Figure 1. Lood lead concentrations during the entire study. The shaded boxes
represent the two chelation trials.
S. L. Jeffrey
D. C. Borger
L. B. Willett 1
The Ohio State University
Department of Animal Sciences
1 For more information, contact at: The Ohio State University, Ohio Agricultural Research and Development Center, 128 Gerlaugh Hall, 1680 Madison Avenue, Wooster, OH 44691; 330-263-3792; willett.2@osu.edu
According to a survey by the Environmental Protection Agency, 150,000 bovine animals are exposed to toxic concentrations of lead yearly, of which 20,000 die. An effective and practical treatment for mediating the effects and body burdens of lead in food-producing animals is needed. The preferred drug for treatment of lead-exposed cattle has been calcium disodium ethylenediamine tetraacetate (EDTA). Toxic effects related to nonspecific chelation and relative ineffectiveness suggested an alternative agent be sought. Oral meso-2,3-dimercaptosuccinic acid (DMSA) is the preferred drug for humans but would probably not survive rumen fermentation. Intravenous DMSA administrations have been effective chelators in experimental animals, so they may work in cattle. Preliminary experiments with intravenous administrations of DMSA in cattle produced decreased blood calcium concentrations (hypocalcemia) and renal failure. The role of calcium on the tolerance and effectiveness of DMSA in cows chronically exposed to lead was evaluated. Four lactating Jersey cows were dosed daily with 1.0 grams of lead for 30 days. As intended, this dosage of lead was not toxic to the cows. On experimental days 14 and 28, cows were allotted one intravenous dose of 0, 10, 20, or 40 mg/kg body weight DMSA and 0 or 11 grams of calcium. No signs of renal failure or hypocalcemia were observed. No lead was detected in the urine of the cows outside the chelation trials. However, DMSA promoted the excretion of urine lead for 20 hours following the DMSA administration. Transient 30 to 50% declines in blood lead concentrations were also observed with the administration of DMSA. Calcium did not affect the activity of DMSA on lead chelation. The DMSA is beneficial for chelating lead, but sustained administration of DMSA may be needed to remove sufficient quantities of lead.
Lead poisoning is the most common environmentally induced disease in the United States today (Landrigan and Todd, 1994). Lead is toxic to both humans and domestic animals, including cattle, pigs, sheep, and horses. In order to reduce the effects of lead poisoning, the evaluation of chelation procedures is important. Chelating agents are compounds to which a heavy metal, such as lead, will bind so that the metal can be eliminated from the body. These agents have been used for the past 40 years in human and rat models with varying degrees of success, but little progress has been made with chelators in cattle.
The current drug of choice for treatment of lead-exposed cattle is calcium disodium ethylenediamine tetraacetate (EDTA). However, this drug is a nonspecific chelator and will promote the urinary excretion of essential metals such as zinc. In 1991, meso-2,3-dimercaptosuccinic acid (DMSA) was licensed by the Food and Drug Administration for the reduction of blood lead in humans without producing the toxic effects observed with other chelators. Such observations make DMSA worthy for study in lead-burdened cattle.
Preliminary studies of the effects of DMSA in cattle indicated very low concentrations of DMSA administered to lead-dosed cattle had little effect on lead concentrations, whereas concentrations of DMSA similar to those used in other species produced renal failure and symptoms of hypocalcemia in cattle with lead concentrations similar to those found in the environment (Blanford et al., 1997). The objectives of this study were to evaluate the role of calcium on the tolerance and effectiveness of DMSA in cows chronically exposed to lead.
Four Jersey cows were selected to have similar body weights and milk production. These animals were raised in the Krauss Dairy Center at The Ohio State University's Ohio Agricultural Research and Development Center (OARDC) in Wooster, Ohio, with other cattle that have been intensively studied for environmental lead exposure. Throughout the experimental period, the cows were managed individually so that feed intake and milk production could be determined daily.
In order to adequately evaluate the effectiveness of DMSA and supplemental calcium on chelating lead, the cows received a daily gelatin capsule containing 1.83 grams of lead acetate, and 4.17 grams of finely ground corn served as a carrier for the lead acetate for 30 days. Each bolus delivered 1.0 grams of lead, which has previously been shown to not induce toxic effects in cattle (Aronson et al., 1968; Jeffrey, 1996).
Grab samples of blood, urine, and feces were collected daily for the first seven days and then on days 9, 11, 13, 17, 19, 21, and 24 to monitor lead concentrations. Milk weights were recorded, and all milk was discarded. During the two chelation trials, frequent blood samples and health observations were measured along with complete collections of urine and feces. Once lead dosing ceased on experimental day 30, grab samples of blood, urine, and feces were taken at various times for the following 47 days. Blood serum samples were analyzed for calcium, while lead concentrations in whole blood, urine, and feces were determined.
