Figure 1. The concentration of lead in whole blood of the dosed dam and her calf over the entire sampling period.
S.L. Jeffrey, S.M. Whitaker, D.C. Borger, and L.B. Willett
Department of Animal Sciences
Lead is the environmental toxicant most frequently encountered by cattle. An understanding of how lead moves through the bodies of exposed animals helps minimize risk to livestock and reduces contamination of human foods derived from cattle. The amount and behavior of lead transferred from the cow to the calf previously has not been determined. In this study a pregnant Jersey cow was dosed with one gram of lead for the last 15 days of pregnancy. As intended, this dosage of lead was not toxic to the cow or her calf. Concentrations of lead in the blood of the cow and her calf were monitored. During dosing the lead concentration in the cow reached equilibrium approximately 150 hours before parturition. The lead disappeared from the blood of the cow with a half-life (t1/2beta) of 148 hours. Initially, disappearance of lead from the calf was more rapid than from the cow (t1/2alpha = 22 hours), with a terminal disappearance (t1/2beta) of 223 hours. Clearly, lead does cross the placenta to expose the fetus to this toxicant. The mechanisms of transfer are worthy of additional study.
Lead poisoning is the most common environmentally induced disease in the United States today (Landrigan and Todd, 1994). Not only is lead harmful to humans, but it also is harmful to domestic animals such as cattle, pigs, and horses. Studying lead and its effects on cattle is economically important to livestock producers. Once producers know how much lead is toxic to their cattle and the ways in which lead is encountered, they can begin to take preventative measures to reduce further exposure. Because lead is cumulative in body tissue and is toxic in sufficient quantities, preventing further lead exposure would be beneficial.
Once cattle have been exposed to lead, it is important to determine the rate of disappearance. Pharmacokinetic analyses can be used to predict hazardous exposure and the effectiveness of measures to limit livestock exposure or food contamination. Several studies have been conducted to determine the kinetics of lead. They indicated that disappearance rates were dependent on time of exposure, amount of exposure, and the number of previous exposures. Kinetic analyses also have helped predict what food products from lead-tainted animals may be a risk as a source of lead exposure to humans. For example, studies have indicated that a portion of lead consumed by cattle accumulated in muscle (Tahvonen and Kumpulainen, 1994) with some also in milk (Blanford et al., 1996). Knowing the kinetics of lead also allows the prediction of how long contaminated animals may need to be kept off the market after exposure to allow excretion of the metal.
Although several human studies have been conducted on the transfer of lead from mother to fetus via the placenta, few studies of this sort have been performed on animals. Because lead may be transferred to the calf, it is important to evaluate the amount of exposure prior to birth, the mechanisms of transfer, and the long term consequences of in utero exposure. Understanding the transfer kinetics will help develop strategies to protect the fetus.
This study was a preliminary experiment to determine the amount of maternal lead that enters the blood of the calf and to compare the disappearance of lead between dam and calf. This information is basic to the development of studies to define the mechanisms of lead transfer from dam to fetus.
This initial approach to understand lead transfer was to determine the pharmacokinetics of a lead-dosed pregnant cow and her calf. To accomplish this, it was essential to determine how much of a known quantity of lead passed through the dam's placenta to the calf and how much of that lead was associated with the erythrocytes. The study was performed on a pregnant Jersey cow born and raised at the Ohio Agricultural Research and Development Center (OARDC) in Wooster, Ohio. This animal had spent its entire life as a herdmate with other animals that were intensively studied for environmental lead exposure. Basal blood lead concentration for this cow was similar to that of herdmates (Blanford et al., 1996). The whole blood lead concentration of the sample taken prior to the first lead dose represented the basal blood lead concentration.
To measure the specific amount of lead transferred from the dam to her unborn calf, the dam was dosed daily for 15 days with a gelatin capsule that contained the lead dose. Each capsule contained a mixture of 1.55 g of lead acetate and 10.45 g of finely ground corn mix and was administered orally using a stainless steel balling gun. This dose yielded 1.0 g of lead per day. Daily dosing was continuous from the last 15 days of gestation until the time of parturition. After birth, the calf was immediately removed from her mother and fed colostrum and milk from a cow that was not dosed with lead.
During dosing, blood samples were collected at two-day intervals for lead determinations. After parturition, samples of blood were collected from both the dam and calf at 0.5, 2, 4, 8, 16, and 24 hours, then daily on days 2, 3, 4, 5, 6, 7, 10, and 14.
All blood samples were taken from the jugular vein using a 22 gauge needle and collected in 15 ml heparinized Vacutainer tubes. From each sample, 4 ml of whole blood were pipetted into labeled 5 ml polyethylene tubes and frozen at -20oC. The remaining blood was centrifuged to determine packed cell volume, and the plasma was pipetted into labeled 5 ml polyethylene tubes and stored in the freezer at -20oC. The remaining red cells were washed with an equal volume of 0.9% physiological saline, and the washed red cells were pipetted into polystyrene tubes and frozen at -20oC.
