Arthur L. Aronson

Teratogenic effect of calcium edetate (CaEDTA) in rats and the protective effect of zinc.

The calcium chelate of EDTA (CaEDTA) currently is the drug of choice in the treatment of lead intoxication. This study investigated the teratogenic potential of CaEDTA, administered parenterally during periods of organogenesis and determined if incorporating zinc into EDTA would protect against teratogenic effects. Four doses (2, 4, 6, and 8 mmol/m2/day) of CaEDTA, two concentrations (8 and 20 mmol/m2/day) of ZnEDTA and ZnCaEDTA (molar ratio 0.5:0.5:1) were used, and a saline control (0.9% NaCl). Timed-pregnant Long-Evans rats were assigned at random to the treatment groups, 20 per dose for each chelate and 30 to the saline control. Rats were injected with the chelate or saline solution sc, twice daily during the 11th through 15th days of gestation. Pups removed by cesarean section on the 21st day were processed for osseous and visceral examination. Additional animals per treatment group were used for maternal plasma and liver and fetal zinc determinations. Results showed increases in several abnormalities (submucous cleft, cleft palate, adactyly-syndactyly, curly tail, abnormal rib and vertebrae) with increasing amounts of CaEDTA. No malformations were seen with ZnEDTA at either dose or with ZnCaEDTA at 8 mmol/m2/day. However, submucous cleft was seen in 6 of 20 litters from the dams receiving the higher dose of ZnCaEDTA. It was concluded that CaEDTA is teratogenic in rats at concentrations which, except for decreased weight gain, produce no discernible toxicity to the dam, and which are comparable to the recommended therapeutic dosage in humans (1500 mg/m2/day corresponding to 4 mmol/m2/day). Protection is afforded by incorporating zinc in the chelate.

Studies with calcium ethylenediaminetetraacetate in calves; Toxicity and use in bovine lead poisoning

Abstract The purposes of the present study were (1) to determine the most effective therapeutic regimen of CaEDTA for the mobilization of lead in cattle and (2) to evaluate the toxic effects associated with short-term administration of CaEDTA. The studies indicate that optimal conditions of lead mobilization in calves are provided by concentrations in the order of 135 mμmoles of EDTA per milliliter of plasma and above maintained for 10–12 hours. These concentrations can be achieved by the constant intravenous infusion of CaEDTA at 110–220 mg/kg over 12 hours or approximated by two rapid intravenous injections 6 hours apart at 110 mg/kg each. Intraperitoneal administration is not as effective as intravenous administration in mobilizing lead. The manifestations of CaEDTA toxicity in calves were similar to those observed in man and the dog, but were considerably more extensive than those reported for the rat. Daily 12-hour infusions of CaEDTA at 220 mg/kg were not toxic when maintained for 3 successive days and were mildly toxic when maintained for 5 successive days. Macroscopic and microscopic lesions are described.

A comparative study of the toxic effects of calcium and chromium chelates of ethylenediaminetetraacetate in the dog

Abstract The toxicity of calcium ethylenediaminetetraacetate (CaEDTA) resulting from sustained iv infusion was studied in dogs. Signs of toxicity included vomiting, depression, and, occasionally, bloody diarrhea. The dogs were in a shocklike state terminally. Because of the reported nephrotoxicity of the drug, parameters of renal function were measured using conventional steady-state techniques. Glomerular filtration rate, renal plasma flow, and tubular maxima for glucose and PAH were decreased 8–22% following CaEDTA infusion; acetate enhancement of Tm PAH was substantially reduced. Essentially no changes occurred in blood urea nitrogen, plasma electrolyte concentrations, or urinary excretion rates of sodium, potassium, or chloride. Histopathologic changes included hemorrhage and necrosis of intestinal epithelium with loss of villous structure and a necrotizing nephrosis of the proximal convoluted tubules. Evaluation of intestinal function revealed a marked depression of glucose absorption but no significant changes in the net fluxes of sodium, potassium, or chloride from the duodenum as toxicity developed. The permeability of the intestine to a volume marker was greatly increased. Studies of the intestinal diffusion ratios of solutes of various molecular sizes indicated the permeability increase was limited to the relatively large molecules. The results indicate CaEDTA is not primarily nephrotoxic in the dog and, therefore, renal failure probably is not the cause of death. The infusion of a very stable chelate, CrEDTA, was nontoxic. This suggested that CaEDTA toxicity is due to its chelative properties rather than some nonspecific action of the EDTA moiety.

