ToxSci Advance Access originally published online on June 30, 2008
Toxicological Sciences 2008 105(2):260-274; doi:10.1093/toxsci/kfn124
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Dynamic Changes in Lipids and Proteins of Maternal, Fetal, and Pup Blood and Milk during Perinatal Development in CD and Wistar Rats
The Dow Chemical Company, Midland, Michigan 48674
1 To whom correspondence should be addressed at Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Building 1803, Midland, MI 48674. Fax: (989) 638-9863. E-mail: mmarty{at}dow.com.
Received March 9, 2008; accepted May 26, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSUPPLEMENTARY DATAREFERENCES |
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An understanding of the physiological factors that regulate perinatal dosimetry is essential to improve the ability of physiologically based (PB) pharmacokinetic (PK) models to predict chemical risks to children. However, the impact of changing maternal/offspring physiology on PK during gestation and lactation remains poorly understood. This research determined lipid and protein changes in blood, milk and amniotic fluid of CD and Wistar dams, fetuses and neonates to improve the precision of perinatal PBPK modeling. Samples were collected from time-mated CD dams, fetuses, and pups on gestation day (GD) 18 and 20 (sperm positive = GD 0) or lactation day 0 (day of birth), 1, 3, 5, 10, 15, and 20 (n
5 per time point). Fewer time points were sampled in Wistar rats, which showed similar patterns to CDs. Relative to nonpregnant dams, maternal serum protein levels (albumin, total protein and globulin) each decreased by 
20% during late gestation, whereas maternal serum lipids (triglycerides, low density lipoproteins, and phospholipids) increased up to fourfold. These physiological changes can impact maternal PK of both protein-bound and lipophilic chemicals. During lactation, triglycerides in milk were greater than 100-fold higher than maternal serum, favoring the disposition of lipophilic chemicals into milk and potentially increasing neonatal rodent exposure during critical stages of postnatal development. Serum protein levels in pups were two- to threefold lower than adults at birth, which may increase the bioavailability of protein-bound compounds. These data will aid in the interpretation of perinatal toxicity studies and improve the accuracy of predictive perinatal PBPK models. Key Words: pharmacokinetics; perinatal; gestation; lactation; lipids; proteins; pregnancy.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSUPPLEMENTARY DATAREFERENCES |
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Since passage of the Food Quality Protection Act (1996)
and Safe Drinking Water Act (1996), there has been increased attention on children's exposures to environmental chemicals and whether these exposures may cause adverse health effects in infants, children or subsequently, in adults. Of great import to this issue is whether children are more sensitive to chemical exposures than adults. Differential sensitivity between adults and children may occur due to differences in pharmacokinetics (PK), pharmacodynamics, or both. PK differences between children and adults arise when the same exposure results in the presence of a higher or lower internal dose at the target site(s) in children relative to adults. However, it is often difficult to predict these PK differences due to limited data on human exposure, internal dosimetry and dose response. Typically, data from animal studies are used to estimate dose-response relationships and these data, coupled with physiologically based pharmacokinetic (PBPK) models, can estimate the potential risk to humans.
Compared with adult models, perinatal PBPK models require greater complexity to describe the internal dosimetry in the mother, fetus and child. To improve the accuracy of these perinatal PBPK models, consideration must be given to the dynamic physiological changes occurring in animal models during the perinatal period. For example, limited data indicate that serum chemistry parameters, amniotic fluid and milk composition vary in the rat dam, fetus and neonate during critical developmental stages (LaBorde et al., 1999; Liberati et al., 2004) and these factors can impact chemical disposition (e.g., Byczkowski et al., 1994; Stock et al., 1980). A greater understanding of perinatal physiological changes is needed to improve the accuracy of existing gestational/lactational PBPK models (Corley et al., 2003; Lee, 2005). Furthermore, increased knowledge of the temporal changes in physiological parameters in the pregnant dam, fetus and neonate will aid in the selection of appropriate kinetic sampling strategies (e.g., optimal sampling times, compartments, etc.). These sampling strategies are critical for PK-based testing strategies, such as the International Life Sciences Institute Health and Environmental Sciences Institute agricultural chemical safety assessment program that incorporates PK sampling at various life stages (Cooper et al., 2006).