The DMSA (Aldrich, Milwaukee, Wisc.) was weighed and dissolved in sterile 5% sodium bicarbonate solution to make 400 ml. The sham contained only 500 ml of 5% sterile sodium bicarbonate solution. Cal-Dextro C (Fort Dodge Laboratories, Fort Dodge, Ind.) was the calcium source and provided 11 g of calcium in 500 ml of 15% sterile dextrose solution. The sham was 500 ml of 15% sterile dextrose solution. The treatments allotted to each cow are located in Table 1. The one-time dosages of DMSA and calcium were administered on days of 14 and 28 during Trials 1 and 2 respectively.
|
Table 1. The Treatments Administered to the Experimental Cows During the Two Chelation Trials. | ||
|---|---|---|
| Cow Number | DMSA (grams) | Calcium (grams) |
| Trial I | ||
| 3938 | 0.00 | 0 |
| 3539 | 4.78 | 0 |
| 3408 | 0.00 | 11 |
| 3643 | 4.08 | 11 |
| Trial II | ||
| 3938 | 9.34 | 11 |
| 3539 | 18.52 | 11 |
| 3408 | 9.56 | 0 |
| 3643 | 15.96 | 0 |
The blood and urine assay procedures used in this study were obtained from Varian (Flajnik and Shrader, 1993a; Flajnik and Shrader, 1993b). The matrix modifier for both blood and urine samples consisted of 0.56% Triton X-100 (New England Nuclear, Boston, Mass.), 0.25% ammonium phosphate (Lab Chem, Inc., Pittsburgh, Pa.), and 0.2% nitric acid. The lead concentrations of both blood and urine were determined using a Varian SpectraAA 200 Atomic Absorption Spectrophotometer Graphite Furnace (Mulgrave, Victoria, Australia). The dried feces samples were ground, digested, and measured for lead content by an Inductively Coupled Spectrograph.
Calcium concentrations were determined using a Varian SpectraAA 200 Flame Atomic Absorption Spectrophotometer (Mulgrave, Victoria, Australia). The serum samples were diluted 1:50 with 0.1% lanthanum chloride solution.
Milk production followed that of a typical lactation curve, indicating that the cows were not adversely affected by the daily lead doses. Transient declines in milk production prior to each chelation trial were indicative of experimental manipulation of the animals.
Clinical chemistry parameters and skin and rectal temperatures were used as indicators of the clinical health of the cows. Results from the clinical chemistry measurements revealed no renal failure in any of the cows. Based on the observations of Blanford et al. (1997), clinical signs of hypocalcemia in cows receiving DMSA were expected. Subnormal skin temperatures with normal to subnormal rectal temperatures are primary characteristics of hypocalcemia. The administration of DMSA and (or) calcium did not produce declining skin temperatures. Instead, transient increases in both the skin and rectal temperatures were observed in control and DMSA-dosed cows. The DMSA did not appear to induce hypocalcemia in the cows of this study.
Further evidence that DMSA did not produce hypocalcemia in cows was provided by the serum calcium concentrations. As expected, the cows receiving 11 grams of calcium had increasing serum calcium concentrations, but they returned to normal by the end of the 48-hour sampling period. The DMSA did not appear to have an effect on the serum calcium concentrations in either chelation trial because little change in the serum calcium concentrations was observed among control and DMSA-dosed cows.
Figure 1 shows the changes in blood-lead concentrations of the four cows over the 71-day experimental period. The rates at which blood-lead concentrations were increasing prior to the chelation trials suggest the cows were close to steady state, or equilibrium, with the lead in the various pools in which it accumulates. Once a cow attains steady state with a given amount of a substance, the concentrations of that substance in the body will remain the same. During the two chelation trials, the DMSA promoted transient declines in blood lead concentrations. Cows that received the 20 and 40 mg/kg body weight of DMSA had greater transient decreases in blood lead concentrations (30 to 50%) than cows that received 10 mg/kg body weight of DMSA. Declines in blood lead concentrations began once daily lead dosing ceased on experimental day 31.
Figure 2 shows the changes in urine lead concentrations over the entire experimental period. The DMSA promoted the excretion of lead with the urine. However, without DMSA, urine did not appear to be an important excretion route for lead. Based on the complete collections of urine from cows who did not receive DMSA in the first chelation trial (3938 and 3408), the daily excretion of lead was 1.25 mg. The concentrations of lead per ml of urine were similar to the pre- and post-chelation trial periods. During the first chelation trial, lead excretion for cows receiving DMSA peaked within the first two hours after the administration of DMSA and declined quickly thereafter. Clearly, the effectiveness of the four to five grams of DMSA were of short duration with urine lead concentrations approaching predose concentrations within eight hours after the DMSA was administered. During the second chelation trial, greater urine lead concentrations were observed. Again, the effectiveness of nine to 19 grams of DMSA were of short duration with the urine lead concentrations approaching predose concentrations 12 hours after the DMSA was administered. Only small percentages of the total accumulated lead dose were excreted with urine and feces (Table 2).
