Arch Toxicol (1996) 70:237-243

9 Springer-Veflag 1996

Ira P a l m i n g e r H a l l r n 9 L e i f N o r r g r e n Agneta O s k a r s s o n

Distribution of lead in lactating mice and suckling offspring with special emphasis on the mammary gland

Received 8 May 1995 / Accepted 16 July 1995

The distribution of lead in lactating mice and suckling offspring was studied with whole body autoradiography at 4 and 24 h after a single intravenous injection of 203pb (50 nmol Pb/kg) to the dams. In the lactating mice on day 14 of lactation, the highest uptake of radioactivity at 4 h after administration was recorded in renal cortex, skeleton and liver. A high uptake was also evident in the mammary gland. At 24 h after administration, the radioactivity had decreased in most organs except in the skeleton. In the suckling pups, exposed to lead only via dams' milk for 24 h, the highest level of radioactivity was present in the intestinal mucosa and a much lower level of radioactivity was present in the skeleton. The mammary glands from mice given three daily intravenous injections of 240 I.tmol Pb/kg were examined with X-ray microanalysis. At 4 h after the last injection, lead was found associated with casein micelles both inside the alveolar cell and in the milk lumen, indicating that lead is excreted into the milk, bound to casein, via the Golgi secretory system.

Abstract

Key words

Lead 9 Autoradiography 9 Sucklings 9

Mammary gland 9 X-ray microanalysis

Introduction During the neonatal period, breast milk is the most important food for the infant due to its nutritional and immunological properties. Unfortunately, it can also be a source of

I. Palminger Hallrn ( ~ ) Toxicology Division, Swedish National Food Administration, Box 622, S-751 26 Uppsala, Sweden L. Norrgren Department of Pathology, Swedish University of Agricultural Sciences, Faculty of Veterinary Medicine, S-750 07 Uppsala, Sweden A. Oskarsson Department of Food Hygiene, Swedish University of Agricultural Sciences, Faculty of Veterinary Medicine, S-750 07 Uppsala, Sweden

exposure to undesired pollutants. Lead has been found in milk of several species, including humans (Rabinowitz et al. 1985; Namihira et al. 1993). In general, the levels of lead in milk are low and may not pose a health risk to the breast fed infant, except in certain contaminated areas where high lead levels have been found in human milk (Namihira et al. 1993). The effects on neonates exposed to lead via milk is not known, although pre- and postnatal lead exposure has been connected with effects on neurobehavioural development in children (Hammond 1991). Lactation represents an altered physiological condition which may influence the kinetics of lead in the body. Lactating mice have a higher elimination of lead from plasma compared with nonlactating mice, partly due to an additional route of elimination, i.e. milk (Palminger Hallrn et al. 1995). Consequently, high levels of lead can be transferred to the suckling offspring via milk (Kostial 1974; Keller and Doherty 1980; Palminger HalMn and Oskarsson 1993). The distribution of lead between plasma and blood was also different in lactating compared to nonlactating mice. A lower binding capacity of lead in erythrocytes was evident in lactating mice compared with the nonlactating animals. The consequences on the disposition of lead in the whole body due to these changes have not been examined. Although the location and external form of the mammary gland differ among species, the mechanisms of milk production and secretion are remarkably similar (Neville and Neifert 1983). The excretion of chemical substances into milk can occur via different mechanisms (Walsh and Neville 1994). Many drugs enter the milk by passive diffusion of the unionised forms (Wilson et al. 1980). Essential trace elements are suggested to be transferred to milk by carrier proteins (Neville and Neifert 1983). The manner in which lead is transferred into milk is not known and the amount of lead transferred seems to vary considerably among different species. A similar transport mechanism for lead as for calcium has been suggested for lead by Keller and Doherty (1980), since they found similar milk: plasma ratios of lead and calcium in lactating mice (Keller and Doherty 1980). In many biological systems calcium

238 and lead interact (Simons 1986). In addition, the major part of lead in milk after in vitro or in vivo labelling is b o u n d to the casein fraction (Beach and H e n n i n g 1988; Palminger Hallrn and Oskarsson 1995), as is calcium. In the present study, the localisation of lead in m a m m a r y alveolar cells was studied in lead exposed mice with X-ray micro analysis. In addition, the disposition of 203pb in lactating mice and in their suckling offspring, exposed to lead via dams milk, was examined by whole body autoradiography.