Effect of calcium and chromium chelates of ethylenediaminetetraacetate on intestinal permeability and collagen metabolism in the rat

The parenteral administration of a toxic dose of the calcium chelate of ethylenediaminetetracetate (CaEDTA) to rats caused a marked increase in the permeability of the intestinal wall to normally nonpermeating solutes. By contrast, the administration of an equim dose of CrEDTA, a highly stable and inert chelate that does not undergo metal exchange in vivo, did not produce any observable toxic effects, and the permeability of the intestine was not affected. The possibility was investigated that either deposition or depletion of metals or a derangement of the connective tissue matrix might explain the enhanced permeability of the intestinal wall. Although no significant changes in metal concentrations of the intestinal wall were measured, the study did not rule out the possibility of an inactivation of metals in situ due to the formation of ternary complexes. The urinary excretion of hydroxyproline, an imino acid virtually specific for collagen, increased approximately 6-fold in rats treated with CaEDTA, When proline-14C was administered 1 mo preceding treatment with CaEDTA, the urinary excretion of hydroxyproline-14C increased, but its specific activity remained unchanged. The urinary excretion of hydroxyproline was not affected by the administration of CrEDTA. Thus it seems that CaEDTA effects collagen degradation by virtue of its chelating property.

The mechanism of renal transport and excretion of ethylenediaminetetraacetate with interspecies comparisons

Abstract The renal transport and excretion of ethylenediaminetetraacetate (EDTA) was studied by conventional steady-state techniques in conscious dogs, calves, and rats. Concentrations of 250–400 μ m EDTA were maintained in the plasma during the period of study. The average ratio C EDTA /C inulin measured for the 3 species ranged from 0.94 to 0.97. The clearance ratio was unaffected by alterations in urine pH or rate of urine flow or by the administration of p -aminohippurate (PAH), probenecid, or N 1 -methyl nicotinamide. The intravenous injection of CaEDTA, producing initial plasma concentrations in excess of 1 m m , did not affect the renal clearance of either EDTA or inulin. Likewise, the administration of PAH was without effect on the distribution volume of EDTA in the body or its rate of disappearance from the plasma. The data indicate that the mechanism for the renal excretion of EDTA is glomerular filtration.

Calcium ethylenediaminetetraacetate toxicity in the rat: Sequential light- and electron-microscopic studies on chelate-induced enteropathy☆☆☆

Abstract Sequential morphologic and functional studies of CaEDTA-induced enteropathy revealed that morphologic changes are present before an increase in permeability occurs. Light microscopy revealed degenerative changes in duodenal crypt cells and electron microscopy revealed highamplitude swelling of mitochondria as early as 12 hr following the start of CaEDTA infusion. The mitotic index and cell population density was markedly diminished by hour 24. At this time the villus structure was still intact. Enhanced permeability of the intestine occurred between hour 24 and 36 and correlated with the loss of villous epithelium. This study supports the work of others suggesting that inhibition of DNA synthesis is an important biochemical lesion in CaEDTA-induced enteropathy. The occurrence of high-amplitude mitochondrial swelling suggests that interference with the energy supply in the cell may be a contributing factor toward inhibition of the synthesis of DNA.

Comparative effects of Ca-ethylenediaminetetraacetic acid (EDTA), ZnEDTA, and ZnCaEDTA in mobilizing lead.

Male Long-Evans rats weighing approximately 240 g were given (Pb) at 14 mg/kg as the acetate by slow iv infusion 17 days prior to chelate treatment. The chelating agents were administered by continuous iv infusion at either 1 mmol/kg over 6 hr or 6 mmol/kg over 24 hr or at 0.16 mmol/kg/day by sc injections. ZnEDTA was 60% and ZnCaEDTA 76% as effective as CaEDTA in promoting urinary Pb excretion at 1 mmol/kg over 6 hr, iv. At 6 mmol/kg/24 hr, iv, ZnEDTA was 76% and ZnCaEDTA 98% as effective as CaEDTA. Mean urinary Pb excretion for each chelate via the sc route and the lowest iv route of administration was the same. Blood delta-aminolaevulinic acid dehydratase (ALAD) activity (an indicator of Pb toxicity) was enhanced approximately twofold by CaEDTA, two and one-half-fold by ZnCaEDTA, and fivefold by ZnEDTA treatment. It is suggested that the safety of EDTA can be markedly enhanced if administered as a ZnCaEDTA chelate without appreciably diminishing its efficacy in promoting urinary Pb excretion.