The current study addresses data gaps for several biochemical parameters, including lipid fractions and major proteins, which are likely to impact perinatal dosimetry. Proteins and lipid levels in blood, milk, and amniotic fluid, in addition to pH levels in milk and amniotic fluid, were determined throughout late gestation and the early postnatal period. Data were collected from dams, fetuses and neonates in both CD and Wistar rats, two strains routinely used in reproductive and developmental toxicity testing. Data collection was designed to replicate time points reported in previous studies to verify their reproducibility as well as new time points and parameters not examined previously.
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METHODS |
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Animals and husbandry.
Adult, time-mated and nonpregnant (NP) female Crl:CD(SD) (CD; Sprague-Dawley derived) and Wistar (Han) rats were obtained from Charles River Laboratories, Inc. (Portage, MI and Raleigh, NC, respectively). CD dams were 10–11 weeks of age and weighed 200–250 g. Wistar (Han) dams were 8–10 weeks of age and weighed 175–225 g. Pregnant dams were received on gestation day (GD) 4–9 (sperm positive = GD 0) and allowed to acclimate to the laboratory until the designated GD when samples were collected. Dams used for lactation time points were allowed to naturally deliver their litters (day of birth was designated as postnatal day [PND] 0). On PND 4, CD litters were culled to 8 pups/litter, retaining four male and four female offspring whenever possible. Wistar litters were not culled. NP rats and pregnant dams up to GD 19 were singly housed in stainless steel cages. On GD 19 and thereafter, time-mated dams were housed one per cage (with their litter) in plastic cages provided with ground corn cob nesting material until terminal blood samples were collected. Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St Louis, MO) in pelleted form. Feed and municipal water were provided ad libitum. The animal room was maintained at 22 ± 3°C, 40–70% humidity and had a 12-h light:dark cycle. The animal activities required for the conduct of this study were approved by the Institutional Animal Care and Use Committee. The laboratory is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
Maternal, fetal, and pup blood and tissue collection.
Adult and neonatal CD and Wistar rats were anesthetized by isoflurane inhalation. Fetuses were anesthetized concurrent with the isoflurane-exposed dams (Wixom and Smiler, 1997). Once deep anesthesia was reached, terminal blood samples were collected from dams by cardiac puncture or via the inferior vena cava. Fetal and pup blood were collected by nicking the left ventricle of the heart. Fetuses/pups were tilted slightly anteriorly to keep pooled blood in the thoracic cavity while it was collected in heparin-coated capillary tubes. Samples of fetal and neonatal blood were pooled by litter to obtain sufficient volume for analysis. Blood from dams and fetuses/pups were collected on GD 18, 20, lactation day (LD)/PND 1 and 5 (Wistar rats) and GD 18, 19, 20, LD/PND 0, 1, 3, 5, 10, 15, and 20 (CD rats) (Table 1). Fetal/pup samples were collected in the morning from 7 to 11 AM with the exception of PND 0 pups. Timing of sample collection may be important as adult rats eat primarily at night (Saghir et al., 2006), which would impact fetal nutrition and milk composition for pups. PND 0 pups were euthanized within 10 h of birth to optimize potential differences between PND 0 and 1 samples. Dam, fetal and pup sera were separated from whole blood by centrifugation using serum separator tubes. After terminal blood collection, fetal and pup brain, liver, kidney and lung tissues were harvested and weighed.
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Amniotic fluid collection.
Amniotic fluid from pregnant CD and Wistar dams was collected on GD 18 and 20 by anesthetizing the animals, exteriorizing the uterine horns and penetrating the amniotic cavity with a 25-gauge needle attached to a syringe. Fluid was gently drawn into the syringe and transferred to sample tubes for analyses.
Milk collection.
Milk was collected on LD 1 and 5 from Wistar dams to confirm previously published values for milk components. Milk was collected from CD dams on LD 0, 1, 3, 5, 10, 15, and 20 (Table 1). Four hours before milk collection, the pups were separated from the lactating dams and euthanized for blood/tissue collection. This allowed the dams to accumulate milk in the mammary compartments for greater sample yields. Approximately 10–15 min before milk collection, dams were administered 8 US pharmacopeia oxytocin i.p. in two split doses, 5–10 min apart. Approximately five minutes before milk collection (after the second oxytocin dose), the dams were anesthetized by inhalation of isoflurane. Milk was collected from the nipples of the animals by gently squeezing the gland and the nipple area. Approximately, 500–1000 µl of milk were collected per animal in nonheparinized hematocrit tubes. After milk collection, dams were euthanized and blood samples were obtained.
