Renal damage induced by cadmium-metallothionein: effects on biochemical indicators.

Time dependent changes in urinary biochemical indicators for renal tubular injury and dysfunction were determined in female Wistar rats after an intraperitoneal injection of cadmium-metallothionein (Cd-MT) (50, 150 or 300 micrograms Cd/kg body wt) to further characterize the tubular damage caused by Cd. The Cd-MT injection caused dose-dependent increases in urinary activities of the enzymes (alkaline phosphatase; gamma-glutamyl transpeptidase; lactate dehydrogenase, LDH; N-acetyl-beta-D-glucosaminidase) on day 1, which appeared to reflect the tubular injury. The rate of increase in LDH, a cytosolic enzyme, was the largest among those of the enzymes. This result coincided with the data reported for repeated administration of ionic Cd to rats, suggesting that the feature of tubular injury caused by an injection of Cd-MT is similar to that by chronic exposure to ionic Cd. Changes in urinary glucose and total protein, indicators of tubular dysfunction, and metals (Cd, zinc and copper) were accompanied with those in urinary enzymes. Hydrocarbons in breath of rats injected with Cd-MT at a dose of 300 micrograms Cd/kg body weight were also determined as an indicator of in vivo lipid peroxidation. The levels of ethane and propane were significantly increased at 12 h after injection, which suggests that lipid peroxidation is partly involved in the tubular damage reflected by the increases in urinary enzymes.

Significance of increase in urinary metallothionein of rats repeatedly exposed to cadmium.

Cadmium chloride was injected subcutaneously (s.c.) into female Wistar rats at a dose of 1 mg Cd/kg body weight, 5 times a week up to 10 weeks. At specified intervals, 24-h urine was collected and the excreted amounts of metallothionein (MT), cadmium, copper, zinc and several indicators of renal damage were determined. Concentrations of cadmium and MT in the livers and kidneys of rats were also determined. Both cadmium and MT in the livers and kidneys were increased upon cadmium exposure. The urinary MT excretion started to increase within a week after the start of exposure. This increased excretion preceded those of enzymes and total protein as well as histopathological abnormalities in the proximal tubular cells. After the occurrence of tubular damage that disturbs reabsorption of MT, MT in urine was drastically increased. These results indicate that urinary MT levels may be an indicator not only of cadmium exposure but also of tubular damage.

Renal discrimination between strontium and calcium in rats. Effects of renal damage caused by cadmium-metallothionein.

Renal functions were damaged at the reabsorptive site by the injection of cadmium-metallothionein (Cd-MT) and its effect on discrimination of chemically similar elements was examined for the two alkaline-earth elements, calcium (Ca) and strontium (Sr). Tubular damage was induced in female Wistar rats, 7 wk old, body wt 145.1 +/- 3.5 g (mean +/- S.D.), by an intraperitoneal injection of Cd-MT (400 micrograms Cd/kg body wt). The Cd-MT injection caused increases in urinary enzymes (lactate dehydrogenase; alkaline phosphatase), glucose, and total protein. Urinary Ca and Sr increased and urinary Sr/Ca ratio decreased rapidly after the Cd-MT injection. Both changes remained at significant levels throughout the experiment. On the other hand, renal Ca and Sr levels increased with time after 18 or 24 h and changed similarly. Although plasma levels of Ca and Sr slightly increased after 30 or 36 h post-injections, the plasma Sr/Ca level remained constant. A close exponential relationship formed between the relative clearances of Ca and Sr. There was no significant difference between the exponent of the following equation (K) for the Cd-MT-injected group and that for the control group. Sr clearance/creatinine clearance = (Ca clearance/creatinine clearance)K where K = 0.408 from experimental data for the two groups. This suggests that the discrimination mechanism between Sr and Ca during the reabsorptive step in the kidney is strictly regulated.

Discrimination between strontium and calcium in suckling rats.

Discrimination between strontium (Sr) and calcium (Ca) was examined in suckling rats and compared with that in older rats after weaning. Concentrations of Sr and Ca and the Sr/Ca ratios in serum and femur of 10-d old and 21-d old rats were determined. The Sr concentrations and Sr/Ca ratios in the serum and femur of 10-d old rats were lower than those of 21-d old rats, that could be explained by the fact that 10-d old rats ingested only maternal milk in which the Sr/Ca ratio was much lower than the laboratory diet. The relative ratios of Sr/Ca in serum and femur to that in the diet were found to be higher in 10-d old rats compared with those in 21-d old rats, and also higher than those in the older rats after weaning, as described in our previous publication. This result may reflect that discrimination between Sr and Ca during intestinal absorption is lacking in very young animals before weaning and develops after this age. Renal discrimination between Sr and Ca in the suckling rats at 10 d of age was evaluated by determining the relationship between the relative clearances of Ca and Sr. The mathematical model proposed by Walser and Robinson was applied on these results and the parameter for the equation, that is, the discrimination constant, was shown to be higher in 10-d old rats compared to those in young (7 wk of age) and adult (25 wk of age) rats. This result suggests that the discrimination of Sr in favor of Ca during the tubular reabsorptive process may not be fully developed in the very young rats before weaning.

