- Email: [email protected]
Toxicokinetics of Cadmium in Lactating and Nonlactating Ewes after Oral and Intravenous Administration
ENVIRONMENTAL RESEARCH ARTICLE NO.
72, 140–150 (1997)
ER963690
Toxicokinetics of Cadmium in Lactating and Nonlactating Ewes after Oral and Intravenous Administration P. HOUPERT, B. FEDERSPIEL,
AND
G. MILHAUD
Service de pharmacie et toxicologie, e´quipe associe´e INRA-ENVA Cadmium et Aliment d’Origine Animale, Ecole Nationale Ve´te´rinaire d’Alfort, 7 avenue du ge´ne´ral De Gaulle, 94704 Maisons-Alfort cedex, France Received January 3, 1996
must be quantified. In a previous experiment we studied the toxicokinetics of cadmium in several nonlactating ewes (Houpert et al., 1995). Ewes were selected as a model farm animal because humans consume their meat and milk. Using a toxicokinetic model we estimated absorption and elimination without slaughtering the animals. The present experiment aimed to confirm our previous results and to quantify the elimination of cadmium through milk. Two groups of ewes, a lactating group and a nonlactating group, were kept under similar conditions. We studied the toxicokinetics of cadmium after a single intravenous and a single oral dose of cadmium chloride.
We studied the toxicokinetics of cadmium on two groups of ewes, a lactating group and a nonlactating group, after single intravenous and oral administrations of cadmium chloride using a semisimultaneous method and a three-compartment model. The nonlactating ewes showed a low cadmium bioavailability (0.12–0.22%), a large steady-state volume of distribution (23.8 ± 5.4 liter/kg), and a low blood clearance (0.20 ± 0.03 liter/kg/day). Their mean residence time was 113 ± 28 days. The lactating ewes had a higher bioavailability (0.33–1.7%). Their mean residence time was close to that in nonlactating ewes despite a greater blood clearance (0.46 ± 0.013 liter/kg/day) because the volume of distribution of cadmium in the body was larger (VSS = 48.8 ± 10.3 liter/kg). Their cadmium clearance in milk, changing with time, remained low and could not explain their higher blood clearance. In one nonlactating ewe, a greater cadmium bioavailability (5%) increased cadmium in the body. Increased cadmium amounts could induce renal damage and shorten the mean residence time (78 days). © 1997
MATERIALS AND METHODS
Animals Twelve Lacaune–Prealp cross-breed ewes were used. Throughout the study, the animals’ mean weight was 60 ± 4 kg. Eleven had no clinical troubles. The 12th ewe died a few days after the beginning of the study for reasons unrelated to the study. Six ewes (Ewes 1 to 6) had had their lambs 5 to 9 days before the experiment started, and the other 5 ewes (Ewes 7 to 11) were nonlactating and nonpregnant. The animals were kept in the sheepfold of the experimental farm Brouessy (INRA, France). Every morning, they were given ammoniated straw, dehydrated lucern, lucern hay, and a mineral additive including vitamins dosed according to the physiological state of the animals. They drank water ad libitum. The amounts of cadmium, calcium, zinc, iron, magnesium, manganese, and copper in the diet and the water were determined. The amount of phosphorus in the diet was also determined.
Academic Press
INTRODUCTION
The more cadmium contaminates the agricultural environment, the more it contaminates the food chain and endangers human consumers’ health. Levels in soils and plants are increased by the application of phosphate fertilizers and sewage sludge (WHO, 1992). Cadmium also contaminates tissues and, to a lesser extent, the milk of grazing animals. As a result, humans, exposed mainly through food, can be more and more exposed in the future (WHO, 1992). To reduce contamination of animal products, one objective is to lower cadmium absorption or speed up cadmium elimination in animals. To use this strategy, cadmium absorption and elimination 140 0013-9351/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.
141
TOXICOKINETICS OF CADMIUM IN EWES
Administration of Cadmium The ewes received two successive doses of cadmium chloride (CdCl2, H2O, Merck, Nogent-surMarne, France) at a 21-day interval in a crossover design. One of the doses contained 25 mg/kg of cadmium and was given orally. The cadmium was enclosed in gelatin capsules and placed on the base of the tongue. The capsules were immediately swallowed. The oral dose was given to Ewes 1 to 3 and 10 to 12 on Day 0, and to Ewes 4 to 8 on Day 21. The other dose contained 0.1 mg/kg of cadmium diluted to 1 mg/ml. The solution was then sterilized in an autoclave for 20 min in sealed tubes. The solution was injected in the left jugular vein via a catheter (Intraflon, 2/Vygon). The iv dose was administered to Ewes 4 to 8 on Day 0, and to Ewes 1 to 3 and 10 to 12 on Day 21. Sampling Blood samples of 2 ml each were collected from the right jugular vein into vacutainer tubes guaranteed free of any trace of heavy metals (Ref. 367735 Vacutainer tubes, Beckton–Dickinson, Mayland, France). These tubes were then stored at 4°C. On the days that iv injections were given (Day 0 and Day 21), blood was collected 2, 4, 8, 15, and 30 min and 1, 2, 3, 4, and 6 hr after the injection. On the days that cadmium was administered orally (Day 0 or Day 21), the blood was collected 5, 10, 15, and 30 min and 1, 2, 3, 4, and 6 hr after the administration. The blood of the six ewes receiving first the iv injection was collected 1, 2, 3, 6, 9, 13, 16, 20, 21, 22, 23, 24, 27, 30, 34, 37, 41, 48, 62, 76, 90, 106, 120, 134, 148, 181, 212, 241, and 272 days after the injection. The blood of the five ewes receiving first the oral administration was collected 1, 2, 3, 4, 7, 10, 14, 17, 21, 22, 23, 24, 25, 28, 31, 35, 38, 42, 49, 63, 77, 91, 107, 121, 135, 149, 182, 213, 242, and 273 days after the administration. Milk was collected the same day blood was collected until the ewes naturally stopped giving milk, i.e., 150 to 180 days from the beginning of the experiment. Milk was homogenized and samples (50 ml) were collected and stored in polyethylen flasks at 4°C until cadmium was dosed in the following 24 hr. The daily volume of milk was measured. Cadmium Measurement The samples were first diluted 1:10 for blood and 1:5 for milk with 0.05 N nitric acid in the presence of Triton. The cadmium concentrations in blood and
milk were then measured directly by graphite furnace atomic absorption spectrometry with a Perkin– Elmer 1100B spectrophotometer AGA 700 according to the technique recommended by Lauwerys et al. (1990). The decomposition temperature was 550°C for blood and 600°C for milk; the atomization temperature was 1450°C. The limit of detection was estimated to be 0.3 mg/liter, the limit of quantification 0.8 mg/liter. The intraday coefficient of variation was 25% at 0.8 mg/liter, 13% at 2 mg/liter, 2% at 5 mg/liter in blood and 6% at 1 mg/liter, 5% at 5 mg/liter in milk. The accuracy of the method was controlled with certified material: BCR194: 0.5 ± 0.4 vs 0.5 ± 0.1 mg/liter, BCR195: 5.4 ± 0.5 vs 5.37 ± 0.24 mg/liter; for blood; CRM063: 2.8 ± 1.2 vs 2.9 ± 1.2 ng/liter, CRM150: 21.8 ± 1.6 vs 21.8 ± 1.4 ng/liter for milk of BCR (Commission of European Communities, Community Bureau of Reference, Brussels, Belgium). Toxicokinetic Analyses The toxicokinetic analyses were carried out via a nonlinear regression program adapted from Multi (Yamaoka et al., 1981). The following general polyexponential equations were adjusted to the data set using the superposition principle, according to the semisimultaneous administration method (Wijnand, 1992),
(FY ·e n
C~t! =
F(
−lit
i
+
i=1
n
−
ka · Yi F · Dpo −l T e ka − li Div i
ka · Yi F ? Dpo Div i=1 ka − li
G
G
? e−k T,
(1)
a
when the first administration was an iv injection, and
(FY ?e n
C~t! =
F(
i=1
n
−
i=1
i
−liT
+
ka ? Yi F ? Dpo −l t e ka − li Div
ka ? Yi F ? Dpo ka − li Div
i
G
? e−k t, a
G (2)
when the first administration was given orally. In these equations, T 4 0 if t < 21 and T 4 t-21 if t > 21, c is the cadmium concentration in blood at time t, Y1 is the coefficient of the ith exponential term li, ka is the first-order absorption constant, F is the bioavailability, and Dpo/Div is the ratio of oral to iv administrations. The initial parameters were obtained using linear regression methods from iv administration data. The number of necessary exponents (2 or 3) was defined by the application of
142
HOUPERT, FEDERSPIEL, AND MILHAUD
Akaike’s information criterion (Yamaoka et al., 1978). The volume of the central compartment (Vc) was obtained using the equation Vc =
Div
.
n
(Y
(3)
milk on Day d, C(d) is the cadmium concentration in blood on Day d, and W is the body weight. Maximum clearance in milk (Clm(max)) was the highest value of Clm(d). The mean clearance in milk (Clm) was calculated as Clm 4 Qm/AUC z W,
i
(10)
i=1
The steady-state volume of distribution (Vss) was obtained using the equation Vss 4 Vc(1 + k12/k21 + k13/k31),
(4) Statistical Analyses
where k12, k21, k13, and k31 represent the first-order distribution rate constants between compartment 1 and compartments 2 and 3, respectively. The mean residence time after iv administration (MRT) was obtained using the equation n
MRT =
(
Yi
i=1
/l2i
n
( Y /l
.