Materials and methods Animals BSVS mice with litters, bred at the Toxicology Division, National Food Administration (Uppsala, Sweden) were fed R3 pellets (Ewos AB, Srdertalje, Sweden) and given tap water ad libitum. The mice were housed in individual cages and kept behind strict hygenic barriers at 23 • 1 ~ with 50-60% humidity and a 12 h: 12 h light : dark cycle. Experimental procedure 203pb was obtained from the The Svedberg Laboratory, Uppsala University, Sweden. The specific activity was 0.32 mCi/nmol Pb and the isotope was supplemented with unlabelled lead acetate [(CH3COO)2Pb.31-120]. On day 14 of lactation, eight mice with eight pups per litter were given 50 nmol Pb/kg (20 I-tCi203pb) intravenously in a tail vein. At 4 or 24 h after injection the animals were exsanguinated by cardiac puncture during ether anesthesia. Whole blood and a number of maternal and fetal organs were collected. Erythrocytes and plasma were separated after centrifugation at 1800 rpm for 15 rain. The radioactivity in all samples was measured in a gamma counter (Nuclear Chicago, Model 1185) using the characteristic line of 279 keV photon emission with a counting efficiency of 60%. The activity obtained was corrected for decay using a TV2 value for 203pb of 52.1 h. A 203Pb standard, with known content of radioactivity, was counted at each counting session to check the counting efficiency. Two mice were administered 150 BCi 2~ (50 nmol Pb/kg) and used for whole body autoradiography, according to the method described fly Ullberg (1977). At 4 and 24 b after injection the mice and two pups from the litter exposed for 24 h were killed with CO2 asphyxia and embedded in an aqueous gel of carboxymethyl cellulose and frozen in hexane cooled with dry ice. Sagittal whole body sections (20 p.m) attached onto tape were taken through the whole animals in a cryostat at -20 ~ The tape sections were freeze dried at -20 ~ whereafter the sections were apposed against X-ray films (Stukturix D4, Agfa Geavert, Belgium) for 9 days at 4 ~ Sections and films were then separated and the films were developed at standarized conditions, fixed, rinsed and dried.

Results Distribution of lead in lactating mice and suckling pups The distribution pattern of 203pb at 4 h after an i. v. injection to lactating mice was characterized by a high uptake of radioactivity in the renal cortex, skeleton and liver. Radioactivity was also present in the m a m m a r y gland (Fig. 1 a). The lungs, which are rich in blood, showed a similar level of radioactivity as the blood. Table 1 shows that erythrocytes had a high and similar concentration of lead as levels found in femur and liver. At 24 h after injection the activity had decreased in most of the organs except in the skeleton. Also, in m a m m a r y gland a significant decrease in lead concentration was seen (Table 1, Fig. 1 b). In the neonates exposed continuously via milk, an increase of radioactivity in all organs was evident between 4 and 24 h, owing to the longer exposure period (Table 1). The autoradiograms of the suckling pups at 24 h revealed a high accumulation of lead in the gastrointestinal tract, mainly the intestinal mucosa, and in the skeleton (Fig. 2a, b). Low concentrations of lead were found in the brain of both dams and offspring.

Ultrastructural morphology and distribution of lead in m a m m a r y gland In the m a m m a r y gland, milk is produced by mammary epithelial cells that line the milk alveolar lumen. In the m a m m a r y gland blood vessels are also present. In the epithelial cells and in milk alveolar lumen casein micelles and milk lipid globules are evident (Fig. 3). In the mammary alveolar cells lead was detected in the casein micelles inside the cells (Fig. 4, arrow). No lead was detected in the cytoplasm outside the micelle vacuole. In the milk alveolar lumen a low amount of lead was also found within the casein micelles (Fig. 4, arrowhead), whereas no lead was Table 1 Distribution of lead in maternal and pups' organ 4 and 24 h after intravenous injection of 20 laCi Pb (50 nmol/kg) in lactating mice. The concentrations are expressed in ng per g or I.tl _+ SD

Maternal organs

Transmission electronmicroscopy (TEM) and X-ray microanalysis On day 11 of lactation two BSVS mice with litters were given three consecutive intravenous injections of 240 I.tmol Pb/kg per day for 3 days. Four hours after the last injection, pieces of mammary gland were dissected and fixed for approximately 4 h in a cold mixture of 15% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M phosphate buffer. After 1 h post-fixation in OsO4 and dehydration up to absolute ethanol, the specimens were passed through propyleneoxide and embedded in Agar 100. Ultrathin sections were cut with a diamond knife, double stained with uranyl acetate and lead citrate and examined in a Philips TEM 420. In order to reveal the presence of lead deposits in the tissue, specimens neither post-fixed in OsO4 nor contrast-stained, were analysed by energy dispersive X-ray analysis (EDXA) with a Philips TEM 400 equipped with a LINK QX 200.