Calcium ethylenediaminetetraacetate (CaEDTA) toxicity: Time- and dose-response studies on intestinal morphology in the rat☆

Abstract The effect of CaEDTA toxicity on intestinal morphology was investigated in rats. Constant iv infusion of CaEDTA was used in time- and dose-response studies. A dosage of 6.0 mmoles/kg/24 hr was needed to induce marked morphological changes. These changes consisted of shortening of the villi, reduced height of epithelium, degeneration of the brush border and decreased mitotic index. After 36 hr of infusion at a dosage of 6.0 mmoles/kg/24 hr the villi were drastically reduced and no mitotic figures were seen. If the CaEDTA infusion was stopped at this point the intestine began to recover. After 24 hr of recovery mitotic figures were again in evidence and by 48 hr the mitotic index was higher than in controls. It is proposed that the pattern of changes observed is consistent with the mode of action of the chelating agent being a block of DNA synthesis in the S or early M phase. All other cellular changes are consistent with an inflammatory response.

Calcium ethylenediaminetetraacetate (CaEDTA) toxicity: Time- and dose-response studies on intestinal DNA synthesis in the rat

Abstract The effect of CaEDTA on intestinal DNA synthesis was studied by measuring the incorporation of [methyl-3H]thymidine. An ip injection of thymidine (0.25 μCi/g body weight) was given 1 hr before sacrifice. DNA was extracted from a 10% (w/v) homogenate of mucosal scrapings by a modification of the Schmidt-Thannhauser procedure. Radioactivity in the DNA fraction was assayed by liquid scintillation counting. Dose-response studies showed that a constant iv infusion of 3.0 mmoles/kg/24 hr for 24 hr was required to produce a significant inhibition of DNA synthesis. Time-response studies revealed that 18 hr of CaEDTA infusion at a dosage of 6 mmoles/kg/24 hr were required for significant inhibition of DNA synthesis; the degree of inhibition occurring arabinoside (250 mg/kg) injection. Although folic acid (250 mg/kg) did not protect, the infusion of ZnEDTA, CoEDTA, or ZnCaEDTA afforded complete protection against inhibition of DNA synthesis. Even when the rats were moribund following CaEDTA administration, the intestine was capable of recovering its ability to synthesize DNA. This recovery was significantly enhanced by supplementation with zinc, but not by cobalt or manganese. When parenteral alimentation was employed, the survival of the rats was markedly improved. It is proposed that zinc serves as a low-molecular weight nutrient essential to initiation of DNA synthesis and that the mechanism of inhibition of DNA synthesis during the administration of CaEDTA may be the blocking of normal zinc uptake into the cell. Since the intestine is rapidly regenerating at a time when the rat is moribund it is suggested that inhibition of DNA synthesis in the intestine is not the direct cause of death.

Calcium ethylenediaminetetraacetate (CaEDTA) toxicity--effect of lysosomal stabilizers and labilizers on CaEDTA-induced collagen degradation in the rat.

Abstract Degradation of collagen accompanies the progression of CaEDTA toxicity in the rat. After the parenteral administration of CaEDTA (6 m-moles/kg/24 hr, constant i.v. infusion for 48 hr), the urinary excretion of hydroxyproline is markedly increased. Radioisotope studies indicated that the source of this enhanced urinary hydroxyproline is the degradation of both mature and immature collagen. The present study was concerned with the effect of known lysosomal stabilizers and labilizers on CaEDTA-induced collagen breakdown. Massive doses of cortisol (50 mg/kg/8 hr, i.m.), as well as other anti-inflammatory agents believed to stabilize lysosomal membranes, were administered together with CaEDTA to rats. The concurrent administration of cortisol partially antagonized the degradative action of CaEDTA as evidenced by a significant reduction in urinary hydroxyproline excretion. Radioisotope labeling experiments traced this reduction in urinary hydroxyproline to inhibition of CaEDTA-induced degradation of mature (insoluble) collagen. In contrast, the concurrent administration of CaEDTA and vitamin A palmitate (250,000 I.U./kg/12 hr, s.c.), a lysosomal labilizer, produced a synergistic increase in urinary hydroxyproline excretion which was the result of increased breakdown of newly formed (soluble, immature) collagen. These results suggest that lysosomal mechanisms play a role in the degradation of collagen produced by CaEDTA.