Pine oil effects on chemical and thermal injury in mice and cultured mouse dorsal root ganglion neurons.

A commercial resin-based pine oil (PO) derived from Pinus palustris and Pinus elliottii was the major focus of this investigation. Extracts of pine resins, needles, and bark are folk medicines commonly used to treat skin ailments, including burns. The American Burn Association estimates that 500,000 people with burn injuries receive medical treatment each year; one-half of US burn victims are children, most with scald burns. This systematic study was initiated as follow-up to personal anecdotal evidence acquired over more than 10 years by MH Bhattacharyya regarding PO's efficacy for treating burns. The results demonstrate that PO counteracted dermal inflammation in both a mouse ear model of contact irritant-induced dermal inflammation and a second degree scald burn to the mouse paw. Furthermore, PO significantly counteracted the tactile allodynia and soft tissue injury caused by the scald burn. In mouse dorsal root ganglion neuronal cultures, PO added to the medium blocked adenosine triphosphate-activated, but not capsaicin-activated, pain pathways, demonstrating specificity. These results together support the hypothesis that a pine-oil-based treatment can be developed to provide effective in-home care for second degree burns.

Liquid scintillation based quantitative measurement of dual radioisotopes (3H and 45Ca) in biological samples for bone remodeling studies.

Acute and prolonged bone complications associated with radiation and chemotherapy in cancer survivors underscore the importance of establishing a laboratory-based complementary dual-isotope tool to evaluate short- as well as long-term bone remodeling in an in vivo model. To address this need, a liquid scintillation dual-label method was investigated using different scintillation cocktails for quantitative measurement of (3)H-tetracycline ((3)H-TC) and (45)Ca as markers of bone turnover in mice. Individual samples were prepared over a wide range of known (45)Ca/(3)H activity ratios. Results showed that (45)Ca/(3)H activity ratios determined experimentally by the dual-label method were comparable to the known activity ratios (percentage difference ∼2%), but large variations were found in samples with (45)Ca/(3)H activity ratios in range of 2-10 (percentage difference ∼20-30%). Urine and fecal samples from mice administered with both (3)H-TC and (45)Ca were analyzed with the dual-label method. Positive correlations between (3)H and (45)Ca in urine (R=0.93) and feces (R=0.83) indicate that (3)H-TC and (45)Ca can be interchangeably used to monitor longitudinal in vivo skeletal remodeling.

Increased metallothionein in mouse liver, kidneys, and duodenum during lactation.

Lactation-induced increases in cadmium absorption and retention have been demonstrated in mid-lactating mice, but no systematic measurements of endogenous metal-binding protein concentrations during lactation have been reported. Using Cd/hemoglobin radioassay, this study detected significant increases in metallothionein (MT) concentrations in liver (4-fold), kidneys (2-fold), and duodenum (2-fold), but not jejunum, of mouse dams on days 13 and 20 of lactation. These increases occurred in the absence of cadmium exposure and were specific to the lactation period; dams 5 days after weaning showed MT levels that were similar to those of nonpregnant (NP) mice. Similarly, Northern blot analyses of livers from lactating mice demonstrated that MT mRNA concentrations in maternal liver during mid-lactation were 6-fold higher than those observed 5 days after pups were weaned. Gel filtration of final supernatants from the Cd/hemoglobin assay confirmed that the Cd-binding molecule induced during lactation was indeed metallothionein. In addition, chromatographic analyses of cytosols from tissues taken from dams administered small amounts of Cd (66 ng/mouse) showed that the trace amounts of Cd absorbed through the maternal gastrointestinal tract during mid-lactation were also bound to the MT. These results indicate MT induction in mouse dams occurs as a physiological consequence of lactation, requiring no external stimulus. This induced MT participates in binding low levels of dietary cadmium consumed by the dam. During lactation, elevated maternal MT may affect pathways for essential trace metals as well as sequester toxic metals harmful to the neonate. Multiparous humans may have increased risk of accumulating environmental Cd.