(5)
i
The mean residence time in the central compartment (MRTc was obtained using the equation MRTc 4 1/k10,
(6)
where k10 is the first-order elimination rate constant from compartment 1. The blood clearance (Cl) was calculated from the equation Cl =
Div n
( Y /l
.
(7)
i
i=1
Student’s t test was used to get an overview of significant differences between the groups (lactating and nonlactating) for the kinetic parameters. RESULTS
Daily Mineral Intake
i
i=1
where Qm is the total cadmium excreted into milk, W is the weight of the ewe, and AUC is the area under the blood concentration–time curve calculated by trapezoidal rule from Time 0 to the end of lactation.
i
The cadmium amount excreted in milk on Day d was calculated using the equation Qm(d) 4 Cm(d) z Vm(d),
(8)
where Cm(d) is the cadmium concentration in milk on Day d and Vm(d) is the milk volume produced on Day d. The clearance in milk on Day d was calculated using the equation Clm(d) 4 Qm (d)/C(d) z W,
(9)
where Qm(d) is the cadmium amount excreted in
The daily mineral intake is shown in Table 1. Cadmium content of the diet was below 5 mg/kg/day. Calcium content was 20 to 44 g/day, depending on the physiological state of the animals. Cadmium Levels in Blood Cadmium concentrations in the blood and in the milk of the ewes before the first cadmium administrations were below the limit of quantification of the analytical method (0.8 mg/liter). After cadmium was administered orally on Day 0, levels in blood differed widely among animals. They could be measured 5 min to 3 hr after administration and they reached maximum levels, ranging from 2.5 to 80 mg/liter, on the first or second day. Levels then slowly decreased until the 21st day, but remained above the quantification limit. The blood cadmium levels in nonlactating ewes (Ewes 10 and 11) were similar to those of lactating ewes (Ewes 2 TABLE 1 Total Mineral Intake per Day during the Study
Cadmium (mg) Calcium (g) Phosphorus (g) Zinc (mg) Iron (mg) Magnesium (g) Manganese (mg) Copper (mg)
Nonlactating ewe
Start of lactation
End of lactation
0.15 20.3 4.05 150 437 2.65 157 15.1
0.29 43.6 8.07 181 1086 5.29 231 22.6
0.23 29.6 5.85 164 727 3.88 192 18.6
TOXICOKINETICS OF CADMIUM IN EWES
and 3; Figs. 1A and 1B). The blood cadmium level of lactating Ewe 1 was substantially higher. After cadmium was injected on Day 21 levels in blood were more similar in all animals, as compared to oral administration on Day 0. Its levels rose rapidly, reaching maximum peaks of 350 to 950 mg/liter. They then decreased rapidly in the first hours and
143
then more slowly in the following days and weeks. The average cadmium concentrations observed in nonlactating ewes were within the range of to those observed in lactating ewes. The cadmium levels in blood observed in the five ewes receiving an iv injection on Day 0 were very similar to those observed in the ewes receiving the iv
FIG. 1. (A) Average and (B) individual cadmium levels observed in blood after oral administration (on Day 0) and iv administration (on Day 21) of cadmium in nonlactating and lactating ewes.
144
HOUPERT, FEDERSPIEL, AND MILHAUD
injection on Day 21 (described above). After the iv injection, the cadmium levels of the lactating ewes were below those of the nonlactating ewes. After cadmium was administered orally on Day 21, the cadmium levels in the blood of one nonlactating ewe (Ewe 7) were much higher than those of the other ewes (Figs. 2A and 2B); in the latter the difference of blood cadmium levels between lactating and nonlactating ewes was very weak (in lactating ewes they remained slightly below those in the nonlactating ewes). Amount of Cadmium Eliminated through Milk After cadmium was orally or intravenously administered, its levels in milk increased rapidly and could be detected as soon as the evening milking (6 hr after administration). Levels in milk reached a maximum value on the second or third day after administration (Fig. 3). These values then decreased, quickly at first and then slowly. Seventy days after the second administration, 1.1 ± 0.8 mg/ day of cadmium was being excreted in milk. Pharmacokinetic Parameters According to Akaike’s criterion, the disposition of cadmium in all ewes was best modeled by a triexponential equation corresponding to a three-compartment open model (Fig. 4). The average toxicokinetic parameters after the analysis of blood concentrations are listed in Table 2 for the six lactating ewes and the four nonlactating ewes. The parameters of nonlactating Ewe 7, whose cadmium levels were very different from those of the other ewes, are given separately. The bioavailability (F) of cadmium given orally to the lactating ewes was 0.51, 0.36, and 0.33% with the iv preadministration and 1.7, 0.59, and 0.44% without it. In the nonlactating ewes, it was 0.12 and 0.19% with the iv preadministration and 0.22 and 0.20% without it. In Ewe 7, it was 5%. The ratio of distribution constants between the central compartment and the deep compartment (k13/k31) was 272 ± 176 in the nonlactating group, 493 ± 106 in the lactating group, and 923 in Ewe 7. The steady-state volume of distribution ranged from 19.4 to 31.6 liter/ kg in the nonlactating group and from 37.3 to 62.3 liter/kg in the lactating group. The blood clearance was 0.2 ± 0.03 liter/kg/day in the nonlactating group and 0.46 ± 0.13 liter/kg/day in the lactating group. It reflected respectively, 0.18–0.25 and 0.38–0.67% of the cardiac flow (0.37% for Ewe 7). The mean residence time in the central compartment (MRTc) was 0.22 ± 0.06 days in the
lactating ewes and 0.59 ± 0.37 days in the nonlactating ewes (0.08 day in Ewe 7). The mean residence time in the body (MRT) varied slightly according to the physiological state of the animals. The total amounts of cadmium eliminated through milk during lactation ranged from 175 to 900 mg. The clearance in milk was not linear (Fig. 5); it reached a maximum value 7 to 14 days after the first cadmium administration, whatever the administration route. The maximum value (Cl m(max) ) ranged from 0.03 to 0.08 liter/kg/day (Table 3). It decreased rapidly in the following 2 to 3 weeks and then decreased slowly. The mean clearance in milk (Clm) was calculated over the entire lactation period. It ranged from 0.0066 to 0.0237 liter/kg/day. DISCUSSION
We studied the toxicokinetics of cadmium after single iv and single oral bolus doses using the semisimultaneous or sequential method (Wijnand, 1992). Because cadmium is eliminated slowly (Robards and Worsfold, 1991; WHO, 1992), the sampling period following an administration and the washout intervals between two administrations are too long to determine the cadmium bioavailability with a classical method. Using the semisimultaneous method, we could administer the iv and oral doses with a 21-day interval and calculate all the kinetic parameters. We chose to give single doses of cadmium, although environmental exposures do not occur in single doses, because this permitted us to administer the oral and the iv boluses in comparable situations. Although the oral dose was much higher than environmental exposures, the single-dose exposure model results are qualitatively valid in relation to prolonged dietary exposure to ionic cadmium. Interexperimental comparisons should be used quantitatively only with great care, but intraexperimental comparisons are valid (Andersen et al., 1992). Experimental administration of cadmium chloride can reflect environmental exposures because animal studies indicate that the bioavailability of cadmium is less influenced by the chemical form of cadmium than by the diet composition (Andersen et al., 1992). The bioavailability of cadmium was low in the nonlactating ewes. In four nonlactating ewes it ranged from 0.12 to 0.22%. The bioavailabilities estimated with the toxicokinetic approach were similar to those obtained with other approaches. The total cadmium body retention was estimated to be 0.3– 0.4% in goats (Miller et al., 1969), 0.09% in steers (Johnson et al., 1981), and 0.035–0.75% in dairy
TOXICOKINETICS OF CADMIUM IN EWES
145
FIG. 2. (A) Average and (B) individual cadmium levels observed in blood after iv administration (on Day 0) and oral administration (on Day 21) of cadmium in nonlactating and lactating ewes.
cows (Neathery et al., 1974; Van Bruwaene et al., 1982). To assess the bioavailability of cadmium, the toxicokinetic approach is easier to use than approaches based on measuring cadmium in the whole body which requires either animal slaughtering or
the use of radioactive cadmium. We could more accurately estimate the bioavailability of cadmium using the toxicokinetic approach based on the amount of cadmium in blood, i.e., of absorbed cadmium, than using methods based on the amount of
146
HOUPERT, FEDERSPIEL, AND MILHAUD
FIG. 3. Amounts of cadmium excreted daily in the milk of three ewes after iv administration (on Day 0) and oral administration (on Day 21) of cadmium.
cadmium found in feces and urine, i.e., of nonabsorbed cadmium, because the bioavailability of cadmium is very low. The bioavailability of cadmium administered orally in a single dose was not much different than that calculated after daily administrations of lower dosages. In our previous study the nonlactating ewes absorbed 0.15–0.50% of the daily administered cadmium (Houpert et al., 1995). The bioavailability in one nonlactating ewe, Ewe 7, was 20 to 40 times higher than that of the other nonlactating ewes. Compared to the oral administration dosages, in our previous experiments we never observed cadmium levels as high in blood in the other ewes (Houpert et al., 1995). However, Ewe 7 showed no clinical sign of intoxication. Its bioavailability was close to that observed in man and in pig (Van Bruwaene et al., 1984). The human intestinal absorption of cadmium can vary from one person to another by 20 times (Andersen et al., 1992). We do not know why Ewe 7 absorbed so much cadmium. The lactating ewes showed higher bioavailabilities than the four nonlactating ewes (0.33 to 1.7% vs 0.12 to 0.22%). A similar phenomenon was observed in mice where systemic availability was higher (0.60%) in lactating mice, with a peak in the middle of the lactation period, as compared to nonlactating or nonpregnant mice (0.26%) (Bhattacharyya et al., 1981).
