David J. Thomas

Lipopolysaccharide induces double-stranded DNA fragmentation in mouse thymus : protective effect of zinc pretreatment

Intraperitoneal injection of female NAW/W1 mice with 5 mg of Salmonella typhimurium lipopolysaccharide/kg results in decreased body and thymus weight. Reduced thymic weight is accompanied by fragmentation of DNA into multimers of about 200 bp size. This effect is consistent with the induction of intranucleosomal cleavage of double-stranded DNA in thymus. Maximal fragmentation of DNA occurs between 18 and 24 h after treatment; by 48 h post lipopolysaccharide treatment, there is little evidence of thymic DNA fragmentation. Pretreatment of mice with Zn protects against lipopolysaccharide-induced DNA fragmentation. This effect is maximal at about 72 h after Zn treatment (24 h after lipopolysaccharide treatment) and persists until about 96 h after Zn treatment. At 72 h after pretreatment, the antagonism of thymic DNA fragmentation by Zn is dose-dependent. To examine the role of the acute phase inflammatory response elicited by lipopolysaccharide treatment in the production of changes in thymic weight and DNA integrity, the effects of treatment with casein, a well-characterized inducer of the acute phase inflammatory response in mice, were examined. In contrast to the effect of lipopolysaccharide, casein treatment did not produce a similar pattern of DNA fragmentation in thymus. Taken together, these data suggest that lipopolysaccharide induces DNA fragmentation in thymus by a mechanism which does not occur during the pathophysiological changes which accompany the casein-induced acute phase response. Further, the antagonism by Zn of lipopolysaccharide-induced fragmentation of thymic DNA is consistent with earlier findings that Zn can prevent dexamethasone-induced DNA fragmentation in vitro.

Osteotoxicity of cadmium and lead in HOS TE 85 and ROS 17/2.8 cells: relation to metallothionein induction and mitochondrial binding.

Epidemiological, experimental and clinical data indicate that cadmium and lead are osteotoxins in man and other species. The relative sensitivities of a clonal human osteosarcoma cell line (HOS TE 85) and a clonal rat osteosarcoma cell line (ROS 17.28) to the cytotoxic effects of cadmium and lead were tested in serum-free media without added growth factors. The rat osteosarcoma cells were more sensitive to cadmium with cytotoxicity and inhibition of proliferation at 0.25 versus 0.75 and 1.0 μmol l− cadmium, respectively, for human osteosarcoma cell lines. The lower sensitivity to cadmium of human osteosarcoma cells is attributed, at least partly, to induction of metallothionein synthesis by cadmium and zinc in this cell line; in the rat osteosarcoma cell line, they do not induce metallothionein synthesis. Human osteosarcoma cells were more sensitive than rat osteosarcoma cells to lead with inhibition (IC50) of proliferation at 4 μmol l− lead and cytotoxicity at 20 versus 6 and over 20 μmoll− lead, respectively, for these variables in rat osteosarcoma cells. Both cells lines attained the highest lead concentration in the 15 000 × g (mitochondrial) fraction. The lead in the mitochondrial, microsomal, nuclear and cytosolic fractions of the human cell line did not decrease during 24 h post-washout. Binding of lead was much less stable in the less sensitive rat cells, with 50–100% loss of mitochondrial, microsomal and nuclear lead during 24 h post-washout.

Lead inhibits the basal and stimulated responses of a rat osteoblast-like cell line ROS 17/2.8 to 1α,25-dihydroxyvitamin D3 and IGF-I

Low-level exposure to lead impairs longitudinal growth in children and in experimental animals. The proposed mechanisms include decreased osteocalcin secretion in response to 1 alpha,25-(OH)2 vitamin D3 and decreased response to insulin-like growth factor-I. The interaction of lead, 1 alpha,25-(OH)2 vitamin D3, and insulin-like growth factor-I was investigated in an osteoblast-like cell line from rat osteosarcoma, ROS 17/2.8. Cells were cultured 24 hr in a serum-free medium with lead, 1 alpha,25-(OH)2 vitamin D3, and insulin-like growth factor-I. 1 alpha,25-(OH)2 vitamin D3 (10 nM) evoked a 4-5 X increase in osteocalcin secretion and a 100% increase in cellular alkaline phosphatase activity but no increase in DNA/cell layer. Insulin-like growth factor-I (92.5 ng/ml) evoked a 100% increase of osteocalcin secretion and a 20% increase in cellular DNA contents but no change in cellular alkaline phosphatase activity. Basal and stimulated cellular osteocalcin secretion, cellular alkaline phosphatase activity, and DNA contents were significantly inhibited by addition of 1-10 microM lead. The data are consistent with a toxic effect of lead on osteoblastic function and the cellular responses to 1 alpha,25-(OH)2 vitamin D3 and insulin-like growth factor-I. This interaction may be relevant to impaired childhood growth at low levels of lead exposure.