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Table 2. Amounts of Lead Excreted with Urine and Feces and the Percent of the Total Administered Lead Dose Excreted with Urine and Feces During the Two 48-Hour Chelation Trials. A Total of 16 and 30 Grams of Lead Were Administered by the End of the First and Second Chelation Trials, Respectively. | ||||||
|---|---|---|---|---|---|---|
| Cow | DMSA | Calcium | Excreted Lead in Urine | Excreted Lead in Feces | Lead Dose in Urine | Lead Dose in Feces |
| Trial I | g | g | mg | g | % | % |
| 3938 | 0.00 | 0 | 3.08 | 1.58 | 0.019 | 9.904 |
| 3539 | 4.78 | 0 | 14.91 | 1.45 | 0.093 | 9.052 |
| 3408 | 0.00 | 11 | 1.90 | 1.49 | 0.012 | 9.330 |
| 3643 | 4.08 | 11 | 26.91 | 1.34 | 0.168 | 8.352 |
| Trial II | ||||||
| 3938 | 9.34 | 11 | 53.66 | 1.65 | 0.179 | 5.507 |
| 3539 | 18.52 | 11 | 43.76 | 1.26 | 0.146 | 4.203 |
| 3408 | 9.56 | 0 | 53.14 | 0.75 | 0.177 | 2.510 |
| 3643 | 15.96 | 0 | 57.12 | 1.78 | 0.190 | 5.928 |
During both chelation trials, calcium did not appear to affect the chelation of blood or urine lead. Both the blood and urine lead concentrations themselves and the fluctuations of blood and urine lead concentrations observed during the two chelation trials differed among cows that received calcium.
Although lead has been recognized as a hazard in the environment and human food chain for centuries, regulation and use restrictions have not eliminated this element of concern in the general population and food-producing animals. Botts (1977) has estimated that more than 150,000 bovine animals are exposed to toxic amounts of lead annually. An effective and practical treatment for mediating the effects and body burdens of lead in food-producing animals is needed. The current drug of choice for treatment of lead-exposed cattle is EDTA. However, the toxic side effects related to nonspecific chelation (Aposhian and Aposhian, 1990; Chisolm, 1990; Goyer et al., 1995) and relative ineffectiveness suggest that an alternative agent be sought.
Oral preparations of DMSA are the current drug of choice for humans, but these compounds would probably not survive rumen fermentation. Intravenous administrations have been effective chelators in experimental animals, so they may work equally well in cattle. Unfortunately, preliminary experiments suggested that DMSA may produce hypocalcemia and renal failure (Blanford et al., 1997). Additional research was warranted. The objectives of this study were to evaluate the role of calcium on the tolerance and effectiveness of DMSA in cows chronically exposed to lead. Such information is beneficial to consumers, agriculturalists, and to the well being of lead-burdened animals.
In contrast to the report of Blanford et al. (1997) where cows exposed to background concentrations of environmental lead were intoxicated by intravenous doses of DMSA, the lead-burdened cows in this study that received an intravenous administration of 10, 20, or 40 mg/kg DMSA with no calcium supplement did not experience renal failure or produce clinical signs of parturient paresis. In addition, calcium supplements did not affect the activity of DMSA on the chelation of lead. Therefore, the administration of DMSA with no calcium supplement to lead-burdened dairy cows was a beneficial method for chelating lead. This study has raised the interesting concept that a source of chelatable metal needs to be present to protect the animal from toxicity derived from the DMSA as the animals that were intoxicated averaged 60 ng/ml of blood lead compared to 400 to 600 ng/ml in the present study.
The results from this study revealed that 20 and 40 mg/kg of infused DMSA were effective in chelating lead. The administration of DMSA promoted the excretion of lead through the urine, but only a small percentage (< 1%) of the accumulated lead dose was associated with the DMSA chelations. In addition, a transient 30 to 50% decrease in blood-lead concentrations was observed with doses of 20 and 40 mg/kg DMSA shortly after infusion. As might be expected, the blood-lead concentrations rebounded because the total amount of lead actually cleared in urine was so small. Continued infusions, repeated doses, or a slow release vehicle for the DMSA may be needed to prolong the excretion of lead.
The results from this study provided evidence that further study of DMSA as an alternative to EDTA for chelating lead in cattle is worthwhile. As long as the problems of renal failure and hypocalcemia do not occur with DMSA, the toxicoses associated with mineral imbalances induced with EDTA can be avoided.
The authors wish to thank OARDC Krauss Dairy Center employees John Durst and Kevin Snyder for their animal assistance and colleagues Beth Amiet, Suzanne Whitaker, and Heather Keller. This work was the basis for The Ohio State University Master of Science Thesis of S. L. Jeffrey.
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