Factors affecting the absorption of ingested cadmium include animal species, type of compound, dose, frequency of administration, age of experimental animals, pregnancy and lactation, presence or absence of drugs, and interactions of cadmium with various nutrients (Nomiyama, 1978). In this study, the bioavailability of cadmium was influenced only by lactation. The ewes received the same diet except for calcium. The only changing factor was the amount of food, depending on the production of milk. The diet composition was controlled, because it is known that the gastrointestinal availability of cadmium is increased by zinc, copper, lead, and calcium deficiencies (Fox, 1979; Abdulla and Chmielnicka, 1990; Andersen et al., 1992) and decreased by manganese supplementation (Sarhan et al., 1986). The diet was balanced in phosphorus, zinc, lead, magnesium, manganese, and copper and supplemented in calcium: three times more than needed in the lactating ewes and four times more in the nonlactating ewes. It is unlikely that the greater calcium supplementation in the nonlactating ewes decreased the cadmium bioavailability. The higher cadmium bioavailability of lactating ewes might be due to lactation itself. In the rat, the gastrointestinal tract increases both in weight and in total surface area during lactation (Campbell and Fell, 1964; Boyne et al., 1966). This proliferation during lactation is accom-
TOXICOKINETICS OF CADMIUM IN EWES
147
FIG. 4. Blood concentrations of cadmium in time after intravenous administration (on Day 0) and oral administration (on Day 21) in one nonlactating ewe. The solid line represents the computer simulation data.
panied by an increased absorption of calcium, iron, and lead (Halloran and De Luca, 1980; Batey and Gallagher, 1977, Kostial and Momcilovic, 1972). It is possible that an increase in the gastrointestinal abTABLE 2 Toxicokinetic Parameters Describing the Disposition of Cadmium in Six Lactating Ewes, Four Nonlactating Ewes, and Nonlactating Ewe 7 after Single Oral and Intravenous Administrations of Cadmium Ewe
k12/k21 k13/k31 Vc (1iter/kg) Vss (liter/kg) Cl (liter/kg/day) MRT (days) MRTc (days) F (%)
Nonlactating (n 4 4)
Lactating (n 4 6)
Ewe 7
3.61 ± 6.26 272 ± 176* 0.124 ± 0.099 23.8 ± 5.4* 0.20 ± 0.03** 113 ± 28 0.59 ± 0.37* 0.18 ± 0.04
0.57 ± 0.19 493 ± 106* 0.097 ± 0.013 48.8 ± 10.3* 0.46 ± 0.013** 110 ± 29 0.22 ± 0.06* 0.65 ± 0.52
1.30 923 0.03 27.6 0.35 78 0.08 5
Note. k12, k21, k13, k31 are first-order distribution rate constants between compartment 1 and compartments 2 and 3, respectively; Vc, central compartment volume; Vss, steady-state volume of distribution; Cl, blood clearance; MTR, mean residence time in the body after iv administration; MRTc, mean residence time in the central compartment; F, bioavailability. Values are ± SD. * P < 0.05, ** P < 0.005 by Student’s ± test.
sorption of cadmium may also occur, as it does for calcium and iron (Bhattacharyya et al., 1981, 1982). The iv preadministration of cadmium may have decreased oral cadmium bioavailability. In the lactating and the nonlactating ewes, the oral cadmium bioavailabilities were slightly lower with iv preadministration of cadmium (0.51, 0.36, 0.33% and 0.12, 0.19%, respectively) than without it (1.7, 0.59, 0.44% and 0.22, 0.20%, respectively). One reason for this could be induction of metallothionein synthesis by the iv preadministration of 0.1 mg Cd/kg. Injection of 0.25 mg Cd/kg intraperitoneally induces metallothioncin in rats (Brady, 1991). In mice, intravenous injection of 1 mg Cd/kg is enough to induce it (Jonah and Bhattacharyya, 1989). Metallothionein has been reported to decrease gastrointestinal absorption of cadmium by decreasing the release of cadmium from the intestine and its transport to organs (Foulkes and McMullen, 1986; Sugawara and Sugawara, 1987; Ohta and Cherian, 1991). The kinetic parameters on the distribution of cadmium (k21, k31, VSS) showed a great cadmium accumulation in the body. They are very close to those calculated in previous studies (Houpert et al., 1995). The steady-state volume of distribution (VSS 4 23.8 ± 5.4 liter/kg) showed a large distribution of cadmium. The k13 to k31 ratio (272 ± 175) showed a high
148
HOUPERT, FEDERSPIEL, AND MILHAUD
FIG. 5. Mean cadmium clearance in milk (solid circles) and mean milk production (open circles) of six ewes after cadmium administrations on Days 0 and 21.
amount of cadmium in the deep compartment. It is known that cadmium is concentrated in certain tissues, notably in the liver and in the kidney (Miller et al., 1969; Houpert et al., 1995). The steady-state volume of distribution and the k13 to k31 ratio were significantly higher in the lactating ewes than in the nonlactating ewes. Cadmium is more widely distributed in the body and accumulated in the deep compartment of lactating ewes. In mice, a single dose of cadmium administered during lactation increases the retention of cadmium in the kidneys three to four times as compared to nonlactating mice (Bhattacharyya et al., 1981). The blood clearance of cadmium was low. Cad-
mium concentrates in red cells rather than in plasma and therefore is less available for elimination (Nordberg and Nordberg, 1987). It reached, on average, 0.21 ± 0.04% of the cardiac flow in the nonlactating ewes. This value was close to our previous results (Houpert et al., 1995). On average, the clearance of cadmium in the lactating ewes was higher (0.50 ± 0.13% of the cardiac flow) than in the nonlactating ewes. This suggests that cadmium can be eliminated from blood more quickly during lactation. However the mean residence time did not change during lactation (MRT 4 113 ± 28 days in nonlactating ewes, 110 ± 29 days in lactating ewes). Cadmium was not eliminated more quickly from
TABLE 3 Parameters Describing the Elimination of Cadmium through Milk in Six Lactating Ewes after Both Oral and Intravenous Cadmium Doses Ewe Parameters Qm (mg) Clm (liter/kg/day) Clm(max) (liter/kg/day) Clm/Cl (%)
1
2
3
4
5
6
900 0.0148 0.0411 4.2
350 0.0183 0.0749 3.3
285 0.0197 0.0571 3.1
175 0.0066 0.0335 2.0
400 0.0179 0.0513 4.9
350 0.0237 0.0808 4.2
Note. Qm, total amount of cadmium excreted in milk during lactation; Clm, mean clearance in milk during lactation; Clm(max), maximum clearance in milk; Cl, blood clearance.
149
TOXICOKINETICS OF CADMIUM IN EWES
the whole body during lactation, possibly because its higher retention in the deep compartment made it less available for elimination. Because cadmium was eliminated more quickly from blood and was distributed more widely in the body during lactation, its mean residence time in the central compartment, in blood, was shorter in lactating ewes than in nonlactating ewes (MRTc 4 0.22 ± 0.06 vs 0.59 ± 0.37 days). Ewe 7 disposed cadmium differently than in the other nonlactating ewes. Despite higher retention in the deep compartment (k13/k31 4 923, VSS 4 27.6 liter/kg), elimination was quicker and mean residence time was shorter (78 days). The higher bioavailability of cadmium (F 4 5%) in this ewe could have increased the amount of cadmium in the body. Because cadmium is nephrotoxic (Murakami et al., 1983; Nordberg, 1984), the high amount of cadmium could have induced renal damage which could have sped up cadmium elimination. In man, the renal tubular damage induced by cadmium shortens the half-life of cadmium (7 years instead of 16 years (Ja¨rup et al 1983)). The clearance of cadmium in milk was low and changed over time. It was the highest during a short period at the beginning of lactation. Its maximum (Clm(max)) was 9.1 to 14.5% of the blood clearance. It then decreased rapidly for a few weeks, reaching values close to the milk production. In milk, cadmium is bound preferentially to protein (casein and albumin) (Van Bruwaene, 1982). The number of proteins in milk decreases in the first weeks of lactation (Ruckebush, 1974; Coulon, 1994), which could explain why the clearance of cadmium in milk decreased rapidly. After these first weeks, the clearance of cadmium in milk decreased slowly. Over the entire lactation period, the clearance in milk averaged 2 to 5% of the blood clearance. The clearances in milk, either maximum or average, were low compared to total clearances. The elimination of cadmium through the ewes’ milk did not explain the higher blood clearance observed during lactation. The clearances other than in milk (Cl − Clm(max)) of these ewes remained significantly, higher (P < 0.01) than blood clearance of the nonlactating ewes. The lactating ewes had greater capacities to clear their blood than nonlactating ewes besides the elimination of cadmium through milk. These parameters allowed us to estimate the percentage of orally administered cadmium excreted in milk during the entire lactation period, which was 0.017 ± 0.015%. In the cow, this percentage was estimated to be less than 0.02% (Task Group, 1973).