The blood assay procedure used in this study was obtained from The Wadsworth Center Lead Poisoning Laboratory, New York State Department of Health (Albany, NY) (Parsons, 1991). The matrix modifier consisted of 10% Triton X-100 (New England Nuclear, Boston, MA), concentrated (69.0 to 71.0%) nitric acid (Sigma, Fair Lawn, NJ), and 20% ammonium dihydrogen phosphate (Sigma, St. Louis, MO). The lead concentrations of whole blood, plasma, and red cells were measured on a Perkin Elmer 3030B atomic absorption spectrophotometer with an HG400 graphite furnace and 400-G automatic sampler. As recommended by similar lead studies (deSilva, 1981), the red cell lead concentrations were corrected for hematocrit and compared to the whole blood lead concentration. Curve peeling analysis, using the program Peel 2, was performed on the whole blood lead concentrations of the dosed dam and calf to calculate disappearance rates of lead from the blood.
No signs of lead toxicity were apparent during and after the dosing of the cow or calf despite the chronic exposure of 15 g of lead as acetate. The cow's behavior, weight, and eating habits remained stable. The calf also did not exhibit signs of toxicity during the data collection period. The calf exhibited normal behavior and weight increase. Over the 15-day period, the calf gained 1.8 kg.
Environmental lead uptake from feed and water was derived using data from another lead study (Blanford et al., 1996) conducted at the OARDC for which comparable feeds were measured (Table 1). Therefore, intake of environmental lead by the Jersey cow was estimated as 17 mg/day.
Whole blood, red cells, and plasma were analyzed individually on the graphite furnace atomic absorption spectrophotometer. Lead concentrations of plasma were too low for accurate comparisons between dam (< 20 ng/ml) and calf (< 10 ng/ml). Lead concentrations in most plasma samples were very close to the limit of analytical sensitivity of the instrument used.
The lead was associated with the red blood cells of both the cow and her calf. In this study, whole blood was a more reliable measure of blood lead content than were the harvested and washed red blood cells. Variability in the lead content of red cells was probably a reflection of the additional
manipulation of the red cells plus storage in polystyrene tubes, which may irreversably bind a portion of the lead. When data were corrected for hematocrit content, the coefficient of determination (r2) for the lead content of whole blood and red cells was 0.61 for the dam and 0.77 for the calf.
| Table 1. The lead concentration in the feed components and water consumed by cattle at the Ohio Agricultural Research and Development Center. | |||
| Feed component |
Lead
(micro-g/g) |
Proportion of ration | Lead
contribution to feed
(micro-g/g) |
| Corn silage | 0.352 | 0.40 | 0.141 |
| Grain mix | 0.792 | 0.37 | 0.293 |
| Alfalfa | 0.540 | 0.09 | 0.049 |
| Alfalfa hay | 0.492 | 0.14 | 0.069 |
| Total | . . . | . . . | 0.552 |
| Water | 0.070 | . . . | . . . |
The whole blood concentration of lead of the dam prior to dosing was 60 ng/ml and steadily increased once dosing began. Steady state, or equilibrium, was reached approximately 150 hours before birth (Figure 1). The lead concentration reached its peak of 485 ng/ml 0.5 hours after parturition. Dosing ceased with parturition, and whole blood concentrations of lead of the dam decreased rapidly. The final sample, taken 328 hours after birth, contained a lead concentration of 110 ng/ml (Figure 1).
At 0.5 hours after parturition before colostrum feeding, the lead concentration in whole blood of the calf was 375 ng/ml (Figure 1). The concentration of lead in whole blood of the calf was 77.3% that of the dam. The lead in the blood of the calf also decreased after birth and at 328 hours was 80 ng/ml, approximately 72.7% that of the dam.
The disappearance of lead from whole blood of the dam and calf are shown in Figures 2 and 3. Whole blood lead in the dam had a first-order disappearance of t1/2beta = 148 hours (Figure 2). The lead concentration of whole blood in the calf exhibited two disappearance curves, an initial () and terminal () disappearance of lead from whole blood. The t1/2 = 22 hours and t1/2 = 223 hours (Figure 3).
Figure 1. The concentration of lead in whole blood
of the dosed dam and her calf over the entire sampling period.
Figure 2. The first-order disappearance of lead in
whole blood of the dosed dam beginning at parturition and continuing
until the end of the sampling period (r2 = 0.973).
Cr = whole blood concentration of lead (ng/g); B = zero time
intercept of the terminal disappearance; beta = rate constant for the
terminal disappearance; t1/2beat = half-life of lead
disappearance.