Analyte determination.
Upon collection, blood from dams, NP rats, fetuses and pups, amniotic fluid and milk specimens were placed on ice and shipped overnight to Anilytics, Inc. (Gaithersburg, MD). The parameters listed in Table 1 were analyzed using a Hitachi 717 analyzer. In both blood and milk, lipoproteins (high density lipoproteins—HDL, low density lipoproteins—LDL) and phospholipids were characterized only in late gestation and the early postnatal period (PND 0–5) to coincide with marked lipid changes in maternal blood and milk over this period. LDL and HDL cholesterol and triglycerides were converted from mg/dl to mmol/l using the average molecular weights of 390 and 890 g/mol, respectively. The pH values were determined on a Corning Model 278 blood gas analyzer (MidMichigan Regional Medical Center, Midland, MI) within 2 h of collection. Samples used for pH measurements were collected directly into capillary tubes (milk) or into a syringe then transferred into capillary tubes (amniotic fluid) to minimize exposure to atmospheric gases that could alter pH.
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RESULTS |
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Impact of Rat Strain on Perinatal Physiological Parameters
In most cases, data in Wistar rats did not differ appreciably from data collected in CD rats and/or published data; thus, the limited Wistar data are included online as supplemental data. In cases where differences were noted, these differences are highlighted in the text of this manuscript. Tables of Wistar and CD data also are available online as supplemental data.
Clinical Chemistry Parameters in Maternal, Fetal, and Pup Serum throughout Late Gestation and Lactation
Trends for changing serum lipid and protein levels in dams and fetuses/pups are summarized in Table 2. Graphs illustrating the temporal changes of individual parameters are illustrated in Figures 1 and 2.
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Maternal serum.
Over the time points analyzed, the largest changes in maternal serum parameters compared with NP dams occurred in lipid and protein levels during late gestation (Fig. 1). Generally, maternal serum lipid levels increased toward the end of gestation and decreased to NP levels around the time of delivery. Cholesterol levels in mated CD rats were similar to NP levels over the entire course of late gestation and lactation (Fig. 1A), whereas Wistar rat serum cholesterol increased by 41% during late gestation (GD 20; supplemental data). The increased cholesterol levels in Wistar dams returned to NP levels shortly after delivery. Peak triglyceride levels in dams of both strains were elevated fourfold on GD 18 compared with NP levels (3.77 ± 1.51 vs. 0.99 ± 0.41 mmol/l in CD rats; Fig. 1B). Similarly, LDL values increased approximately twofold over the GD 18–20 interval in both CD and Wistar rats, reaching peak values of 0.26 mmol/l in pregnant CD rats compared with 0.12 mmol/l in NP CD rats (Fig. 1C). Triglycerides and LDL returned to pregestational levels around the time of delivery and remained similar to NP levels throughout lactation. Relative to NP values, HDL levels were not altered in either strain during gestation and showed only minimal increases (
20%) early in lactation (Fig. 1D). Phospholipids increased by 23% by GD 20 in CD dams (Fig. 1E) but decreased (Wistars) or were decreasing (CDs) toward NP values on LD 5, the last time point for phospholipid measurements.
Generally, serum protein levels were decreased in CD rats toward the end of gestation, a result that is consistent with the gestational increase in blood volume and subsequent hemodilution of blood proteins (Barron, 1987; Krauer, 1987). By GD 20, each of the measured proteins (albumin, total protein and globulin) decreased by 19% in CD rats, indicating that less protein may be available for binding xenobiotics during gestation compared with a NP animal (Figs. 1F–H). On LD 0–1, these proteins quickly reestablished levels similar to NP animals and then slightly decreased again during the remainder of the lactation period relative to NP values. Serum protein levels showed slightly different responses in Wistar rats, where minimal protein changes (total protein, albumin and globulin) occurred from GD 18 to LD 5.
Fetal and pup serum.
Generally, serum lipids (cholesterol, LDL, and HDL) increased during late gestation (Figs. 2A–E), an event that coincides with the high energy demand required for fetal growth as well as energy and substrates for milk synthesis (Herrera, 2000; Knopp et al., 1970). At birth, serum lipids (cholesterol, LDL, HDL, phospholipids) generally decreased. This change coincides with the transition from the high carbohydrate diet of the fetus to the high fat diet of the neonate.