CONCLUSION
In the nonlactating ewe the bioavailability of cadmium was very low, the distribution of cadmium in the body was very large, and cadmium was slowly eliminated. In the lactating ewe the bioavailability was higher. The mean residence time in the body was close to that of nonlactating ewes despite greater blood purification capacities because the distribution of cadmium in the body was larger. The elimination of cadmium through milk was low and did not explain the higher blood purification capacities observed during lactation. Using a semisimultaneous toxicokinetic approach, we could accurately estimate cadmium absorption, distribution, elimination through milk, and body elimination in these two groups of ewes. In future studies we can use this approach to test methods which would decrease cadmium availability, retention in tissues, and excretion in milk, to reduce the contamination of animalbased food products. ACKNOWLEDGMENTS We thank Professor P.L. Toutain for his contribution to the toxicokinetic analysis of the results. We are very grateful to M. Mirman and his team who supervised the herd during the study period.
REFERENCES Abdulla, M., and Chmielnicka, J. (1990). New aspects on the distribution and metabolism of essential trace elements after dietary exposure to toxic metals. Biol. Trace Elem. Res. 23, 25– 53. Andersen, O., Nielsen, J. B., and Nordberg, G. G. (1992). Factors affecting the intestinal uptake of cadmium from the diet. In ‘‘Cadmium in the Human Environment: Toxicity and Carcinogenicity’’ (G. F. Nordberg, R. F. M. Herber, and L. Alessio, Eds.), Vol. 118, pp. 173–187. IARC Scientific Publications, Lyon. Batey, R., and Gallagher, N. (1977). Effect of iron stores and hysterectomy on iron absorption and distribution in pregnant mice. Am. J. Physiol. 232, 57–61. Bhattacharyya, M. H., Whelton, B. D., and Peterson, D. P. (1981). Gastrointestinal absorption of cadmium in mice during gestation and lactation: I. Short-term exposure studies. Toxicol. Appl. Pharmacol. 61, 335–342. Bhattacharyya, M. H., Whelton, B. D., and Peterson, D. P. (1982). Gastrointestinal absorption of cadmium in mice during gestation and lactation: II: Continuous exposure studies. Toxicol. Appl. Pharmacol. 66, 368–375. Boyne, R., Fell, B. F., and Robb, I. (1966). The surface area of the intestinal mucosa in the lactating rat. J. Physiol. 183, 570–575. Brady, F. O. (1991). Induction of metallothionein rats. In ‘‘Methods in Enzymology: Metallobiochemistry,’’ Part B, ‘‘Metallothionein and Related Molecules’’ (J. F. Riordan and B. L. Vallee, Eds.), Vol. 205, pp. 559–567 Academic Press, San Diego. Campbell, R. M., and Fell, B. F. (1964). Gastrointestinal hyper-
150
HOUPERT, FEDERSPIEL, AND MILHAUD
trophy in the lactating rat and its relation to food intake. J. Physiol. 171, 90–97. Coulon, J. B. (1994). Effet du stade physiologique et de la saison sur la composition chimique du lait de vache et ses caracte´ristiques technologiques. Rec. Med. Vet. 170, 367–374. Foulkes, E. C., and McMullen, D. M. (1986). Endogenous metallothionein as determinant of intestinal cadmium absorption: A reevaluation. Toxicology 38, 285–291. Fox, M. R. S. (1979). Nutritional influences on metal toxicity: Cadmium as a model toxic element. Environ. Health Perspect. 29, 95–104. Halloran, B. P., and De Luca, H. F. (1980). Calcium transport in the small intestine during pregnancy and lactation. Am. J. Physiol. 239, 64–68. Houpert, P., Mehennaoui, S., Joseph-Enriquez, B., Federspiel, B., and Milhaud, G. (1995). Pharmacokinetics of cadmium following intravenous and oral administration to non-lactating ewes. Vet. Res. 26, 145–154. Ja¨rup, L., Rogenfelt, A. R., Elinder, C. G., Nogawa, K., and Kjellstro¨m, T. (1983). Biological half-time of cadmium in the blood of workers after cessation of exposure. Scand. J. Work Environ. Health. 9, 327–331. Johnson, D. E., Kienholtz, E. W., Baxter, J. C., Spangler, E., and Ward, G. M. (1981). Heavy metal retention in tissues of cattle fed high cadmium sewage sludge. J. Anim. Sci. 52, 108–114. Jonah, M. M., and Bhattacharyya, M. H. (1989). Early changes in the tissue distribution of cadmium after oral but not intravenous cadmium exposure. Toxicology 58, 325–338. Kostial, K., and Momtilovic, B. (1972). The effect of lactation on the absorption of 209Pb and 47Ca in rats. Health Phys. 23, 383– 384. Lauwerys, R., Amery, A., Bernard, A., Bruaux, P., Buchet, J. P., Clayes, F., De Plaen, P., Ducoffre, G., Fagard, R., Lijnen, P., Nick, L., Roels, H., Rondia, D., Saint-Remy, A., Sartor, F., and Staessen, J. (1990). Health effects of environmental exposure to cadmium: Objectives, design and organization of the cadmibell study: A cross-sectional morbidity study carried out in Belgium from 1985 to 1989. Environ. Health Perspect. 87, 283–289. Miller, W. J., Blackmon, D. M., Gentry, R. P., and Pate, F. M. (1969). Effect of dietary cadmium on tissue distribution of cadmium following a single oral dose in young goats. J. Dairy Sci. 52, 2029–2035. Murakami, M., Cain, K., and Webb, M. (1983). Cadmiummetallothionein-induced nephropathy: A morphological and autoradiographic study of cadmium distribution, the development of tubular damage and subsequent cell regeneration. J. Appl. Toxicol 3, 237–244. Neathery, N. W., Miller, W. J., Gentry, R. P., Stake, P. E., and Blackman, D. M. (1974). Cadmium-109 and methyl mercury-
203 metabolism, tissue distribution and secretion into milk of cows. J. Dairy Sci. 57, 1177–1183. Nomiyama, K. (1978). Experimental studies on animals: In vivo experiments. In ‘‘Cadmium Studies in Japan: A Review’’ (K. Tsuchiya, Ed.), pp. 47–86 Elsevier Science, Amsterdam/Oxford/ New York. Nordberg, M. (1984). General aspects of cadmium: transport, uptake, metabolism by the kidney. Environ. Health. Perspect. 54, 13–20. Nordberg, G. F., and Nordberg, M. (1987). Different binding forms of cadmium-implications for distribution and toxicity. Univ. Occup. Environ. Health. 20, 153–164. Ohta, H., and Cherian, M. G. (1991). Gastrointestinal absorption of cadmium and metallothionein. Toxicology 107, 63–72. Robards, K., and Worsfold, P. (1991). Cadmium: Toxicology and analysis. Analyst 116, 205–214. Ruckebusch, Y. (1977). ‘‘Physiologie Pharmacologie The´rapeutique Animales.’’ Maloine SA, Paris. Sarhan, M. J., Roels, H., Lauwerys, R., Reyners, H., and Gianfelici de Reyners, E. (1986). Influence of manganese on the gastrointestinal absorption of cadmium in the rats. J. Appl. Toxicol. 5, 313–316. Sugawara, N., and Sugawara, C. (1987). Role of mucosal metallothionein preinduced by oral Cd or Zn on the intestinal absorption of a subsequent Cd dose. Bull Environ. Contam. Toxicol. 38, 295–299. Task Group on Metal Accumulation (1973). Accumulation of toxic metal with special reference to their absorption, excretion and biological half-times. Environ. Physiol. Biochem. 3, 65–107. Van Bruwaene, R., Gerber, G. B., Kirchmann, R., and Colard, J. (1982). Transfert and distribution of radioactive cadmium of dairy cows. Int J. Environ. Stud. 19, 47–51. Van Bruwaene, R., Kirchmann, R., and Impens, R. (1984). Cadmium contamination in agriculture and zootechnology. Experencia (Basel) 40, 43–52. W.H.O. (1992). ‘‘Cadmium Environmental Health Criteria 134,’’ World Health Organization, Geneva. Wijnand, H. P. (1992). The determination of the absolute bioavailability for drug substances with long elimination half-lives (with PC-programs for the method of truncated areas). Comput. Methods Programs Biomed. 39, 61–73. Yamaoka, K., Nakawaga, T., and Uno, T. (1978). Application of Akaike’s information criterion (ALC) in the evaluation of the mean pharmacokinetic equations. J. Pharmacokinetic Biopharm. 6, 165–175. Yamaoka, K., Tanigawara, Y., and Nakawaga, T. (1981). A pharmaco-kinetic analysis program (Multi) for microcomputer. J. Pharmacobio-Dyn. 4, 879–889.