Ovariectomy-induced changes in aged beagles: histomorphometry of rib cortical bone.

Bone loss associated with estrogen depletion is well documented in cancellous bone but less well characterized in cortical bone. The effects of ovariectomy on the aged beagle skeleton were studied by histomorphometric analysis of the cortical bone in sequential rib biopsies. Biopsies were taken from each ovariectomized or sham-operated dog at the time of surgery and at 1, 4, and 8.5 months after surgery. Just prior to each postoperative biopsy, tetracycline, calcein, and xylenol orange, respectively, were administered by a fluorochrome labeling procedure (2d-10d-2d) to provide markers of bone formation. Analysis of sequential rib biopsies provided a means to follow the ovariectomy response over time and to compare each animal against its own baseline. Though ovariectomy did not influence histomorphometric indices at 1 month after surgery, a transient increase in cortical bone formation occurred thereafter, with a sixfold increase over that of sham-operated dogs at 4 months (P < 0.001) and a return to near control levels at 8.5 months. Cortical porosity increased by the fourth month after ovariectomy and remained high at 8.5 months. These data demonstrate for the first time that rib cortical bone is a responsive site for the effects of ovariectomy in aged female dogs.

Effects of cadmium on bone: an in vivo model for the early response.

Cadmium (Cd) exposure induces bone resorption in vitro and in vivo that can lead to low bone mass and increased incidence of fracture. We have developed an animal model for following the early skeletal response to Cd. A low-calcium (but not calcium-deficient) diet is used to increase gastrointestinal absorption of calcium so that the endogenous fecal calcium excretion is essentially the total fecal calcium excretion. The bone response is followed by quantitation of stable fecal calcium and does not require a radioactive label. After mice were adjusted to a low-calcium diet, Cd was administered by a single gavage and fecal calcium was monitored to determine the magnitude of the calcium release from bone. Fecal calcium excretion (microg Ca/hr; mean +/- SE) remained at the background level for 8 hr (13.6 +/- 1.8, n = 18) but increased during the 8- to 24-hr and 24- to 56-hr collection periods (43.8 +/- 6.8, n = 12; 50.75 +/- 3.7, n = 6, respectively). The bone response was transient and dropped to nearly background levels during the 56- to 104-hr collection period. Blood calcium levels were normal throughout the time course. Bone resorption occurred at Cd levels of 7.9 +/- 0.7 microg/liter blood (mean +/- SE, n = 6), which is in the range of occupational exposure levels. The transient nature of the bone response contrasted to the slow but continuing rise observed in blood Cd levels. These results suggest that a threshold level of Cd is required for a bone response but that chronic levels of Cd in blood do not necessarily indicate the occurrence of continuous active bone resorption. This model can be used to probe early gene changes (prior to the bone response) that may be occurring in response to Cd exposure.

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%).

Effects of cadmium on osteoclast formation and activity in vitro.

Chronic exposure to cadmium has been linked to bone loss, low bone mass, and increased incidence of fracture. To determine if Cd could directly increase the formation of cells responsible for bone resorption, we cultured normal canine bone marrow cells containing the progenitor cells for osteoclasts. Cultures were evaluated for the number of multinucleate osteoclast-like cells (MNOCs) formed. Exposure to Cd (10-100 nM) increased the number of MNOCs formed over control values when cultured in the presence but not in the absence of a bone wafer. The MNOCs formed were functional, evidenced by pits excavated on the bone wafers included in the cultures. By 12 days, MNOCs formed in the presence of 50 nM Cd excavated significantly more pits and a greater pit area than did untreated MNOCs. By 14 days, the control values were similar to those of the Cd-exposed MNOCs, but pit formation was enhanced by Cd in that the ratio of pit complexes to single pits was increased twofold over that for untreated cultures. Mature osteoclasts, isolated from the long bones of rat neonates and cultured for 1-3 days on bone slices, provided a direct method to assess the effect of Cd on osteoclast activity. Exposure of osteoclast cultures to 100 nM Cd increased the number of osteoclasts present over that for untreated osteoclasts by a factor of 1.7 +/- 0.1, the number of pits excavated by 2.8 +/- 0.6, the area excavated by 3.2 +/- 0.8, and the area excavated per osteoclast by 1.8 +/- 0.4 (mean +/- SE; n = six experiments). These data suggest that Cd accelerates the differentiation of new osteoclasts from their progenitor cells and activates or increases the activity of mature osteoclasts.