Postnatally, serum lipid levels progressively increased throughout the remaining postnatal period. At birth, the body of a rat pup has stored fuel available for mobilization, particularly with respect to carbohydrates and proteins (Girard et al., 1973). The high fat content of milk, the sole source of nutrition in pups, coupled with the high rate of lipolysis and increased liver lipoprotein lipase activity in neonates, likely contribute to the increase in serum lipids (Harris et al., 1966; Herrera, 2000). Peak cholesterol values in CD pups were 4.27 ± 0.44 mmol/l on PND 15 compared with 1.44 ± 0.20 mmol/l at birth (Fig. 2A). HDL, LDL, and phospholipids in CD pups increased from PND 0 with the smallest increase in LDL (1.5x), an intermediate increase in phospholipids (1.9x) and the greatest increase in HDL levels (2.8x) (Figs. 2C–E). Peak postnatal levels for HDL and phospholipids exceeded the peak prenatal values. Wistar pups showed similar increases in HDL (1.5x) and phospholipids (1.5x) from PND 1 to PND 5, whereas LDL levels remained relatively stable. Serum triglyceride values, which were relatively stable in CD fetuses during late gestation, were more variable in pups over the course of lactation.
Unlike lipid profiles, serum binding proteins (albumin and globulin) and total protein progressively increased from GD 18 to LD 20 in CD pups, with each parameter increasing 3.0- to 3.5-fold over that period (Figs. 2F–H). These results are consistent with previous reports (Papworth and Clubb, 1995) and reflect protein intake from milk. Proteins protect against infection, serve as carriers for bioactive molecules, have enzymatic activity, and are needed throughout the neonatal period to support pup growth (LaKind et al., 2004). Clinical chemistry parameters in Wistar pups were qualitatively similar to the CD pups, although fewer time points were examined.
Clinical Chemistry Parameters in Amniotic Fluid during Late Gestation
Means and ranges for clinical chemistry parameters in amniotic fluid during late gestation are presented in Table 3 for both CD and Wistar rats. Amniotic fluid lipid (cholesterol and triglycerides) and protein (albumin and total protein) increased 1.8-fold and 4-fold, respectively, from GD 18 to GD 20 in CD pups. Similar increases in lipid and protein levels occurred in Wistar dams. The impact of amniotic fluid parameters on chemical disposition is not well understood.
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Clinical Chemistry Parameters in Milk during Lactation
Changes in CD dam milk composition occurred primarily in the early postnatal period (Fig. 3). Colostrum (milk collected on LD 0 within 10 h of birth) had higher levels of cholesterol, HDL, phospholipids, albumin, and total protein to support pup growth. Levels of these constituents decreased in milk collected on LD 1. The lipids in colostrum originate from maternal circulation (Herrera, 2000
). At LD 1, milk triglyceride, LDL, and phospholipid levels remained high and gradually declined thereafter. Minimal changes occurred in the milk parameters examined throughout the remaining period of lactation (LD 1–20) with the exception of cholesterol. Cholesterol progressively increased throughout lactation, slowly between LD 5 and 15 followed by a more rapid increase between LD 15 and 20. Peak milk levels of cholesterol were attained on LD 20.
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Milk composition in Wistar rats was only evaluated at LD 1 and 5 in order to verify milk composition values previously reported in this strain (Nicholas and Hartmann, 1991
). Milk composition in Wistar rats was qualitatively similar to CD rats.
Fetal and Pup Tissue Weight Changes
Body weights and selected tissue weights for late term fetuses and preweanling CD and Wistar pups are shown in Figure 4. Fetal body and tissue weights on GD 18 and 20 were consistent with previously reported values (O'Flaherty et al., 1992; Olanoff and Anderson, 1980). Regression equations were formulated to describe the time-course changes in body and tissues weights of CD pups from birth to PND 20 (R2 = 0.96–0.99 for all tissues). Equations to describe the time-course changes in body and tissue weights of CD pups as a function of age from birth to PND 20 are provided in Appendix A. Using these equations, body and liver weights were best described as increasing exponentially from birth to PND 20 (Figs. 4A, B), whereas kidney weights were described as increasing linearly (Fig. 4C). Lung and brain weights rapidly increased from birth to PND 10 (lung) or 15 (brain) and then began to plateau through PND 20 (Figs. 4D, E).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSUPPLEMENTARY DATAREFERENCES |
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Several data gaps exist in the current knowledge of normal strain and life-stage specific changes in physiology of the rodent dam, fetus and neonate during perinatal development. These uncertainties provide unique challenges to developing predictive models, such as PBPK models, that sufficiently describe the dynamic physiological and biochemical changes regulating tissue dosimetry. This research determined the time-course changes of specific lipids, binding proteins and other clinical chemistry parameters in blood, milk and amniotic fluid of the CD and Wistar Han dam, fetus, and neonate throughout perinatal development.