72, 140–150 (1997)
ER963690
Toxicokinetics of Cadmium in Lactating and Nonlactating Ewes after Oral and Intravenous Administration P. HOUPERT, B. FEDERSPIEL,
AND
G. MILHAUD
Service de pharmacie et toxicologie, e´quipe associe´e INRA-ENVA Cadmium et Aliment d’Origine Animale, Ecole Nationale Ve´te´rinaire d’Alfort, 7 avenue du ge´ne´ral De Gaulle, 94704 Maisons-Alfort cedex, France Received January 3, 1996
must be quantified. In a previous experiment we studied the toxicokinetics of cadmium in several nonlactating ewes (Houpert et al., 1995). Ewes were selected as a model farm animal because humans consume their meat and milk. Using a toxicokinetic model we estimated absorption and elimination without slaughtering the animals. The present experiment aimed to confirm our previous results and to quantify the elimination of cadmium through milk. Two groups of ewes, a lactating group and a nonlactating group, were kept under similar conditions. We studied the toxicokinetics of cadmium after a single intravenous and a single oral dose of cadmium chloride.
We studied the toxicokinetics of cadmium on two groups of ewes, a lactating group and a nonlactating group, after single intravenous and oral administrations of cadmium chloride using a semisimultaneous method and a three-compartment model. The nonlactating ewes showed a low cadmium bioavailability (0.12–0.22%), a large steady-state volume of distribution (23.8 ± 5.4 liter/kg), and a low blood clearance (0.20 ± 0.03 liter/kg/day). Their mean residence time was 113 ± 28 days. The lactating ewes had a higher bioavailability (0.33–1.7%). Their mean residence time was close to that in nonlactating ewes despite a greater blood clearance (0.46 ± 0.013 liter/kg/day) because the volume of distribution of cadmium in the body was larger (VSS = 48.8 ± 10.3 liter/kg). Their cadmium clearance in milk, changing with time, remained low and could not explain their higher blood clearance. In one nonlactating ewe, a greater cadmium bioavailability (5%) increased cadmium in the body. Increased cadmium amounts could induce renal damage and shorten the mean residence time (78 days). © 1997
MATERIALS AND METHODS
Animals Twelve Lacaune–Prealp cross-breed ewes were used. Throughout the study, the animals’ mean weight was 60 ± 4 kg. Eleven had no clinical troubles. The 12th ewe died a few days after the beginning of the study for reasons unrelated to the study. Six ewes (Ewes 1 to 6) had had their lambs 5 to 9 days before the experiment started, and the other 5 ewes (Ewes 7 to 11) were nonlactating and nonpregnant. The animals were kept in the sheepfold of the experimental farm Brouessy (INRA, France). Every morning, they were given ammoniated straw, dehydrated lucern, lucern hay, and a mineral additive including vitamins dosed according to the physiological state of the animals. They drank water ad libitum. The amounts of cadmium, calcium, zinc, iron, magnesium, manganese, and copper in the diet and the water were determined. The amount of phosphorus in the diet was also determined.
Academic Press
INTRODUCTION
The more cadmium contaminates the agricultural environment, the more it contaminates the food chain and endangers human consumers’ health. Levels in soils and plants are increased by the application of phosphate fertilizers and sewage sludge (WHO, 1992). Cadmium also contaminates tissues and, to a lesser extent, the milk of grazing animals. As a result, humans, exposed mainly through food, can be more and more exposed in the future (WHO, 1992). To reduce contamination of animal products, one objective is to lower cadmium absorption or speed up cadmium elimination in animals. To use this strategy, cadmium absorption and elimination 140 0013-9351/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.
141
TOXICOKINETICS OF CADMIUM IN EWES
Administration of Cadmium The ewes received two successive doses of cadmium chloride (CdCl2, H2O, Merck, Nogent-surMarne, France) at a 21-day interval in a crossover design. One of the doses contained 25 mg/kg of cadmium and was given orally. The cadmium was enclosed in gelatin capsules and placed on the base of the tongue. The capsules were immediately swallowed. The oral dose was given to Ewes 1 to 3 and 10 to 12 on Day 0, and to Ewes 4 to 8 on Day 21. The other dose contained 0.1 mg/kg of cadmium diluted to 1 mg/ml. The solution was then sterilized in an autoclave for 20 min in sealed tubes. The solution was injected in the left jugular vein via a catheter (Intraflon, 2/Vygon). The iv dose was administered to Ewes 4 to 8 on Day 0, and to Ewes 1 to 3 and 10 to 12 on Day 21. Sampling Blood samples of 2 ml each were collected from the right jugular vein into vacutainer tubes guaranteed free of any trace of heavy metals (Ref. 367735 Vacutainer tubes, Beckton–Dickinson, Mayland, France). These tubes were then stored at 4°C. On the days that iv injections were given (Day 0 and Day 21), blood was collected 2, 4, 8, 15, and 30 min and 1, 2, 3, 4, and 6 hr after the injection. On the days that cadmium was administered orally (Day 0 or Day 21), the blood was collected 5, 10, 15, and 30 min and 1, 2, 3, 4, and 6 hr after the administration. The blood of the six ewes receiving first the iv injection was collected 1, 2, 3, 6, 9, 13, 16, 20, 21, 22, 23, 24, 27, 30, 34, 37, 41, 48, 62, 76, 90, 106, 120, 134, 148, 181, 212, 241, and 272 days after the injection. The blood of the five ewes receiving first the oral administration was collected 1, 2, 3, 4, 7, 10, 14, 17, 21, 22, 23, 24, 25, 28, 31, 35, 38, 42, 49, 63, 77, 91, 107, 121, 135, 149, 182, 213, 242, and 273 days after the administration. Milk was collected the same day blood was collected until the ewes naturally stopped giving milk, i.e., 150 to 180 days from the beginning of the experiment. Milk was homogenized and samples (50 ml) were collected and stored in polyethylen flasks at 4°C until cadmium was dosed in the following 24 hr. The daily volume of milk was measured. Cadmium Measurement The samples were first diluted 1:10 for blood and 1:5 for milk with 0.05 N nitric acid in the presence of Triton. The cadmium concentrations in blood and
milk were then measured directly by graphite furnace atomic absorption spectrometry with a Perkin– Elmer 1100B spectrophotometer AGA 700 according to the technique recommended by Lauwerys et al. (1990). The decomposition temperature was 550°C for blood and 600°C for milk; the atomization temperature was 1450°C. The limit of detection was estimated to be 0.3 mg/liter, the limit of quantification 0.8 mg/liter. The intraday coefficient of variation was 25% at 0.8 mg/liter, 13% at 2 mg/liter, 2% at 5 mg/liter in blood and 6% at 1 mg/liter, 5% at 5 mg/liter in milk. The accuracy of the method was controlled with certified material: BCR194: 0.5 ± 0.4 vs 0.5 ± 0.1 mg/liter, BCR195: 5.4 ± 0.5 vs 5.37 ± 0.24 mg/liter; for blood; CRM063: 2.8 ± 1.2 vs 2.9 ± 1.2 ng/liter, CRM150: 21.8 ± 1.6 vs 21.8 ± 1.4 ng/liter for milk of BCR (Commission of European Communities, Community Bureau of Reference, Brussels, Belgium). Toxicokinetic Analyses The toxicokinetic analyses were carried out via a nonlinear regression program adapted from Multi (Yamaoka et al., 1981). The following general polyexponential equations were adjusted to the data set using the superposition principle, according to the semisimultaneous administration method (Wijnand, 1992),
(FY ·e n
C~t! =
F(
−lit
i
+
i=1
n
−
ka · Yi F · Dpo −l T e ka − li Div i
ka · Yi F ? Dpo Div i=1 ka − li
G
G
? e−k T,
(1)
a
when the first administration was an iv injection, and
(FY ?e n
C~t! =
F(
i=1
n
−
i=1
i
−liT
+
ka ? Yi F ? Dpo −l t e ka − li Div
ka ? Yi F ? Dpo ka − li Div
i
G
? e−k t, a
G (2)
when the first administration was given orally. In these equations, T 4 0 if t < 21 and T 4 t-21 if t > 21, c is the cadmium concentration in blood at time t, Y1 is the coefficient of the ith exponential term li, ka is the first-order absorption constant, F is the bioavailability, and Dpo/Div is the ratio of oral to iv administrations. The initial parameters were obtained using linear regression methods from iv administration data. The number of necessary exponents (2 or 3) was defined by the application of
142
HOUPERT, FEDERSPIEL, AND MILHAUD
Akaike’s information criterion (Yamaoka et al., 1978). The volume of the central compartment (Vc) was obtained using the equation Vc =
Div
.
n
(Y
(3)
milk on Day d, C(d) is the cadmium concentration in blood on Day d, and W is the body weight. Maximum clearance in milk (Clm(max)) was the highest value of Clm(d). The mean clearance in milk (Clm) was calculated as Clm 4 Qm/AUC z W,
i
(10)
i=1
The steady-state volume of distribution (Vss) was obtained using the equation Vss 4 Vc(1 + k12/k21 + k13/k31),
(4) Statistical Analyses
where k12, k21, k13, and k31 represent the first-order distribution rate constants between compartment 1 and compartments 2 and 3, respectively. The mean residence time after iv administration (MRT) was obtained using the equation n
MRT =
(
Yi
i=1
/l2i
n
( Y /l
.