Figure 3. The first-order disappearance of lead in
whole blood of the calf of the dosed dam beginning at parturition and
continuing until the end of the sampling period (r2 = 0.889).
Cr = whole blood concentration of lead (ng/g); A = zero time
intercept of the initial disappearance; B = zero time intercept of the
terminal disappearance; alpha = rate constant for the initial disappearance;
= Rate constant for the terminal disappearance; t = time in hours;
t1/2 = half-life of lead disappearance.
The results of this study supported the hypothesis that lead is transferred from dam to calf. Although there is no existing literature discussing this topic for cattle models, similar theories have been established in both rodent and human studies (McClain and Becker, 1974; Kimmel et al., 1980; Gulson et al., 1995).
The daily lead dose of 1.0 g of lead as acetate (approximately 2.0 mg/kg body weight) did not produce toxic effects on either the cow or calf. According to Hammond and Aronson (1964), the approximate cumulative fatal dose of lead for cattle is 6 to 7 mg/kg. Several studies have been conducted on the amount of lead necessary for acute lead toxicity in calves. Allcroft (1951) found that 200 to 400 mg/kg was lethal to calves fed lead-burdened paint, carbonate, oxide, or phosphate, whereas Buck (1975) determined the dose to be 400 to 600 mg/kg. Because of the rapid growth rate of calves, it may be difficult to estimate the cumulative toxic dose of lead for calves. The work of Allcroft (1951), Buck (1975), and Logner (1984) differed from this study in that they dosed the calves themselves, whereas the calf in this study obtained lead from the mother.
This study showed that, even with a significant daily dose of lead, plasma was below the limits of sensitivity of the graphite furnace and therefore could not be used as a reliable estimate of the lead burden in the blood of the cow and calf. Lead concentrations in plasma only represented a small fraction of the total blood lead burden and have been reported to have a much shorter disappearance rate than other components of the blood (deSilva, 1981). Because 90 to 95% of blood lead was stored in the erythrocytes (Skerfving et al., 1993; Landrigan and Todd, 1994), red cells were a good indicator of the lead burden in blood. The disappearance rate of lead in red cells, 36 + 5 days (Landrigan and Todd, 1994), was much longer than that of plasma. However, the data from this study indicated the lead content of red cells was not as reliable as whole blood. Whole blood concentrations of lead linearly increased with dosing and steadily decreased after dosing, whereas red cell concentrations of lead followed a similar increasing and decreasing trend but were not linear. One reason for this may have been the difference in cell manipulation between whole blood and erythrocytes. The whole blood was removed directly from the collection tube and placed in the freezer, whereas the red cells were centrifuged twice and washed with saline. These extra steps may have influenced the distribution of lead in the sample. Therefore, because the most accurate values were found in the whole blood, whole blood concentrations of lead were used for the comparison of distribution of lead between dam and calf.
This study has established that lead was transferred from the dam to the calf, although the mechanisms of how the lead was transferred are still in question. Studies of the bovine placenta indicated that only certain substances such as some nutrients from the dam and waste products can penetrate the five cell layers separating the dam from the calf. Lead did cross the placenta to the fetus, yet the mechanism remains unclear. Further analysis of the lead composition of the placenta itself may be helpful. Red blood cells do not exchange between the dam and fetus, so exchange of plasma components may be important. This, too, has not been demonstrated and is an area for future study.
When the calf was born, dosing of the dam ceased, and the concentration of lead began to decrease. The whole blood concentration of lead was the highest for both dam and calf 0.5 hours after birth with concentrations of 485 ng/ml and 375 ng/ml, respectively. The sample taken two hours after birth indicated that the concentration of lead had already begun to decrease. Not only was this due to the completion of dosing, but it also may be related to physiological and metabolic changes as a result of the stress of birthing.
The final blood samples of both dam and calf were taken 328 hours after parturition. The whole blood concentrations of lead in the dam and calf were 110 ng/ml and 80 ng/ml, respectively. Although the sampling was discontinued, blood concentration of lead in the dam probably would continue to decrease until reaching a basal concentration of approximately 60 ng/ml, which should be in equilibrium with environmental exposure. The environmental exposure of the calf was unknown because the lead content of the whole milk fed was not determined.
In order to determine the quantities of lead transferred to calves of typical environmentally exposed cows, another study is ongoing. The kinetics of lead in 27 cows and their calves currently is being determined.
The authors wish to thank OARDC Krauss Dairy Center employees John Durst, Alan Griffiths, Nancy Oliver, Kevin Synder, and Ken Wise for their animal assistance and student colleagues Joe Allen, Heather Keller, and Rachel Kosa. This work was the basis for the College of Wooster Independent Study Thesis of S.L. Jeffrey.
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