A summary of the changes in maternal, fetal, and pup serum is presented in Table 2. Briefly, serum triglycerides, LDL, and phospholipids increased, whereas proteins decreased during late gestation in dams relative to NP females. These maternal serum parameters rapidly returned to pregestational values by birth and remained similar to NP levels throughout lactation. In CD fetuses/pups, serum lipid and protein levels increased during late gestation; however, most lipids decreased just prior to birth. All measured serum parameters increased in pups postnatally to levels greater than those at birth. Wistar fetal and pup data were consistent with responses in the CD rats.
Gestational/Lactational Physiology and Changes in Perinatal Parameters
Lipids.
The results of this study are consistent with the changing physiology of the dam, fetus and pup that occurs during gestation and lactation (Herrera, 2002; Herrera et al., 2000). Rat dams undergo an initial period of anabolism during the first two weeks of gestation. This period is marked by hyperphagia and endocrine changes that support fat deposition (Lopez-Luna et al., 1986). During the third trimester, dams revert to a catabolic state to support rapid fetal growth. Lipolysis is increased, releasing free fatty acids and glycerol from adipose tissue (Knopp et al., 1970). These substrates are metabolized in the liver to triglycerides and ketone bodies; hence, the elevated maternal levels of triglycerides during late gestation. Triglycerides, primarily located in the very low density lipoprotein (VLDL) blood fraction, are elevated as are other higher density lipoproteins involved in their transport (e.g., LDL and HDL) (Alvarez et al., 1996; Montelongo et al., 1992). In the present study, maternal LDL increases were more prominent than those of HDL.
Maternal lipid profiles during late gestation did not necessarily translate to corresponding changes in the fetal lipid profiles. Maternal lipids levels of triglycerides increased in late pregnancy whereas fetal triglyceride values remained constant. Consistent with the results from our study, triglycerides do not cross the placenta intact (Herrera and Lasuncion, 2004); therefore, high maternal triglyceride levels do not translate to high fetal triglyceride values. Instead, the placenta takes up circulating maternal triglycerides, which are metabolized via lipoprotein lipase, and release free fatty acids to the fetus (Herrera and Lasuncion, 2004). Once the placenta releases free fatty acids, these substrates are transported to the fetal liver and converted to triglycerides. In addition, the placenta takes up HDL2 cholesterol and has LDL receptors, consistent with the increase in fetal HDL, cholesterol, and LDL levels prior to term.
After birth, levels of fatty acids from triglycerides associated with VLDL and LDL decline markedly in the dam. Furthermore, circulating triglycerides are transported to the mammary gland where increased lipase activity facilitates milk synthesis (Herrera et al., 2000). Consistent with these changes, our data indicate that maternal serum triglycerides and LDL levels decreased postpartum relative to peak levels in the third trimester of gestation. In contrast, phospholipids and cholesterol undergo only minor changes during the postpartum period. Similar findings for phospholipids and cholesterol have been previously reported (Geursen et al., 1987; Green et al., 1981; LaBorde et al., 1999). Although serum triglycerides decreased postnatally in the dam, triglyceride levels progressively increased in the pup throughout development. This increase in triglycerides likely corresponds to increasing uptake of fatty acids from circulating triglycerides in milk as the pup grows and increased endogenous synthesis of triglycerides during the postnatal period.
Proteins.