(5)
i
The mean residence time in the central compartment (MRTc was obtained using the equation MRTc 4 1/k10,
(6)
where k10 is the first-order elimination rate constant from compartment 1. The blood clearance (Cl) was calculated from the equation Cl =
Div n
( Y /l
.
(7)
i
i=1
Student’s t test was used to get an overview of significant differences between the groups (lactating and nonlactating) for the kinetic parameters. RESULTS
Daily Mineral Intake
i
i=1
where Qm is the total cadmium excreted into milk, W is the weight of the ewe, and AUC is the area under the blood concentration–time curve calculated by trapezoidal rule from Time 0 to the end of lactation.
i
The cadmium amount excreted in milk on Day d was calculated using the equation Qm(d) 4 Cm(d) z Vm(d),
(8)
where Cm(d) is the cadmium concentration in milk on Day d and Vm(d) is the milk volume produced on Day d. The clearance in milk on Day d was calculated using the equation Clm(d) 4 Qm (d)/C(d) z W,
(9)
where Qm(d) is the cadmium amount excreted in
The daily mineral intake is shown in Table 1. Cadmium content of the diet was below 5 mg/kg/day. Calcium content was 20 to 44 g/day, depending on the physiological state of the animals. Cadmium Levels in Blood Cadmium concentrations in the blood and in the milk of the ewes before the first cadmium administrations were below the limit of quantification of the analytical method (0.8 mg/liter). After cadmium was administered orally on Day 0, levels in blood differed widely among animals. They could be measured 5 min to 3 hr after administration and they reached maximum levels, ranging from 2.5 to 80 mg/liter, on the first or second day. Levels then slowly decreased until the 21st day, but remained above the quantification limit. The blood cadmium levels in nonlactating ewes (Ewes 10 and 11) were similar to those of lactating ewes (Ewes 2 TABLE 1 Total Mineral Intake per Day during the Study
Cadmium (mg) Calcium (g) Phosphorus (g) Zinc (mg) Iron (mg) Magnesium (g) Manganese (mg) Copper (mg)
Nonlactating ewe
Start of lactation
End of lactation
0.15 20.3 4.05 150 437 2.65 157 15.1
0.29 43.6 8.07 181 1086 5.29 231 22.6
0.23 29.6 5.85 164 727 3.88 192 18.6
TOXICOKINETICS OF CADMIUM IN EWES
and 3; Figs. 1A and 1B). The blood cadmium level of lactating Ewe 1 was substantially higher. After cadmium was injected on Day 21 levels in blood were more similar in all animals, as compared to oral administration on Day 0. Its levels rose rapidly, reaching maximum peaks of 350 to 950 mg/liter. They then decreased rapidly in the first hours and
143
then more slowly in the following days and weeks. The average cadmium concentrations observed in nonlactating ewes were within the range of to those observed in lactating ewes. The cadmium levels in blood observed in the five ewes receiving an iv injection on Day 0 were very similar to those observed in the ewes receiving the iv
FIG. 1. (A) Average and (B) individual cadmium levels observed in blood after oral administration (on Day 0) and iv administration (on Day 21) of cadmium in nonlactating and lactating ewes.
144
HOUPERT, FEDERSPIEL, AND MILHAUD
injection on Day 21 (described above). After the iv injection, the cadmium levels of the lactating ewes were below those of the nonlactating ewes. After cadmium was administered orally on Day 21, the cadmium levels in the blood of one nonlactating ewe (Ewe 7) were much higher than those of the other ewes (Figs. 2A and 2B); in the latter the difference of blood cadmium levels between lactating and nonlactating ewes was very weak (in lactating ewes they remained slightly below those in the nonlactating ewes). Amount of Cadmium Eliminated through Milk After cadmium was orally or intravenously administered, its levels in milk increased rapidly and could be detected as soon as the evening milking (6 hr after administration). Levels in milk reached a maximum value on the second or third day after administration (Fig. 3). These values then decreased, quickly at first and then slowly. Seventy days after the second administration, 1.1 ± 0.8 mg/ day of cadmium was being excreted in milk. Pharmacokinetic Parameters According to Akaike’s criterion, the disposition of cadmium in all ewes was best modeled by a triexponential equation corresponding to a three-compartment open model (Fig. 4). The average toxicokinetic parameters after the analysis of blood concentrations are listed in Table 2 for the six lactating ewes and the four nonlactating ewes. The parameters of nonlactating Ewe 7, whose cadmium levels were very different from those of the other ewes, are given separately. The bioavailability (F) of cadmium given orally to the lactating ewes was 0.51, 0.36, and 0.33% with the iv preadministration and 1.7, 0.59, and 0.44% without it. In the nonlactating ewes, it was 0.12 and 0.19% with the iv preadministration and 0.22 and 0.20% without it. In Ewe 7, it was 5%. The ratio of distribution constants between the central compartment and the deep compartment (k13/k31) was 272 ± 176 in the nonlactating group, 493 ± 106 in the lactating group, and 923 in Ewe 7. The steady-state volume of distribution ranged from 19.4 to 31.6 liter/ kg in the nonlactating group and from 37.3 to 62.3 liter/kg in the lactating group. The blood clearance was 0.2 ± 0.03 liter/kg/day in the nonlactating group and 0.46 ± 0.13 liter/kg/day in the lactating group. It reflected respectively, 0.18–0.25 and 0.38–0.67% of the cardiac flow (0.37% for Ewe 7). The mean residence time in the central compartment (MRTc) was 0.22 ± 0.06 days in the
lactating ewes and 0.59 ± 0.37 days in the nonlactating ewes (0.08 day in Ewe 7). The mean residence time in the body (MRT) varied slightly according to the physiological state of the animals. The total amounts of cadmium eliminated through milk during lactation ranged from 175 to 900 mg. The clearance in milk was not linear (Fig. 5); it reached a maximum value 7 to 14 days after the first cadmium administration, whatever the administration route. The maximum value (Cl m(max) ) ranged from 0.03 to 0.08 liter/kg/day (Table 3). It decreased rapidly in the following 2 to 3 weeks and then decreased slowly. The mean clearance in milk (Clm) was calculated over the entire lactation period. It ranged from 0.0066 to 0.0237 liter/kg/day. DISCUSSION
We studied the toxicokinetics of cadmium after single iv and single oral bolus doses using the semisimultaneous or sequential method (Wijnand, 1992). Because cadmium is eliminated slowly (Robards and Worsfold, 1991; WHO, 1992), the sampling period following an administration and the washout intervals between two administrations are too long to determine the cadmium bioavailability with a classical method. Using the semisimultaneous method, we could administer the iv and oral doses with a 21-day interval and calculate all the kinetic parameters. We chose to give single doses of cadmium, although environmental exposures do not occur in single doses, because this permitted us to administer the oral and the iv boluses in comparable situations. Although the oral dose was much higher than environmental exposures, the single-dose exposure model results are qualitatively valid in relation to prolonged dietary exposure to ionic cadmium. Interexperimental comparisons should be used quantitatively only with great care, but intraexperimental comparisons are valid (Andersen et al., 1992). Experimental administration of cadmium chloride can reflect environmental exposures because animal studies indicate that the bioavailability of cadmium is less influenced by the chemical form of cadmium than by the diet composition (Andersen et al., 1992). The bioavailability of cadmium was low in the nonlactating ewes. In four nonlactating ewes it ranged from 0.12 to 0.22%. The bioavailabilities estimated with the toxicokinetic approach were similar to those obtained with other approaches. The total cadmium body retention was estimated to be 0.3– 0.4% in goats (Miller et al., 1969), 0.09% in steers (Johnson et al., 1981), and 0.035–0.75% in dairy
TOXICOKINETICS OF CADMIUM IN EWES
145
FIG. 2. (A) Average and (B) individual cadmium levels observed in blood after iv administration (on Day 0) and oral administration (on Day 21) of cadmium in nonlactating and lactating ewes.
cows (Neathery et al., 1974; Van Bruwaene et al., 1982). To assess the bioavailability of cadmium, the toxicokinetic approach is easier to use than approaches based on measuring cadmium in the whole body which requires either animal slaughtering or
the use of radioactive cadmium. We could more accurately estimate the bioavailability of cadmium using the toxicokinetic approach based on the amount of cadmium in blood, i.e., of absorbed cadmium, than using methods based on the amount of
146
HOUPERT, FEDERSPIEL, AND MILHAUD
FIG. 3. Amounts of cadmium excreted daily in the milk of three ewes after iv administration (on Day 0) and oral administration (on Day 21) of cadmium.
cadmium found in feces and urine, i.e., of nonabsorbed cadmium, because the bioavailability of cadmium is very low. The bioavailability of cadmium administered orally in a single dose was not much different than that calculated after daily administrations of lower dosages. In our previous study the nonlactating ewes absorbed 0.15–0.50% of the daily administered cadmium (Houpert et al., 1995). The bioavailability in one nonlactating ewe, Ewe 7, was 20 to 40 times higher than that of the other nonlactating ewes. Compared to the oral administration dosages, in our previous experiments we never observed cadmium levels as high in blood in the other ewes (Houpert et al., 1995). However, Ewe 7 showed no clinical sign of intoxication. Its bioavailability was close to that observed in man and in pig (Van Bruwaene et al., 1984). The human intestinal absorption of cadmium can vary from one person to another by 20 times (Andersen et al., 1992). We do not know why Ewe 7 absorbed so much cadmium. The lactating ewes showed higher bioavailabilities than the four nonlactating ewes (0.33 to 1.7% vs 0.12 to 0.22%). A similar phenomenon was observed in mice where systemic availability was higher (0.60%) in lactating mice, with a peak in the middle of the lactation period, as compared to nonlactating or nonpregnant mice (0.26%) (Bhattacharyya et al., 1981).