Kidney Femur Liver Lung Brain Mammary gland Erythrocytes Plasma Pups' organs

Kidney Femur Liver Lung Brain Blood

4h

24h

(n = 4) 86.1 +_5.4 28.7 + 4.4 23.6 _+2.5 8.5 +-0.9 0.3 +_0.06 10.1 _+1.7 26.9 +_1.3 0.2 ___0.02

(n = 4) 29.7 __+4.0 31.7 +_5.7 9.9 +_1.5 3.9 _+1.1 0.3 _+0.08 1.7 +-0.3 9.3 +-0.9 0.08 +-0.003

(n = 8) 0.2 +_0.04 0.3 • 0.2 +0.04 0.07 +_0.02 0.01 +_0.004 0.08 +_0.01

(n = 8) 1.3 +_0.2 9.1 • 2.0 +_0.6 0.5 _0.1 0.06 ----0.02 1.5 +_0.5

239

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Liver detected in the surroundings. No lead inclusion bodies were found in the epithelial cells of the mammary gland. An Xray spectrum is shown in Fig. 5, displaying three peaks of lead.

Discussion In the present study the disposition of lead in lactating mice and suckling offspring was examined. A high uptake of lead in the skeleton of lactating mice was shown at 4 h after an intravenous injection and no decrease was evident after 24 h indicating a high retention of lead in the skeleton. This has previously been shown also in pregnant mice (Danielsson et al. 1984). At 4 h after administration a high uptake of lead was found in the mammary gland. After 24 h the level of radioactivity in mammary gland had markedly decreased, indicating that there was no obvious retention of

Mammary gland Fig. 1 Whole-bodyautoradiograms of lactating BSVS mice on day 14 of lactation at 4 (a) and 24 (b) h after intravenous injection of 203Pb (50 nmol Pb/kg per 150 laCi) lead in the mammary gland. Cadmium, in contrast, is known to be accumulated in the mammary gland (Lucis et al. 1972) which might explain why the excretion of cadmium to breast milk is very low, as shown by a milk: plasma ratio below 1 (Pietrzak-Flies et al. 1978). The milk:plasma ratio of lead have been reported to be about 100 in rodents (Palminger Hali6n and Oskarsson 1993) showing that the lead is effectively transported to milk, rather than being retained in the mammary gland. In liver and kidney, nuclear inclusion bodies of lead have been shown in previous studies at similar exposure levels (Oskarsson and Johansson 1987). The absence of nuclear inclusion bodies in the mammary gland indicate an efficient transport of lead into milk as well.

240

A

Brain

Skeleton

Skeleton

Liver

Gastrointestinal tract

Gastrointestinal tract

B

~.~ ~dL~ .~-.

Fig. 2 Whole-body autoradiogram of two suckling mice exposed to lead via their dams milk during 24 h (a). Details of the gastrointestinal tract of a suckling mouse illustrating the activity of 2o3Pb in intestinal mucosa (b)

Danielsson et al. (1984) studied the placental transfer and fetal distribution of lead by autoradiography. They found lower levels of lead in fetal tissues than in maternal tissues, with the highest uptake in the fetal skeleton and liver. In the present study, the highest radioactivity in the suckling offspring was found in intestinal mucosa and in skeleton. In a previous study in infant rats, a high accumulation of lead was found in the distal parts of intestinal mucosa, i.e. the ileum, after exposure to lead in milk (Palminger Hall6n and Oskarsson 1995). Ileum has a high capacity for nonspecific pinocytosis and lead associated to milk casein seems to be accumulated at this site. It is not known if the lead is subsequently absorbed into the blood or if it is retained in the epithelial ceils until their desquamation, but it has been proposed that this site of accumulation does not represent a site of lead absorption into the circulation (Mushak 1990). However, this is in contradic-

+:+.

+1~

Intestinal mucosa tion by previous results in suckling mice, where a lead absorption at the ileal site is strongly supported (Palminger Hall6n et al. 1996). After an intravenous injection of lead to lactating mice, the highest dose of lead via milk was delivered within 24 h, but the highest blood concentrations of lead in suckling pups did not occur until 74 h after administration to the dams, supporting a mechanism of delayed absorption of lead in the suckling due to an accumulation of lead, bound to casein, in the intestine. The central nervous system is especially sensitive to lead. Neurobehavioural disorders due to perinatal exposure to low levels of lead have been reported in several human studies (Bellinger et al. 1987, 1990; Leviton et al. 1993). In immature rodents a high uptake of lead in the brain has been demonstrated (Momcilovic and Kostial 1974; Livesey et al. 1986). In the present study, no specific accumulation of lead in the central nervous system was found in the