Age-related changes in strontium to calcium ratios in rat tissues.

The Sr/Ca ratios in plasma, urine, bone, and soft tissues for various ages after weaning in male and female rats were determined to examine the effects of aging on the discrimination between strontium (Sr) and calcium (Ca) under physiological conditions. Age-related changes in the Sr/Ca ratios were similar in all tissues; the Sr/Ca ratios decreased rapidly until about 25-wk-old and then slowly, from that period on, reaching much lower values than in the diet. When the logarithm of the Sr/Ca ratio in each tissue was plotted against the logarithm of age, a linear relationship was observed with statistically significant (p less than 0.05) regression lines. The higher levels of Sr/Ca ratios in all tissues of the younger rats could be explained by the high efficiency of Sr absorption by the small intestine early in life. Parameters for the equations between age and Sr/Ca ratio differed with tissues, suggesting the existence of specific discrimination mechanisms in each tissue.

Biological discrimination between calcium and strontium in kidneys and bone of young and adult rats.

Effects of aging on the biological discrimination between calcium (Ca) and strontium (Sr) by the kidneys and bone were studied in male and female rats of 5 to 50 wk of age by examining Sr/Ca ratios in the plasma, urine, and bone. The Ca-Sr discrimination at the reabsorption process in the kidneys was not affected by aging in male or female rats. On the other hand, discrimination between the two elements was shown to be age-related at the absorption process in the digestive tract, and became more strict with age. The reverse situation was observed in the discrimination of Ca and Sr in the femur; younger rats discriminated the two elements more strictly than older animals.

ATP-dependent strontium uptake by basolateral membrane vesicles from rat renal cortex in the absence or presence of calcium.

ATP-dependent Sr2+ transport was examined in vitro using basolateral membrane (BLM) vesicles isolated from rat renal cortex to clarify the discrimination mechanisms between strontium (Sr) and calcium (Ca) in renal tubules during reabsorption. ATP-dependent Sr2+ uptake and Ca2+ uptake were observed in renal BLM vesicles and were inhibited by vanadate. Hill plots indicate similar kinetic behavior for Ca2+ and Sr2+ uptake. The apparent Km and Vmax of ATP-dependent Sr2+ uptake were both higher than those for Ca2+ uptake. ATP-dependent Sr2+ uptake by BLM vesicles diminished in the presence of 0.1 microM Ca2+ and was more markedly inhibited by 1 microM Ca2+. Hill plots of Sr2+ uptake data with and without 0.1 microM Ca2+ showed that the cooperative behavior of Sr2+ uptake was not changed by Ca2+. In the presence of 0.1 microM Ca2+, the affinity of the transport system for Sr2+ and the velocity of Sr2+ uptake in the BLM were both decreased. However, the rate of Ca2+ uptake was not diminished by Sr2+ concentrations of less than 1.6 microM. These results suggest that Ca2+ is preferentially transported in the renal cortex BLM when Ca2+ and Sr2+ are present at the same time.

[DNA flow cytometry in smooth muscle tumors of the gastrointestinal tract].

DNA ploidy of 45 smooth muscle tumors of the G.I. tract was determined by flow cytometry and correlated with clinical features and prognosis. The sites of the tumors were: esophagus (1), stomach (24), small intestine (12), large intestine (6), liver (1) and pancreas (1). The histologic type was leiomyoma in 14, leiomyosarcoma in 29, and leiomyoblastoma in 2. DNA aneuploidy was more frequent in leiomyosarcoma (17/29) than leiomyoma (5/14), but the difference was not statistically significant. One leiomyoblastoma was diploid and the other was aneuploid. No patients with leiomyoma died. In patients with leiomyosarcomas, 5-year survival was significantly poorer in those with aneuploid tumors (38%) than in those with diploid tumors (83%). There was no correlation between DNA ploidy and clinico-pathological features of tumors. The present study disclosed that DNA ploidy is a prognostic variable, independent of other variables.

Prognostic value of DNA ploidy in primary gastric leiomyosarcoma.

The prognostic value of DNA ploidy was evaluated in 25 patients with primary gastric leiomyosarcoma, using paraffin-embedded archival specimens analysed by DNA flow cytometry. Ploidy patterns were diploid in ten tumours and non-diploid in 15. They did not correlate with clinicopathological features of the lesions such as tumour size and grade or mitotic index. A non-diploid pattern was associated with a significantly reduced patient survival rate (P < 0.05), as was higher tumour grade (P < 0.01). Neither tumour size nor mitotic index correlated with patient survival.