Toxicity of cadmium to rat osteosarcoma cells (ROS 172.8): Protective effect of 1α,25-dihydroxyvitamin D3

Abstract Inadequate vitamin D intake is an important cofactor in clinical and experimental bone disease induced by chronic cadmium exposure. The interaction was investigated by culture of rat osteoblastic osteosarcoma cells (ROS 17 2.8 ) in a serum-free medium with equimolar concentrations of cadmium chloride and 1α,25-(OH) 2 vitamin D 3 . After addition of cadmium alone to culture medium, the unstimulated secretion of osteocalcin and cellular alkaline phosphatase activity were inhibited at 10 p m , and of DNA synthesis and proline incorporation into collagen at 500 n m . In the presence of equimolar amounts of cadmium and 1α,25-(OH) 2 vitamin D 3 , all four responses paralleled those of 1α,25-(OH) 2 vitamin D 3 alone up to the inhibitory concentration of 500 m m cadmium. Neither 10 n m 1α,25-(OH) 2 vitamin D 3 nor 1 μ m cadmium induced synthesis of metallothionein in these cells indicating that the protective effect of D 3 was not related to the induction of a metallothionein-like protein in ROS 17 2.8 cells. In the presence or absence of D 3 , cadmium inhibited osteoblastic function at concentrations below the whole-organ concentration of cadmium in bone as reported in experimental and clinical cadmium-induced osteotoxicity. The extreme sensitivity of ROS 17 2.8 cells to cadmium may relate to the absence of metallothionein synthesis.

Distribution and retention of organic and inorganic mercury in methyl mercury-treated neonatal rats

Seven-day-old Long Evans rats received one mumol of 203Hg-labeled methyl mercury/kg sc and whole body retention and tissue distribution of organic and inorganic mercury were examined for 32 days postdosing. Neonates cleared mercury slowly until 10 days postdosing when the clearance rate abruptly increased. During the interval when whole body clearance of mercury was extremely slow, methyl mercury was metabolized to inorganic mercury. Peak concentration of mercury in kidney occurred at 2 days postdosing. At 32 days postdosing, 8% of mercury in kidney was in an organic from. Liver mercury concentration peaked at 2 days postdosing and organic mercury accounted for 38% at 32 days postdosing. Brain concentrations of mercury peaked at 2 days postdosing. At 10 days postdosing, organic mercury accounted for 86% of the brain mercury burden, and, at 32 days postdosing, for 60%. The percentage of mercury body burden in pelt rose from 30 to 70% between 1 and 10 days postdosing. At 32 days postdosing pelt contained 85% of the body burden of mercury. At all time points, about 95% of mercury in pelt was in an organic form. Compartmental analysis of these data permitted development of a model to describe the distribution and excretion of organic and inorganic mercury in methyl mercury-treated neonatal rats.

Effect of sodium butyrate on metallothionein induction and cadmium cytotoxicity in ROS 17/2.8 cells

ROS 17/2.8 cells, a cloned rat osteosarcoma cell line, are exceptionally sensitive to the cytotoxic effects of cadmium. This sensitivity is associated with the inability of this metal to induce the synthesis of metallothionein, a transition metal-binding protein, which detoxifies this metal by its sequestration. Sodium butyrate induces the synthesis of metallothionein in these cells in a concentration-dependent manner. Treatment with this agent also significantly increases the resistance of these cells to the cytotoxic effects of cadmium and the protective effect of butyrate is reversed upon its removal from culture medium. Butyrate treatment did not significantly alter the accumulation of cadmium by these cells. Hence, the increased synthesis of metallothionein in butyrate-treated cells is not due to increased cellular uptake of cadmium. Inhibition of DNA synthesis due to butyrate was not a sufficient condition to alter metallothionein synthesis or to protect against Cd-induced cytotoxicity. Equivalent inhibition of DNA synthesis with hydroxyurea failed to increase metallothionein synthesis in cadmium-treated cells. These results indicate that modulation of metallothionein gene expression in this cell line is the critical factor in determining cellular sensitivity to the cytotoxic effects of cadmium.