Serum albumin level in the dam decreased during late pregnancy but returned to NP levels by LD 0–1. This decrease is likely attributed to hemodilution, which progresses throughout gestation with the most rapid increase in volume during the last week (Barron, 1987; Krauer, 1987). This decrease in blood albumin levels may alter protein binding capacity and result in increased free xenobiotic levels for compounds that are highly albumin bound. This situation may be exacerbated during the third trimester when increasing levels of free fatty acids can compete with xenobiotics for albumin binding. Stock et al. (1980) documented a decrease in drug-protein binding late in gestation in Long-Evans rats, resulting in increases in the free fraction of several drugs in serum (i.e., salicylic acid, sulfisoxazole, phenytoin, dexamethasone, and diazepam). Plasma volume expansion is required during gestation for normal fetal growth and development (Duvekot et al., 1995); however, there is rapid re-equilibration of these maternal physiological changes after parturition. Thus, total serum protein and albumin returned to NP levels 1 day after parturition. This is consistent with the report by Stock et al. (1980), wherein salicylic acid-, sulfisoxazole-, and phenytoin-protein binding return to NP levels at 2 days postpartum.
Extrapolating rat perinatal physiological data to humans.
Many of these perinatal physiological changes also have been documented in humans, including gestational hyperlipidemia (King, 2000; Long, 1961) and hemodilution of serum proteins (Dean et al., 1980). In rats, milk protein levels remain relatively stable during lactation, whereas milk protein levels slowly decrease in women during the first part of lactation and remain stable thereafter (Lonnerdal et al., 1976). Rats have a higher fat composition in milk (12.6–15% fat by weight) compared with humans (4%) (Ecobichon, 1984), which facilitates greater partitioning of lipophilic chemicals into rat milk (Kimmel et al., 1992). Furthermore, rat pups are altricial at birth relative to humans (e.g., Vidair, 2004; Watson et al., 2006; Zoetis and Hurtt, 2003a, b). Hypothetically, this higher milk lipid content, coupled with a more immature developmental state, may put rat pups at increased risk for toxicity during the early neonatal period, particularly for lipophilic chemicals.
Comparison with Published Data on Perinatal Physiological Parameters
Previous studies have examined some maternal/fetal/pup physiological parameters during gestation and lactation in CD dams, fetuses and neonates (LaBorde et al., 1999), in Wistar rats during late gestation (Liberati et al., 2004) and milk composition in Wistar rats during lactation (Nicholas and Hartmann, 1991). The current study replicated some of the previously collected data and extended the available data sets, including the collection of serum and milk parameters from the same animals.
Although the trends were consistent across studies, results of the current study were not in full agreement with previous reports. The time-course changes in lipids in the pregnant dam are consistent with previously published data in CD (LaBorde et al., 1999) and Wistar Han (Liberati et al., 2004) rats; however, serum triglycerides in NP, pregnant and lactating CD dams were approximately twofold higher in the study by LaBorde et al. (1999) compared with our study. Despite this difference in absolute values, both studies reported a fourfold increase in serum triglyceride levels in pregnant rats during late gestation compared with a NP adult.
Another discrepancy among data sets involves milk lipid changes during early lactation. In our study, levels of triglycerides, a major lipid component in milk, remained stable in CD rats during the early postnatal period (LD 0–5) with a slight decline after LD 5; however, a previous report in Wistar rats indicated that milk triglycerides were fourfold higher on LD 0 with a subsequent rapid decrease over the first 5 days of lactation (Nicholas and Hartmann, 1991). In the current study, other milk lipids (cholesterol, HDL, and phospholipids) declined more sharply on LD 0–5. In both studies, dams were milked only once; however, other methodological differences may have contributed to the differences in results (e.g., dam-pup separation time prior to milk collection, oxytocin dose, maternal diet, etc.—Grigor et al., 1986; Keen et al., 1980, 1981). Lastly, milk fat content was estimated from total esterified fatty acid levels in the study by Nicholas and Hartmann (1991) compared with direct analytical measurement in the current study.
Overall, total protein levels in milk were similar to previously reported studies (e.g., Godbole et al., 1981; Grigor et al., 1986); however, albumin levels during the early postnatal period differed from previously reported values. Nicholas and Hartmann (1991) reported a doubling of serum albumin concentrations in the milk within 3 days of parturition, whereas in the current study, albumin levels in milk declined slightly after LD 0. Milk albumin levels in the current study were approximately sevenfold higher on day 0 than the values reported by Nicholas and Hartmann (1991). This result is more consistent with human data indicating higher levels of total protein and albumin in colostrum (LD 0) than more mature milk (Kulski and Hartmann, 1981).