Factors affecting the absorption of ingested cadmium include animal species, type of compound, dose, frequency of administration, age of experimental animals, pregnancy and lactation, presence or absence of drugs, and interactions of cadmium with various nutrients (Nomiyama, 1978). In this study, the bioavailability of cadmium was influenced only by lactation. The ewes received the same diet except for calcium. The only changing factor was the amount of food, depending on the production of milk. The diet composition was controlled, because it is known that the gastrointestinal availability of cadmium is increased by zinc, copper, lead, and calcium deficiencies (Fox, 1979; Abdulla and Chmielnicka, 1990; Andersen et al., 1992) and decreased by manganese supplementation (Sarhan et al., 1986). The diet was balanced in phosphorus, zinc, lead, magnesium, manganese, and copper and supplemented in calcium: three times more than needed in the lactating ewes and four times more in the nonlactating ewes. It is unlikely that the greater calcium supplementation in the nonlactating ewes decreased the cadmium bioavailability. The higher cadmium bioavailability of lactating ewes might be due to lactation itself. In the rat, the gastrointestinal tract increases both in weight and in total surface area during lactation (Campbell and Fell, 1964; Boyne et al., 1966). This proliferation during lactation is accom-
TOXICOKINETICS OF CADMIUM IN EWES
147
FIG. 4. Blood concentrations of cadmium in time after intravenous administration (on Day 0) and oral administration (on Day 21) in one nonlactating ewe. The solid line represents the computer simulation data.
panied by an increased absorption of calcium, iron, and lead (Halloran and De Luca, 1980; Batey and Gallagher, 1977, Kostial and Momcilovic, 1972). It is possible that an increase in the gastrointestinal abTABLE 2 Toxicokinetic Parameters Describing the Disposition of Cadmium in Six Lactating Ewes, Four Nonlactating Ewes, and Nonlactating Ewe 7 after Single Oral and Intravenous Administrations of Cadmium Ewe
k12/k21 k13/k31 Vc (1iter/kg) Vss (liter/kg) Cl (liter/kg/day) MRT (days) MRTc (days) F (%)
Nonlactating (n 4 4)
Lactating (n 4 6)
Ewe 7
3.61 ± 6.26 272 ± 176* 0.124 ± 0.099 23.8 ± 5.4* 0.20 ± 0.03** 113 ± 28 0.59 ± 0.37* 0.18 ± 0.04
0.57 ± 0.19 493 ± 106* 0.097 ± 0.013 48.8 ± 10.3* 0.46 ± 0.013** 110 ± 29 0.22 ± 0.06* 0.65 ± 0.52
1.30 923 0.03 27.6 0.35 78 0.08 5
Note. k12, k21, k13, k31 are first-order distribution rate constants between compartment 1 and compartments 2 and 3, respectively; Vc, central compartment volume; Vss, steady-state volume of distribution; Cl, blood clearance; MTR, mean residence time in the body after iv administration; MRTc, mean residence time in the central compartment; F, bioavailability. Values are ± SD. * P < 0.05, ** P < 0.005 by Student’s ± test.
sorption of cadmium may also occur, as it does for calcium and iron (Bhattacharyya et al., 1981, 1982). The iv preadministration of cadmium may have decreased oral cadmium bioavailability. In the lactating and the nonlactating ewes, the oral cadmium bioavailabilities were slightly lower with iv preadministration of cadmium (0.51, 0.36, 0.33% and 0.12, 0.19%, respectively) than without it (1.7, 0.59, 0.44% and 0.22, 0.20%, respectively). One reason for this could be induction of metallothionein synthesis by the iv preadministration of 0.1 mg Cd/kg. Injection of 0.25 mg Cd/kg intraperitoneally induces metallothioncin in rats (Brady, 1991). In mice, intravenous injection of 1 mg Cd/kg is enough to induce it (Jonah and Bhattacharyya, 1989). Metallothionein has been reported to decrease gastrointestinal absorption of cadmium by decreasing the release of cadmium from the intestine and its transport to organs (Foulkes and McMullen, 1986; Sugawara and Sugawara, 1987; Ohta and Cherian, 1991). The kinetic parameters on the distribution of cadmium (k21, k31, VSS) showed a great cadmium accumulation in the body. They are very close to those calculated in previous studies (Houpert et al., 1995). The steady-state volume of distribution (VSS 4 23.8 ± 5.4 liter/kg) showed a large distribution of cadmium. The k13 to k31 ratio (272 ± 175) showed a high
148
HOUPERT, FEDERSPIEL, AND MILHAUD
FIG. 5. Mean cadmium clearance in milk (solid circles) and mean milk production (open circles) of six ewes after cadmium administrations on Days 0 and 21.
amount of cadmium in the deep compartment. It is known that cadmium is concentrated in certain tissues, notably in the liver and in the kidney (Miller et al., 1969; Houpert et al., 1995). The steady-state volume of distribution and the k13 to k31 ratio were significantly higher in the lactating ewes than in the nonlactating ewes. Cadmium is more widely distributed in the body and accumulated in the deep compartment of lactating ewes. In mice, a single dose of cadmium administered during lactation increases the retention of cadmium in the kidneys three to four times as compared to nonlactating mice (Bhattacharyya et al., 1981). The blood clearance of cadmium was low. Cad-
mium concentrates in red cells rather than in plasma and therefore is less available for elimination (Nordberg and Nordberg, 1987). It reached, on average, 0.21 ± 0.04% of the cardiac flow in the nonlactating ewes. This value was close to our previous results (Houpert et al., 1995). On average, the clearance of cadmium in the lactating ewes was higher (0.50 ± 0.13% of the cardiac flow) than in the nonlactating ewes. This suggests that cadmium can be eliminated from blood more quickly during lactation. However the mean residence time did not change during lactation (MRT 4 113 ± 28 days in nonlactating ewes, 110 ± 29 days in lactating ewes). Cadmium was not eliminated more quickly from
TABLE 3 Parameters Describing the Elimination of Cadmium through Milk in Six Lactating Ewes after Both Oral and Intravenous Cadmium Doses Ewe Parameters Qm (mg) Clm (liter/kg/day) Clm(max) (liter/kg/day) Clm/Cl (%)
1
2
3
4
5
6
900 0.0148 0.0411 4.2
350 0.0183 0.0749 3.3
285 0.0197 0.0571 3.1
175 0.0066 0.0335 2.0
400 0.0179 0.0513 4.9
350 0.0237 0.0808 4.2
Note. Qm, total amount of cadmium excreted in milk during lactation; Clm, mean clearance in milk during lactation; Clm(max), maximum clearance in milk; Cl, blood clearance.
149
TOXICOKINETICS OF CADMIUM IN EWES
the whole body during lactation, possibly because its higher retention in the deep compartment made it less available for elimination. Because cadmium was eliminated more quickly from blood and was distributed more widely in the body during lactation, its mean residence time in the central compartment, in blood, was shorter in lactating ewes than in nonlactating ewes (MRTc 4 0.22 ± 0.06 vs 0.59 ± 0.37 days). Ewe 7 disposed cadmium differently than in the other nonlactating ewes. Despite higher retention in the deep compartment (k13/k31 4 923, VSS 4 27.6 liter/kg), elimination was quicker and mean residence time was shorter (78 days). The higher bioavailability of cadmium (F 4 5%) in this ewe could have increased the amount of cadmium in the body. Because cadmium is nephrotoxic (Murakami et al., 1983; Nordberg, 1984), the high amount of cadmium could have induced renal damage which could have sped up cadmium elimination. In man, the renal tubular damage induced by cadmium shortens the half-life of cadmium (7 years instead of 16 years (Ja¨rup et al 1983)). The clearance of cadmium in milk was low and changed over time. It was the highest during a short period at the beginning of lactation. Its maximum (Clm(max)) was 9.1 to 14.5% of the blood clearance. It then decreased rapidly for a few weeks, reaching values close to the milk production. In milk, cadmium is bound preferentially to protein (casein and albumin) (Van Bruwaene, 1982). The number of proteins in milk decreases in the first weeks of lactation (Ruckebush, 1974; Coulon, 1994), which could explain why the clearance of cadmium in milk decreased rapidly. After these first weeks, the clearance of cadmium in milk decreased slowly. Over the entire lactation period, the clearance in milk averaged 2 to 5% of the blood clearance. The clearances in milk, either maximum or average, were low compared to total clearances. The elimination of cadmium through the ewes’ milk did not explain the higher blood clearance observed during lactation. The clearances other than in milk (Cl − Clm(max)) of these ewes remained significantly, higher (P < 0.01) than blood clearance of the nonlactating ewes. The lactating ewes had greater capacities to clear their blood than nonlactating ewes besides the elimination of cadmium through milk. These parameters allowed us to estimate the percentage of orally administered cadmium excreted in milk during the entire lactation period, which was 0.017 ± 0.015%. In the cow, this percentage was estimated to be less than 0.02% (Task Group, 1973).