Effect of cadmium on bone calcium and 45Ca in mouse dams on a calcium-deficient diet: evidence of Itai-Itai-like syndrome.

To test whether Cd exposure would increase Ca release from bone during pregnancy and lactation in relation to the etiological mechanism of Itai-Itai disease, virgin female mice with 45Ca prelabeled skeletons (15 microCi/mouse) were subjected to one round of pregnancy/lactation and were exposed to a Ca-deficient diet containing 0, 5, or 25 ppm Cd or 25 ppm Pb for 32 days, from conception until Lactation Day 14. A striking loss of 45Ca was found in the dam's total skeleton (-40%), right femur (-47%), and lumbar vertebrae (L1-L5) (-75%) due only to pregnancy/lactation in conjunction with Ca deficiency. At both 5 and 25 ppm, Cd administered through food induced an additional significant 45Ca loss from the total skeleton (-25% at 5 ppm Cd, -30% at 25 ppm Cd) and right femur (39% at 5 ppm Cd, -32% at 25 ppm Cd) compared to 0 ppm animals. Almost all of the 45Ca lost from the dam's skeleton appeared in the pups, with 80% transferred via the dam's milk during lactation and only 20% transferred during gestation; a very small fraction of the dam's skeletal 45Ca was excreted. Considering stable Ca values, Cd exposure nearly doubled the loss of Ca from the dam's skeleton (-78 mg Ca/mouse at 0 ppm; -146 mg Ca/mouse at 5 and 25 ppm Cd). Paralleling 45Ca losses, a Ca-deficient diet in combination with pregnancy/lactation alone caused significant decreases in weight and mineral content of the right femur and lumbar vertebrae (dry weight, ash weight, ash/dry, Ca content, Ca/dry, and Ca/ash) (-8 to -52%). Cd at both 5 and 25 ppm showed additional decreases (-15 to -32%, Cd groups compared to 0 ppm animals). Responses were specific to Cd in that no significant effect occurred due to 32 days of Pb exposure (25 ppm). This experiment supports the view that Cd exposure in conjunction with Ca deficiency and pregnancy/lactation are key etiological factors of Itai-Itai disease and that Cd at both 5 and 25 ppm in conjunction with one round of gestation/lactation and Ca deficiency can induce an extreme demineralization characteristic of Itai-Itai-like syndrome.

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)

Effect of cadmium on bone calcium and 45Ca in nonpregnant mice on a calcium-deficient diet: evidence of direct effect of cadmium on bone.

