109Cd content in the liver still exceeded that in the kidneys, 109Cd concentration was 4.7-fold greater in renal tissues--an increase not matched by other breeder females consuming nutrient-replete rather than nutrient-deficient (itai-itai) diet. For virgin female mice over the same period, a shift of similar magnitude was observed from remaining carcass to kidneys. Not unlike the breeders, hepatic 109Cd content again exceeded that in renal tissues, while 109Cd concentration was 4.5-fold greater in the kidneys. With respect to renal 109Cd increases, the greater portion of these shifts had occurred by the end of round 3 for both breeder and virgin mice. Comparison of both content and concentration measures for a single, time-matched, virgin male group with those from a virgin female one at the end of round 3 showed only those for mammary tissues to be distinguishable, and by either measure were about fourfold higher in the female group.

Cadmium accelerates bone loss in ovariectomized mice and fetal rat limb bones in culture.

Loss of bone mineral after ovariectomy was studied in mice exposed to dietary cadmium at 0.25, 5, or 50 ppm. Results show that dietary cadmium at 50 ppm increased bone mineral loss to a significantly greater extent in ovariectomized mice than in sham-operated controls. These results were obtained from two studies, one in which skeletal calcium content was determined 6 months after ovariectomy and a second in which 45Ca release from 45Ca-prelabeled bones was measured immediately after the start of dietary cadmium exposure. Furthermore, experiments with 45Ca-prelabeled fetal rat limb bones in culture demonstrated that Cd at 10 nM in the medium, a concentration estimated to be in the plasma of mice exposed to 50 ppm dietary Cd, strikingly increased bone resorption, from 27 +/- 2% (mean +/- SEM) 45Ca release in cultures with no added cadmium to 68 +/- 6% release in cultures containing cadmium (n = 4). These in vitro results indicate that cadmium may enhance bone mineral loss by a direct action on bone. Results of the in vivo studies are consistent with a significant role of cadmium in the etiology of Itai-Itai disease among postmenopausal women in Japan and may in part explain the increased risk of postmenopausal osteoporosis among women who smoke.

Kidney changes in multiparous mice fed a nutrient-sufficient diet containing cadmium.

Female mice were given nutrient-sufficient, purified diets containing cadmium at either 0.25, 5, or 50 ppm, as described in the accompanying publication. One-half of the females were bred for 6 consecutive 42-day rounds of pregnancy/lactation (PL mice); remaining females were non-pregnant controls (NP mice). PL mice and NP controls were sacrificed after 1, 2, 4, or 6 consecutive rounds of pregnancy/lactation. At all levels of dietary cadmium and after all reproductive rounds, kidney cadmium concentrations were 2-5-fold higher in PL than NP mice. After 6 rounds of reproduction, the mean concentration of cadmium in the kidneys of PL mice exposed to dietary cadmium at 50 ppm was 115 micrograms Cd/g kidney, close to the critical concentration for cadmium-induced renal damage (200 micrograms/g). No consistent increases in the concentrations of amino acids, protein, or cadmium in urine were observed in the NP or PL mice in our study, indicating that cadmium-induced renal dysfunction had not yet appeared. Very small increases in kidney concentrations of zinc and copper were observed with large increases in kidney cadmium concentrations. Threshold cadmium concentrations below which the concentrations of zinc and copper were relatively constant and independent of cadmium concentration were identified; they were 7.2 micrograms Cd/g kidney for zinc and 13 micrograms Cd/g kidney for copper. In this study, cadmium-induced decreases in bone-mineral content occurred in the PL mice exposed to cadmium at 5 and 50 ppm (see accompanying publication). Data presented here indicate that the latter bone changes occurred in the absence of cadmium-induced renal dysfunction of the type that results in increased aminoaciduria/proteinuria. They suggest that the bone disease of Itai-Ital patients may also have started prior to the onset of this type of renal dysfunction.

Skeletal changes in multiparous mice fed a nutrient-sufficient diet containing cadmium.

Female mice were given nutrient-sufficient, purified diets containing either 0.25, 5, or 50 ppm Cd. One-half of the females were bred for 6 consecutive 42-day rounds of pregnancy/lactation (PL mice); remaining females were non-pregnant controls (NP mice). PL mice and NP controls were sacrificed after 1, 2, 4, or 6 consecutive rounds of pregnancy/lactation. No consistent, cadmium-dependent decreases in body weight, femur calcium content, or calcium/dry weight (Ca/DW) ratio occurred among the NP mice during the 252 days of cadmium exposure. In contrast, significant, cadmium-dependent decreases in body weight (3-11%), femur calcium content (15-27%), and Ca/DW ratio (5-7%) occurred in the multiparous mice exposed to 50 vs 0.25 ppm Cd. In addition, among the PL mice, the effect of cadmium was dose-dependent, with femur calcium contents decreasing significantly as the cadmium exposure level increased from 0.25 to 5 then 50 ppm Cd (P less than 0.05). Results demonstrate that dietary cadmium exposure had a greater effect on the skeletons of dams exposed to cadmium during the stresses of pregnancy and lactation than in non-pregnant controls. The results provide evidence that the combination of cadmium exposure and multiparity may have played a role in the etiology of Itai-Itai disease in Japan.

Female reproduction and pup survival and growth for mice fed a cadmium-containing purified diet through six consecutive rounds of gestation and lactation.

Female CF1 mice were bred for 6 consecutive, 42-d rounds of gestation-lactation. Their purified diets contained cadmium added at either 0.25, 5.0, or 50.0 ppm Cd; at each cadmium level, the diets were either sufficient or deficient in certain vitamins, minerals, and fat. The deficient diet at 5 ppm cadmium was designed to simulate conditions implicated in the etiology of itai-itai disease among multiparous women in Japan. Fertility, litter size, pup survival, and pup growth (weaning weight) are reported for mice on the six diets during each of the six rounds of gestation/lactation. Except for fertility, decreases in reproductive measures that occurred in response to dietary deficiencies or cadmium during round 1 of reproduction were repeated, unchanged in magnitude, in each successive round. For sufficient diet groups, 50 ppm cadmium had no effect on fertility or pup survival during lactation, but caused a 15% decrease in litter size at birth and a 25% decrease in pup growth. Dietary deficiencies alone decreased all four measures of reproductive performance: fertility by 12%, litter size by 30%, pup survival by 18%, and pup growth by 42%. In addition, dietary deficiencies strikingly decreased the incidence of consecutive pregnancies. Combined effects of 50 ppm cadmium and dietary deficiencies were additive for all reproductive measures except fertility; for fertility, cadmium caused no decrease in the fertility of sufficient-diet animals, but caused a striking 45% decrease in deficient-diet animals. Relating our results to humans, women who contracted itai-itai disease (analogous to mice on the deficient, 5 ppm cadmium diet), in addition to their characteristic bone disease, could have experienced decreases in fertility and in growth of their offspring related to their dietary deficiencies. In addition, their diet-related decreases in fertility could have been enhanced by their combined exposure to cadmium.