Critical concentration of cadmium for renal toxicity in rats.

To evaluate a critical concentration concept of cadmium (Cd) toxicity on the kidney, relationships of renal Cd level with urinary excretion of various substances--i.e., metallothionein, alkaline phosphatase, lactate dehydrogenase, N-acetyl-beta-D-glucosaminidase, total protein, Cd, copper, and zinc--were studied in Cd-injected rats. At the renal Cd concentration of 100-200 micrograms/g tissue, a dramatic increase of all these substances in urine was observed, supporting the idea of the critical concentration proposed by Friberg and co-workers (1974). The significance of increase of urinary metallothionein below this level is also discussed.

Transport and distribution of copper injected into an albumin-deficient (analbuminemic) rat.

Copper (Cu) concentrations in blood, liver, kidney, spleen and pancreas of an albumin-deficient (Nagase analbuminemic) rat (NAR) were compared with those of a control (Sprague-Dawley) rat (SDR). Cu concentrations were significantly higher in the blood and significantly lower in the liver of the NAR strain than those of the SDR strain in female control (saline-injected) groups at 8 weeks old. Female NAR and SDR 8-week-old rats were injected i.p. with Cu at a single dose of 2.0 mg/kg body wt and killed 18 hr later. Concentrations of Cu and other essential elements in the blood, liver, kidney, spleen and pancreas were determined simultaneously by inductively coupled plasma-atomic emission spectrometry. Cu concentration in the liver was significantly lower in the NAR than in the SDR strain suggesting a role for albumin as a carrier protein of free Cu ions in the blood. The effects of Cu loading on other essential elements (Zn, Fe, Ca, Mg, P) were also compared between the NAR and SDR strains.

High-performance liquid chromatography-inductively coupled plasma profiles of cadmium, zinc, sulphur, and other elements in rat liver supernatants after cadmium injection.

The distribution of cadmium, zinc, sulphur and other elements in the liver supernatants of rats and their variation with time following a single intraperitoneal injection of cadmium chloride were characterized by high-performance liquid chromatography (HPLC) coupled with an inductively coupled plasma. Cadmium that was bound to high-molecular-weight-proteins before the induction of metallothionein was analysed more easily using an Asahipak GS-520 column, whereas the distributions of other elements and of cadmium bound to metallothionein were better characterized on a TSK G3000SW column. Cadmium bound primarily to a distinct high-molecular-weight protein before the induction of metallothionein. After the injection its distribution shifted with time to metallothionein. An atomic emission spectrometer, employing an inductively coupled argon plasma and a vacuum UV monochromator was effectively used as a multi-element specific detector for HPLC.

The relationships of urinary metallothionein with other indicators of renal dysfunction in people living in a cadmium-polluted area in Japan.

An epidemiologic investigation was carried out to study the significance of urinary excretion of metallothionein (MT) in people aged 50 years and over living in a cadmium (Cd)-polluted area in Japan. The urinary level of MT was compared with various parameters (age, urinary alpha 1-microglobulin (alpha 1-MG), beta 2-microglobulin (beta 2-MG), total protein, Cd, copper (Cu), and zinc (Zn), and relative clearances to creatinine of alpha 1-MG, beta 2-MG, phosphate and uric acid). It was found that the urinary excretion of MT is closely associated with Cd and the indices of renal dysfunction listed above. This observation was more remarkable in women than men. When subjects with signs of renal dysfunction were compared as a group to those with normal renal functions, the excreted amount of MT in the former is significantly greater. The results support the notion that the urinary excretion of MT reflects not only Cd exposure levels but also renal dysfunction caused by long-term Cd exposure.

Effect of cadmium on T4 outer ring monodeiodination by rat liver.

The effect of cadmium on thyroxine (T4) outer ring monodeiodination was studied in vivo and in vitro in the rat liver. One microgram of T4 was incubated with rat liver homogenates in 50 mM Tris--HCl buffer, pH 7.4, with or without 0.5, 5, and 50 mM dithiothreitol (DTT) for 60 min in the presence of 10(-8) to 10(-3) M CdCl2, and the amount of 3,5,3'-triiodothyronine (T3) produced was determined by a specific radioimmunoassay. Subcutaneous injection of CdCl2, 1 mg/kg BW/day, 5 days a week for 10 weeks, to the rats resulted in a significant reduction in serum T3 concentration (by 37%) and hepatic T3 production from T4 (by 78 to 92%). In vitro addition of 1 microM to 1 mM CdCl2 to liver homogenates caused a concentration-dependent reduction in T3 generation. Without DTT a 50% reduction in the T4 to T3 converting activity was caused by 4 X 10(-6) M CdCl2. DTT (0.5 to 50 mM) partially restored T3 generation roughly in a concentration-dependent manner. These results indicate that cadmium has some effects on the metabolism of thyroid hormone.