241

Fig. 3 Ultrastructural picture of mouse mammary gland at day 14 of lactation after three daily i.v. injections of 240 lamol Pb/kg. Epithelial cells (ec) and blood vessels (by) are shown. In the cells and in milk alveolar lumen (ml) casein micelles (mc) and milk fat globules (mfg) are present. Magnification x 3000

Fig. 4 Ultrastructural picture of mouse mammary epithelial cell at day 14 of lactation after three daily i.v. injections of 240 lamol Pb/kg. Secretory vesicles are present inside the epithelial cell with lead associated (shown by X-ray microanalysis) to casein micelles (arrow). Also in the milk alveolar lumen, lead combined with casein micelles is present (arrowhead), (n. nucleus, c, cytoplasm, sv. secretory vesicle, ml milk lumen). Magnification x 10000

sucklings, on the other hand, maximal tissue concentrations of lead had probably not yet been attained. Lead was associated with casein in the mammary epithelial cell as well as in milk lumen of mouse mammary

gland after three intravenous injections of lead to lactating mice. The high doses of lead were necessary, since some loss of lead was unavoidable during the sample preparation for the X-ray microanalysis. Most likely, lead is transported into milk by the same mechanisms as calcium, since these metal interacts in many biological systems (Simons 1986). Calcium is mainly excreted into milk bound to casein by the Golgi secretory system (Neville and Peaker 1979). Casein is a protein complex containing phosphate groups that bind calcium to a high extent. Casein is synthesised on ribosomes of the endoplasmatic reticulum and transported to the Golgi compartment. In the Golgi system the casein is aggregated into micelles with incorporation of calcium, and these micelles are subsequently secreted into milk by exocytosis (Farrell 1973; Greenberg et al. 1976; McMahon et al. 1984). This phenomenon has been shown ultrastructurally by Dylewska (1983). According to the results of the X-ray microanalysis, a transfer mechanism for transport of lead into milk could be postulated (Fig. 6). In the alveolar cell, lead was found associated with casein micelles inside the Golgi secretory vesicle, indicating that lead can be transferred into the Golgi compartment where it associates with casein and subsequently is excreted into milk. Alternatively, lead might also penetrate water filled pores across the cells and thus reach the milk alveolar lumen, since this is a possible way for small ionised molecules (Wilson et ai. 1980). However, due to the high affinity of lead to casein only low concentrations of unbound lead may be present in the cell cytoplasm and in the milk. In rat milk labelled with 203Pb, in vitro and in vivo, lead was bound to casein to more than 90% (Beach and Henning 1988; Palminger Hall6n and Oskarsson 1995). A third possibility is the transfer with carrier proteins from plasma to milk although this must be considered as a minor mechanism, since the plasma protein content in milk generally is low compared to other milk proteins (Mepham 1987). The binding of lead to casein seems to have a great impact for milk excretion of lead. Thus, the various casein concentrations in milk from different species can explain the observed differences in lead excretion into milk in these species. In rats and mice with a high lead excretion into milk, the casein concentration in milk is indeed very high, about 6 0 - 9 0 mg casein/ml milk (Luckey et al. 1954; Beach and Henning 1988; Palminger Hall6n and Oskarsson 1995). Human milk, in contrast, has a much lower casein content (Ltinnerdal and Forsum 1985; Stebler and Guentert 1990), 2 - 6 mg casein/ml, and the lowest total calcium content of the species studied (Barltrop and Hillier 1974; Vaughan et al. 1979). Human milk has also, as far as is known from the literature, the lowest excretion of lead into milk (Paiminger Hall6n et al. 1996). In conclusion, the present study demonstrated a high disposition of lead in kidney, skeleton, liver and mammary gland of lactating mice. In suckling pups exposed to lead only via milk a high accumulation of lead was evident in the intestinal mucosa that most likely cause a delay in the absorption of lead into the circulatory system in the pups. This study also demonstrated that lead is excreted into milk

242

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References

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Fig. 6 Suggested pathways for excretion of lead into milk. a Inside the mammary alveolar cell, lead are transferred to the Golgi compartment and combined with casein. Casein micelles (cm) are formed and excreted into milk via secretory vesicles (sv). b, Lead is transported across the epithelial cell, reaches the milk and associates with casein

associated to c a s e i n b y the G o l g i secretory system, suggesti n g that the m i l k c o m p o s i t i o n o f various species i n f l u e n c e s the e x c r e t i o n o f lead i n milk. The technical assistance of Mr. L. Ljung is gratefully acknowledged.

Acknowledgement

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