Although the magnitude of changes were consistent with earlier studies, the variability in milk lipid and protein levels across studies highlight the need to standardize milk collection techniques. Standardization would ensure consistency in the measurement of test material and/or metabolites in the milk following chemical exposure. This is especially important when collecting milk samples containing lipophilic chemicals, which will concentrate to a greater extent in milk samples collected during early lactation when lipid levels are highest (e.g., Mattsson et al., 2000). Furthermore, milk levels of lipophilic chemicals may be more variable during early lactation due to the dynamic changes in milk lipids over this period.
Impact of Physiological Changes on Perinatal PK
Given the dynamic physiological processes that occur during gestation and lactation, it is important to consider the impact of these changing variables on the tissue distribution of compounds in the dam, fetus and neonate during perinatal development. Tissue partitioning of compounds is influenced by the characteristics of the compound, including the molecular size and shape, degree of ionization, degree of protein binding, and the relative chemical solubility in the lipid and water fraction of tissues and blood. According to frequently used algorithms to determine tissue partition coefficients, the partitioning into tissues of nonreactive highly lipophilic compounds (i.e., log Poctanol:water [Po:w] > 4) that are not highly protein bound depends on the ratio of neutral lipid fractions in the tissue vs. blood. This ratio determines the tissue partition coefficients rather than the Po:w (Haddad et al., 2000; Poulin and Theil, 1995). As such, the increase that occurs in the neutral lipid fraction of the dam blood during pregnancy will likely decrease the tissue:blood partitioning for highly lipophilic compounds and result in higher blood levels of certain compounds during pregnancy compared with a NP adult. Thus, elevated maternal blood levels of lipophilic chemicals late in gestation may not correspond with total body burden or greater toxicity in the target organs (e.g., Mattsson et al., 2000).
To fully appreciate the impact of perinatal changes on PK, information on the dam and fetal/pup compartments should not be viewed in isolation. Once a chemical has been absorbed systemically by the maternal animal, disposition of that chemical becomes an integrated process with levels fluctuating in maternal serum, fetal/pup serum, and amniotic fluid and/or milk. The characteristics of the chemical, coupled with temporal changes in physiological parameters relative to other compartments, determine the rate of flux of the chemical across compartments. Below are some examples integrating data from different compartments to examine the potential impact of these parameters on chemical disposition during the perinatal period.
Gestation: Maternal and fetal serum
The developing embryo/fetus and the amniotic fluid surrounding it create a unique environment for the conceptus relative to the dam. These differences are evident in the composition of lipids and proteins between maternal serum and fetal serum. For example, serum protein, albumin and globulin levels were two to fivefold higher in maternal serum compared with fetal serum at GD 18–20 (Figs. 5F–H), suggesting an opportunity for greater protein binding of xenobiotics in the dam compared with the fetus. Triglycerides were up to fourfold higher in the dams compared with fetuses, but fetal LDL levels were up to 6.5-fold higher than maternal serum levels (Figs. 5B, D). Differences in other serum lipids were relatively minor, with dams having slightly higher levels of phospholipids and HDL and fetuses having slightly higher levels of cholesterol.
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Data indicate that increased serum lipid levels in dams during late gestation (gestational hyperlipidemia) can impact the partitioning of lipophilic chemicals (Marty et al., 2006
; E. Lowe, personal communication). For example, maternal blood levels of chlorpyrifos, a lipophilic organophosphate pesticide, given at 5 mg/kg/day were 7.5-fold higher prior to birth on GD 20 than after delivery on LD 1 or 5 (Mattsson et al., 2000). Although maternal blood chlorpyrifos levels were higher, cholinesterase inhibition in multiple tissues was not greater in dams on GD 20 than at other postpartum time points, indicating that interaction of chlorpyrifos with its target site had not increased. Fetal blood levels of chlorpyrifos remained 2.5x lower than maternal blood levels on GD 20. These data indicate that gestational hyperlipidemia alters chlorpyrifos PK in dams during the perinatal period, suggesting that changes in blood lipid fractions could be meaningful for other lipophilic chemicals.