To clarify the mechanism of the effect of Cd on bone, virgin female mice with 45Ca prelabeled skeletons (15 microCi/mouse) were exposed to a Ca-deficient diet (0.002%) containing 0, 5, or 25 ppm Cd or 25 ppm Pb for 32 days. During the first 72 hr, the 0 ppm controls showed a 2.5-fold decrease in fecal 45Ca excretion (decreased bone resorption), a 12-fold decrease in total fecal stable Ca, a 5-fold decrease in endogenous fecal Ca excretion, and a significant elevation in both serum 45Ca (108%) and specific activity (92%) due to the Ca-deficient diet. In contrast, Cd immediately and significantly increased fecal 45Ca excretion (55%), total fecal stable Ca (13%), endogenous fecal Ca excretion (32%), as well as serum 45Ca (15%) and specific activity (17%) (25 ppm Cd vs 0 ppm), supporting the hypothesis of an early, direct effect of Cd on bone. Overall, during 32 days, Cd at 25 ppm induced a 60% increase in fecal 45Ca excretion compared to 0 ppm controls, providing a sensitive measure of the Cd-induced increase in bone resorption. In contrast, the effect of Cd was too small to cause a statistically significant 45Ca loss from the right femur and lumbar vertebrae, but a significant 45Ca loss (up to -13%) was seen for the remaining skeleton (25 ppm vs 0 ppm group). In addition, Cd at 25 ppm induced small but statistically significant decreases in ash weight (-12%), ash weight to dry weight (-5%), Ca/dry (-7%), and Ca/ash (-2%) in the right femur and significant decreases in ash/dry (-7%) and Ca/dry (-12%) in the lumbar vertebrae compared with the controls. Our present study supports the hypothesis that Cd at 25 ppm had a significant direct effect on bone, with no effect at 5 ppm Cd or 25 ppm Pb, and no appearance of the extreme demineralization characteristic of Itai-Itai disease, although mice were exposed to a Ca-deficient diet for 32 days. Our results from this and earlier studies support the view that chronic Cd exposure along with nutritional deficiency contributed to the pathogenesis of Itai-Itai disease among multiparous, postmenopausal women in Japan.

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-induced bone loss: increased susceptibility in female beagles after ovariectomy.

Bone resorption, as measured by release of bone 45Ca, was significantly increased in elderly female beagles within 96 h of exposure to 15 mg/l cadmium in drinking-water. The 45Ca response was greater in ovariectomized animals than in sham-operated controls and was not mediated by changes in calciotropic hormone concentrations. Mean blood cadmium concentrations were 3-8 micrograms/l during the earliest bone resorption response and 13-15 micrograms/l at the end of the study. During seven months of cadmium exposure, bone mineral densities decreased most in the ovariectomized animals exposed to cadmium: -15.4 +/- 4.3% for the tibia distal end and -7.2 +/- 1.2% for the lumbar vertebrae (L2-L4) (mean +/- SE, n = 4). The results indicate that cadmium may act directly on bone and that postmenopausal women exposed to cadmium in industry or via cigarette smoke may be at increased risk of cadmium-induced bone loss. They also support a direct role of cadmium in the etiopathology of itai-itai disease among postmenopausal women in Japan.

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).

Early changes in the tissue distribution of cadmium after oral but not intravenous cadmium exposure.

The kinetics of 109Cd distribution in tissues of male and female mice were measured at intervals of 5 min to 15 days after oral (100 micrograms Cd/kg; by gavage) or intravenous (1 micrograms Cd/kg; i.v.) administration of 109CdCl2. Unexpectedly, the ratio of 109Cd in liver to that in kidneys was greater than or equal to 10 within 1 h after administration by either route. However, after 4 h, route-dependent differences in distribution between liver and kidney became apparent. In mice receiving oral cadmium, the liver:kidney 109Cd ratio decreased with time to approximately 4 at 72 h after gavage. In contrast, in mice receiving IV cadmium, the liver:kidney 109Cd ratio remained high and relatively constant during the same time period. The time-dependent decrease in the liver:kidney 109Cd ratio after oral cadmium administration was caused by a 4-5-fold increase in cadmium content of the kidney that occurred between 30 min and 72 h after oral but not i.v. administration. During this time, there was no change in cadmium distribution in subcellular fractions of either liver or kidney. These results could be explained by the existence of 2 separate pathways for cadmium deposition after oral exposure. Early after exposure, cadmium may leave the intestine, bind to serum albumins or other high molecular weight proteins, and accumulate primarily in liver, as is also observed after IV cadmium administration. With time, cadmium may leave the intestinal mucosa bound to metallothionein and deposit primarily in the kidney. The different pathways of deposition after oral vs. i.v. exposure may in part explain why acute parenteral cadmium exposure causes liver toxicity, but chronic oral exposure causes renal toxicity.

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.