CONCLUSION
In the nonlactating ewe the bioavailability of cadmium was very low, the distribution of cadmium in the body was very large, and cadmium was slowly eliminated. In the lactating ewe the bioavailability was higher. The mean residence time in the body was close to that of nonlactating ewes despite greater blood purification capacities because the distribution of cadmium in the body was larger. The elimination of cadmium through milk was low and did not explain the higher blood purification capacities observed during lactation. Using a semisimultaneous toxicokinetic approach, we could accurately estimate cadmium absorption, distribution, elimination through milk, and body elimination in these two groups of ewes. In future studies we can use this approach to test methods which would decrease cadmium availability, retention in tissues, and excretion in milk, to reduce the contamination of animalbased food products. ACKNOWLEDGMENTS We thank Professor P.L. Toutain for his contribution to the toxicokinetic analysis of the results. We are very grateful to M. Mirman and his team who supervised the herd during the study period.
REFERENCES Abdulla, M., and Chmielnicka, J. (1990). New aspects on the distribution and metabolism of essential trace elements after dietary exposure to toxic metals. Biol. Trace Elem. Res. 23, 25– 53. Andersen, O., Nielsen, J. B., and Nordberg, G. G. (1992). Factors affecting the intestinal uptake of cadmium from the diet. In ‘‘Cadmium in the Human Environment: Toxicity and Carcinogenicity’’ (G. F. Nordberg, R. F. M. Herber, and L. Alessio, Eds.), Vol. 118, pp. 173–187. IARC Scientific Publications, Lyon. Batey, R., and Gallagher, N. (1977). Effect of iron stores and hysterectomy on iron absorption and distribution in pregnant mice. Am. J. Physiol. 232, 57–61. Bhattacharyya, M. H., Whelton, B. D., and Peterson, D. P. (1981). Gastrointestinal absorption of cadmium in mice during gestation and lactation: I. Short-term exposure studies. Toxicol. Appl. Pharmacol. 61, 335–342. Bhattacharyya, M. H., Whelton, B. D., and Peterson, D. P. (1982). Gastrointestinal absorption of cadmium in mice during gestation and lactation: II: Continuous exposure studies. Toxicol. Appl. Pharmacol. 66, 368–375. Boyne, R., Fell, B. F., and Robb, I. (1966). The surface area of the intestinal mucosa in the lactating rat. J. Physiol. 183, 570–575. Brady, F. O. (1991). Induction of metallothionein rats. In ‘‘Methods in Enzymology: Metallobiochemistry,’’ Part B, ‘‘Metallothionein and Related Molecules’’ (J. F. Riordan and B. L. Vallee, Eds.), Vol. 205, pp. 559–567 Academic Press, San Diego. Campbell, R. M., and Fell, B. F. (1964). Gastrointestinal hyper-
150
HOUPERT, FEDERSPIEL, AND MILHAUD
trophy in the lactating rat and its relation to food intake. J. Physiol. 171, 90–97. Coulon, J. B. (1994). Effet du stade physiologique et de la saison sur la composition chimique du lait de vache et ses caracte´ristiques technologiques. Rec. Med. Vet. 170, 367–374. Foulkes, E. C., and McMullen, D. M. (1986). Endogenous metallothionein as determinant of intestinal cadmium absorption: A reevaluation. Toxicology 38, 285–291. Fox, M. R. S. (1979). Nutritional influences on metal toxicity: Cadmium as a model toxic element. Environ. Health Perspect. 29, 95–104. Halloran, B. P., and De Luca, H. F. (1980). Calcium transport in the small intestine during pregnancy and lactation. Am. J. Physiol. 239, 64–68. Houpert, P., Mehennaoui, S., Joseph-Enriquez, B., Federspiel, B., and Milhaud, G. (1995). Pharmacokinetics of cadmium following intravenous and oral administration to non-lactating ewes. Vet. Res. 26, 145–154. Ja¨rup, L., Rogenfelt, A. R., Elinder, C. G., Nogawa, K., and Kjellstro¨m, T. (1983). Biological half-time of cadmium in the blood of workers after cessation of exposure. Scand. J. Work Environ. Health. 9, 327–331. Johnson, D. E., Kienholtz, E. W., Baxter, J. C., Spangler, E., and Ward, G. M. (1981). Heavy metal retention in tissues of cattle fed high cadmium sewage sludge. J. Anim. Sci. 52, 108–114. Jonah, M. M., and Bhattacharyya, M. H. (1989). Early changes in the tissue distribution of cadmium after oral but not intravenous cadmium exposure. Toxicology 58, 325–338. Kostial, K., and Momtilovic, B. (1972). The effect of lactation on the absorption of 209Pb and 47Ca in rats. Health Phys. 23, 383– 384. Lauwerys, R., Amery, A., Bernard, A., Bruaux, P., Buchet, J. P., Clayes, F., De Plaen, P., Ducoffre, G., Fagard, R., Lijnen, P., Nick, L., Roels, H., Rondia, D., Saint-Remy, A., Sartor, F., and Staessen, J. (1990). Health effects of environmental exposure to cadmium: Objectives, design and organization of the cadmibell study: A cross-sectional morbidity study carried out in Belgium from 1985 to 1989. Environ. Health Perspect. 87, 283–289. Miller, W. J., Blackmon, D. M., Gentry, R. P., and Pate, F. M. (1969). Effect of dietary cadmium on tissue distribution of cadmium following a single oral dose in young goats. J. Dairy Sci. 52, 2029–2035. Murakami, M., Cain, K., and Webb, M. (1983). Cadmiummetallothionein-induced nephropathy: A morphological and autoradiographic study of cadmium distribution, the development of tubular damage and subsequent cell regeneration. J. Appl. Toxicol 3, 237–244. Neathery, N. W., Miller, W. J., Gentry, R. P., Stake, P. E., and Blackman, D. M. (1974). Cadmium-109 and methyl mercury-
203 metabolism, tissue distribution and secretion into milk of cows. J. Dairy Sci. 57, 1177–1183. Nomiyama, K. (1978). Experimental studies on animals: In vivo experiments. In ‘‘Cadmium Studies in Japan: A Review’’ (K. Tsuchiya, Ed.), pp. 47–86 Elsevier Science, Amsterdam/Oxford/ New York. Nordberg, M. (1984). General aspects of cadmium: transport, uptake, metabolism by the kidney. Environ. Health. Perspect. 54, 13–20. Nordberg, G. F., and Nordberg, M. (1987). Different binding forms of cadmium-implications for distribution and toxicity. Univ. Occup. Environ. Health. 20, 153–164. Ohta, H., and Cherian, M. G. (1991). Gastrointestinal absorption of cadmium and metallothionein. Toxicology 107, 63–72. Robards, K., and Worsfold, P. (1991). Cadmium: Toxicology and analysis. Analyst 116, 205–214. Ruckebusch, Y. (1977). ‘‘Physiologie Pharmacologie The´rapeutique Animales.’’ Maloine SA, Paris. Sarhan, M. J., Roels, H., Lauwerys, R., Reyners, H., and Gianfelici de Reyners, E. (1986). Influence of manganese on the gastrointestinal absorption of cadmium in the rats. J. Appl. Toxicol. 5, 313–316. Sugawara, N., and Sugawara, C. (1987). Role of mucosal metallothionein preinduced by oral Cd or Zn on the intestinal absorption of a subsequent Cd dose. Bull Environ. Contam. Toxicol. 38, 295–299. Task Group on Metal Accumulation (1973). Accumulation of toxic metal with special reference to their absorption, excretion and biological half-times. Environ. Physiol. Biochem. 3, 65–107. Van Bruwaene, R., Gerber, G. B., Kirchmann, R., and Colard, J. (1982). Transfert and distribution of radioactive cadmium of dairy cows. Int J. Environ. Stud. 19, 47–51. Van Bruwaene, R., Kirchmann, R., and Impens, R. (1984). Cadmium contamination in agriculture and zootechnology. Experencia (Basel) 40, 43–52. W.H.O. (1992). ‘‘Cadmium Environmental Health Criteria 134,’’ World Health Organization, Geneva. Wijnand, H. P. (1992). The determination of the absolute bioavailability for drug substances with long elimination half-lives (with PC-programs for the method of truncated areas). Comput. Methods Programs Biomed. 39, 61–73. Yamaoka, K., Nakawaga, T., and Uno, T. (1978). Application of Akaike’s information criterion (ALC) in the evaluation of the mean pharmacokinetic equations. J. Pharmacokinetic Biopharm. 6, 165–175. Yamaoka, K., Tanigawara, Y., and Nakawaga, T. (1981). A pharmaco-kinetic analysis program (Multi) for microcomputer. J. Pharmacobio-Dyn. 4, 879–889.