Effect of 5-azacytidine on metallothionein inducibility and sensitivity to lethality of cadmium in rat osteosarcoma (ROS 172.8) cells

ROS 17/2.8 cells, a cloned rat osteoblastic osteosarcoma cell line, were found to be extremely sensitive to the lethal effects of cadmium and to synthesize little, if any, metallothionein in response to cadmium exposure. Culture of cells for 24 h in the presence of 1 microM 5-azacytidine, a cytidine analog, increased the inducibility of metallothionein by cadmium and significantly reduced (P less than 0.001) cytotoxicity. Anion exchange chromatographic analysis of cadmium binding to low molecular mass cytotoxicity. Anion exchange chromatographic analysis of cadmium binding to low molecular mass cytosolic proteins showed that cells treated with cadmium and 5-azacytidine expressed at least 2 isoforms of metallothionein. One isoform of metallothionein with a low affinity for cadmium was constitutively expressed by these cells. The association of poor inducibility of metallothionein by cadmium with extreme sensitivity of cells to cadmium emphasizes the role of this protein in the cellular response to this toxic metal. The modulation of metallothionein inducibility and sensitivity to cadmium by 5-azacytidine treatment suggest that metallothionein gene structure and regulation are altered in ROS 17/2.8 cells.

Ontogenic variation in acute lethality of cadmium in C57BL/6J mice

Susceptibility of C57BL/6J mice to the lethal effects of parenterally administered Cd declined as a function of age at exposure. The 7-day LD50 increased from 1.65 mg Cd/kg body wt in 7-day-old mice to 4.08 mg/kg in adult mice. Survival time following treatment with Cd also increased as a function of age. High constitutive concentrations of metallothionein, a transition metal-binding protein, in livers of young mice did not protect against the lethality of Cd. These results suggest that, in the mouse, the interaction between Cd and this metal-binding protein may be affected by age at exposure to this toxic metal.

Altered Organ Growth and Zinc and Copper Distribution in Endotoxin-Treated Neonatal Mice

ABSTRACT: Organ weights and the distribution of zinc and copper were compared in HLA/ICR mice that received intraperitoneal injections of 10 μg of Serratia marcescens lipopolysaccharide W or of sterile physiologic saline at 2 d of age. Between 5 and 28 d of age, body weight gains were similar in both groups. At 5 and 7 d of age, lipopolysaccharide W-treated mice had significantly lower thymus weights (p < 0.01). At 7 d of age, liver weight was significantly increased (p < 0.01) in lipopolysaccharide W-treated mice. Compared with tissue copper concentration in coeval saline-treated mice, lipopolysaccharide W treatment significantly increased copper concentration in thymus at 5 d of age (p < 0.05) and significantly decreased concentration of this metal in liver at 7 d of age (p < 0.05) and in spleen at 14 d of age (p < 0.05). Liver zinc concentration was significantly lower (p < 0.05) in 28-d-old mice that had received lipopolysaccharide W. When expressed on the basis of total organ burdens of zinc or copper, only the liver burden of zinc in 5-d-old lipopolysaccharide W-treated mice was significantly increased (p < 0.05). Lipopolysaccharide W treatment consistently decreased copper concentration in liver cytosol and the amounts of zinc and copper bound to metallothionein, a transition metal-binding protein, in liver cytosol. These effects of lipopolysaccharide W on organ size and metal distribution may contribute to the adverse effects that persist after endotoxin exposure in early life.

Influence of age upon the metabolism of zinc in livers of C57BL/6J mice

The kinetics of accumulation and loss of zinc from the liver following subcutaneous administration of 10 mg of zinc per kg were examined in young adult (6 months old) and old (24 months old) male C57BL/6J mice. After zinc treatment, total liver zinc concentrations rose equally in both groups and returned to basal levels at 96 h post treatment. However, differences were found in the subcellular distribution of zinc in these two age groups. The concentration of zinc in the cytosolic fraction (104,000 g supernate) prepared from the livers of old mice attained its maximum at 24 h post treatment. In contrast, the concentration of zinc in the cytosolic fraction of liver from young adult mice peaked at 48 h post treatment. This difference in accumulation of zinc in the cytosol was reflected by differences in the binding of zinc to metallothionein, a cytosolic transition metal binding protein. In old mice the highest amounts of zinc bound to metallothionein were found at 24 h post treatment: in young adults the maximal zinc binding to this protein occurred at 48 h post treatment. Examination of the relationship between cytosolic zinc contents and the binding of zinc to metallothionein in young adult and old mice suggested similar regulatory processes in the two age groups. Thus, age-dependent differences in accumulation and loss of zinc from the cytosolic fraction of liver probably reflect factors other than differences in regulation of the synthesis of metallothionein by this essential metal.