David P. Peterson

Calcium-41 as a long-term biological tracer for bone resorption

Abstract The use of 41 Ca (half-life 1 × 10 5 yr) as a tracer for studying calcium metabolism in living systems is compared to the shorter-lived radionuclides 45 Ca (165 d) and 47 Ca (45 d) and the stable isotopes 42 Ca and 44 Ca. The feasibility of using accelerator mass spectrometry (AMS) measurements of 41 Ca for studying multi-year calcium resorption in humans was tested as part of a companion study that used 45 Ca to measure the effects of dietary cadmium on calcium metabolism in dogs. It was shown that Ca resorbed from prelabeled bones correlates well with 45 Ca for a period of 28 weeks. The advantage of 41 Ca is that, even with a negligible radiation dose, it can be measured by AMS long after the 45 Ca becomes unmeasurable.

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.

Cadmium effects on bone metabolism: Accelerated resorption in ovariectomized, aged beagles

The purpose of this study was to evaluate, in an animal whose skeleton is comparable to humans, the combined effects of estrogen depletion and Cd exposure on bone resorption by monitoring skeletal release of 45Ca and to determine whether Cd-induced bone resorption occurred independent of osteotropic hormone changes and renal dysfunction. Cd exposure following ovariectomy or sham surgery was for 7 months: 1 month by oral ingestion of capsules (1, 5, 15, 50 ppm) and 6 months via drinking water (15 ppm). Serum and fecal 45Ca were increased at 1 week following ovariectomy (OV) (54 +/- 9% and 122 +/- 40%, respectively), but this response was attenuated by 2 weeks. Five of seven exposed dogs had increased serum and fecal 45Ca during the 50-ppm Cd capsule period (15-40% and 15-190%, respectively). Serum 45Ca levels in OV/+Cd dogs showed a significant and consistent increase within 1 week of initiating each of three separate Cd.H2O exposure cycles. Blood Cd levels increased over time from 2 to 15 micrograms/l, coinciding with the elevated serum 45Ca concentrations. No correlation was observed between serum 45Ca increases and parathyroid hormone, 1,25-(OH)2-vitamin D, or calcitonin. No effects of ovariectomy and/or Cd were observed in total serum Ca, calciotropic hormone concentrations, serum or urinary phosphorus and creatinine, creatinine clearance, or urinary specific gravity. Urinary Cd concentrations ranged from 7 to 50 micrograms/l in exposed dogs but were not detectable in nonexposed dogs. Urinary protein concentrations showed no differences between groups. Cd increased bone resorption (skeletal 45Ca release) in ovariectomized and sham-operated dogs without renal dysfunction or calciotropic hormone interaction. Based on our results, Cd is an exogenous factor which exacerbates bone mineral loss in postmenopausal osteoporosis.

Gastrointestinal absorption of cadmium in mice during gestation and lactation. I. Short-term exposure studies.

Abstract The effect on cadmium retention of continuous exposure to drinking water containing low levels of cadmium during pregnancy and lactation was studied in mice. Female mice were provided drinking water ad libitum containing 109CdCl2 (0.03 μCi 109Cd/ml, 0.11 ppb total cadmium) throughout either gestation, lactation, or a combined period of pregnancy and lactation. Nonpregnant control mice were exposed to the same cadmium solution for similar time periods. Dams in all three experimental groups retained two to three times more cadmium (expressed as percentage of ingested dose) than did nonpregnant controls. The 109Cd contents of liver, kidney, mammary tissue, and duodenum increased strikingly in all three groups. Increases in kidney and mammary tissue were particularly apparent during lactation, with increases of fivefold for kidney and at least ninefold for mammary tissue, compared to levels in nonpregnant controls. Increases in 109Cd retention by the duodenum were fivefold during gestation and three- to fourfold during lactation. The kidneys of dams exposed during lactation retained 53% of the whole body 109Cd, while kidneys of nonpregnant controls retained only 27%. Results indicate that pregnant and lactating mice absorb and subsequently retain substantially more cadmium from their diets than do nonpregnant mice.

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.

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.

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%).

Hematological Responses to Arsine Exposure: Quantitation of Exposure Response in Mice

Hematological responses of mice to arsine exposures for 1 hr at 5 to 26 part per million volume (ppmv) are described. Exposure concentrations ranged from a no-effect level for the endpoints studied (5 ppmv) to a concentration lethal to all mice in 4 days (26 ppmv). Hematocrit values at 24 hr after exposure decreased linearly with increasing arsine concentration in the range 5 to 26 ppmv; the hematocrit of the 26-ppmv group reached 10.5% at 24 hr, compared to 48.4% for control mice. Hematocrits of mice from all surviving groups were at or slightly above control values by 11 days after exposure. Changes in numbers of erythrocytes paralleled changes in hematocrit. Significant increases in circulating reticulocytes occurred at 1 and 5 days after exposure; reticulocyte values returned to control levels by 11 days after exposure. Changes in erythrocyte osmotic fragility were observed in mice exposed to 15 and 26 ppmv arsine.

Determination of cadmium in blood, plasma, and urine by electrothermal atomic absorption spectrophotometry after isolation by anion-exchange chromatography☆☆☆

The determination of cadmium in whole blood, urine, or plasma by atomic absorption using electrothermal atomization is described. In preparation for atomic absorption analysis, cadmium was concentrated on an anion-exchange column, significantly lowering the limit of detection and allowing for the first time the accurate and precise determination of plasma cadmium concentrations in persons/animals with low-level cadmium exposures. Recovery of 109Cd from spiked whole blood, plasma, and urine into supernatants of nitric acid-deproteinated samples averaged 99, 100, and 95%, respectively. Anion-exchange isolation of the anionic chlorocadmium complex removed 99.8% of the major elements associated with a deproteinated whole blood sample. The recovery of 109Cd from the anion-exchange column was 92.2 +/- 0.9% (mean +/- SE, N = 35). The separation of cadmium from constituents in blood, urine, or plasma in this manner allowed comparison of unknown samples to aqueous standards with a defined acid matrix using commercially available acids. The mean intra-assay coefficient of variation (CV) was 12 +/- 3% (mean +/- SE, N = 6) for blood, plasma, and urine samples having cadmium concentrations of 0.1-0.8 microgram/liter. The interassay CV was 13% (N = 7) for a blood sample containing 0.6 microgram Cd/liter. The recovery of known amounts of cadmium added to blood, plasma, and urine in the range of 0.2 to 5.0 micrograms Cd/liter was 97 +/- 6% (mean +/- SE, N = 4).

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.