To show this point, the effect of lipid changes on fat:blood partition coefficients for lipophilic compounds were estimated for NP and late-stage pregnant animals (Fig. 7). The increased lipid levels in blood should increase the sequestration of chemicals into the blood, as shown with 1,3-butadiene (Lin et al., 2002). In Figure 7, the algorithm-derived partition coefficient of fat:blood decreased by 35% for highly lipophilic chemicals (chemicals with a log Pow > 4) during late-stage gestation. This change was primarily due to including a twofold increase in the neutral lipids (based on the average changes in cholesterol and triglyceride levels between GD 18 and 20 as described above) into the Poulin and Krishnan algorithm (Poulin and Krishnan, 1995). Using these modified partition coefficients with late-stage gestational PBPK models will lead to an increase in the estimated levels of lipophilic chemicals found in the blood, consistent with the gestational chlorpyrifos data (Mattson et al., 2000). Further work is needed to determine whether the Poulin and Krishnan algorithm or any other structural property relationships accurately predict the changes in partitioning that may occur during late-stage gestation.
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The decrease in maternal protein levels during late gestation has been shown to impact the kinetics of highly protein-bound chemicals. Stock et al. (1980)
reported an increase in the serum free fraction of salicylic acid, sulfisoxazole, phenytoin, dexamethasone and diazepam during late pregnancy (GD 20–21) in Long-Evans dams. Decreased protein binding may facilitate more rapid clearance of chemicals, but it also has been associated with increased biological activity (Gugler et al., 1975; Levy, 1976). This finding also has been shown in our laboratory, where administration of test compound to rabbits during late gestation resulted in increased bioavailability coincident with decreased protein binding (S. Hansen, personal communication). The impact of decreased maternal protein binding on fetal distribution of chemicals must be addressed on a case-by-case basis for highly protein-bound materials.
Maternal serum versus milk.
As the dam transitions into lactation, numerous changes in serum and milk biochemistry occur that could impact the partitioning of compounds between these two compartments in the dam. Lipid levels displayed the most marked differences between milk and serum in CD dams (Fig. 6). Milk triglyceride levels were greater than 100-fold higher than maternal serum levels throughout all of lactation (Fig. 6B) but declined by 25% from LD 3 to 20 (data not easily discernable due to the scale of the graph). Cholesterol also was elevated in milk on LD 0 and LD 20 compared with maternal serum (Fig. 6A). Phospholipid and LDL levels in milk were elevated relative to serum on LD 0 and LD 1 but decreased to levels comparable to serum by LD 5 (Figs. 6C, E). Unlike the other lipid profiles, HDL levels were eight times higher in maternal serum than in milk throughout early lactation (Fig. 6D). Protein levels in milk and serum were similar throughout lactation (Figs. 6F, G).
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The high lipid content of milk favors the partitioning of lipophilic chemicals into milk compared with blood. This increased milk partitioning has been demonstrated for a number of lipophilic chemicals, including polychlorinated biphenyls/polybrominated biphenyls, dioxins, and organochlorine pesticides (LaKind et al., 2004
; Vodicnik and Lech, 1980). However, the temporal changes in milk composition also may affect the amount of chemical transferred during lactation. Few studies have measured lactational transfer of lipophilic chemicals at multiple time points during lactation, including time points prior to LD 5. Mattsson et al. (2000) demonstrated that rat milk contained 1.5 to two times higher concentrations of chlorpyrifos on LD 1 compared with LD 5. The high levels of protein in colostrum also may impact the transfer of protein-bound chemicals during early lactation.
Pup versus NP adult serum.
The lipid and protein changes in developing pup serum throughout the postnatal period were consistent with the maturation of physiological processes occurring during the transition of a pup to adulthood. All serum proteins in the pup were two to three times lower at birth compared with the NP adult but these values increased progressively to adult levels by PND 20. Unlike the protein changes, lipid levels in the pup were dynamic throughout the postnatal period relative to a NP adult. At birth, cholesterol and triglyceride serum levels were similar to the adult and subsequently increased up to fourfold in the late postnatal period compared with the NP adult. Although total cholesterol was similar to the adult during the early postnatal period, HDL was 2.3-fold lower at birth and increased to levels greater than the adult by PND 5. LDL was 5.4- to 9-fold higher in the early postnatal period in the pup compared with adult levels (Figs. 1, 2).
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There are numerous physiological changes in the dam, fetus and pup during the perinatal period and previous studies have shown that these changes can impact chemical PK in a meaningful way. Thus, it is important to consider the impact of these changing variables on tissue dosimetry of chemicals. Ultimately, the relative contribution perinatal changes in lipid and protein levels make to the overall kinetic disposition of compounds in the body will be compound dependent and will need to be evaluated quantitatively using PBPK modeling approaches with specific classes of compounds.
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSUPPLEMENTARY DATAREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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