Transfer of di(2-ethylhexyl) phthalate through rat milk and effects on milk composition and the mammary gland

TOXICOLOGYANDAPPLIEDPHARMACOLOCY

Transfer

91,315-325(1987)

of Di(2-ethylhexyl) Phthalate through Rat Milk and Effects on Milk Composition and the Mammary Gland LORIA.DOSTAL,'ROBERTP.WEAVER,ANDBERNARDA.SCHWETZ National

Toxicology Program, National Institute of Environmental P.O. Box 12233. Research Triangle Park, North Carolina

Received

May

27, 1987; accepted

July

Health 27709

Sciences,

27. 1987

Transfer of Di(2-ethylhexyl) Phthalate through Rat Milk and Effects on Milk Composition andtheMammaryGland. DOSTAL,L. A., WEAVER,R.P.,ANDSCHWETZ,B.A.(~~~~). Toxicol. Appl. Pharmacol. 91,3 1S-325. Five daily oral doses of di(2-ethylhexyl) phthalate (DEHP) (2 g/ kg) given to rats on Days 2-6, 6-10, or 14-18 of lactation caused significant decreases in body weight and increases in hepatic peroxisomal enzymes palmitoyl CoA oxidase and camitine acetyltransferase in the dams and their suckling pups. Plasma cholesterol and triglyceride levels were decreased in the lactating dams. Decreased food consumption, as indicated by pair-fed rats, accounted for the decreased body weight in the pups but not the increases in enzyme activities. To determine whether DEHP and mono(2-ethylhexyl) phthalate (MEHP) were transferred through the milk, milk and plasma were collected from lactating rats 6 hr after the third dose of DEHP. The milk contained 216 f 23 &ml DEHP and 25 + 6 &ml MEHP (mean + SE), while the plasma contained ~0.5 kg/ml DEHP and 75 +_ 12 &ml MEHP. The high milk/ plasma ratio for DEHP (>200) indicates efficient extraction of DEHP from the plasma into the milk. DEHP dosing during lactation also caused a decrease in mammary gland weight and a decrease in mammary gland RNA content which reflects synthetic activity. The water content of the milk was reduced, which probably accounted for the increase in lipid in the milk. Milk lactose was decreased in DEHP-treated and pair-fed rats, consistent with the decrease in milk production. The results show that exposure to high doses of DEHP during lactation in rats can result in changes in milk quality and quantity and can lead to DEHP and MEHP exposure in the suckling rat pups. Q 1987 Academic PW,, 1~.

Although the reproductive effects of environmental chemicals have recently been the subject of intensive research, relatively little is known about chemical toxicity during the early postnatal period. During this time, maternal exposure to toxic substances can lead not only to toxicity in the mother and changes in milk composition or lactational performance, but also can affect the suckling offspring if the substance is secreted or excreted into the milk in sufficient quantities. Although most compounds are secreted into

breast milk in some amount, many lipid soluble drugs and chemicals such as polychlorinated biphenyls and organochlorine pesticides have been found in human and rat milk in very large amounts (Yakushiji et al., 1979; Jensen, 1983; Kornbrust et al., 1986). The plasticizer, di(2-ethylhexyl) phthalate (DEHP), is a highly lipid soluble chemical which is commonly used in the manufacture of soft plastics for medical devices and food wraps. DEHP, like several other compounds including clofibrate, nafenopin, and certain phthalate esters, causes hypolipidemia, hepatic peroxisome proliferation, and liver enlargement in experimental animals (Thomas

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and Thomas, 1984; Albro, 1987). Our recent studies with DEHP (Dostal et al., 1987) have shown that neonatal rats are particularly sensitive to the lethal effects of DEHP when it is administered directly to the pups at doses of 1000 mg/kg, and the neonates show increases in hepatic peroxisomes similar to those of adults, as measured by increases in the peroxisomal enzyme activities palmitoyl CoA oxidase and carnitine acetyltransferase. The hypolipidemic drugs nafenopin and Wy14,643 have been shown to cause hepatic peroxisome proliferation in neonatal rats when their lactating mothers were treated with these compounds (Fahl et al., 1983). In another study, treatment of rats throughout lactation with 2000 mg/kg DEHP caused decreased pup weight gain and decreased hepatic mixed function oxidase activities in 2 lday-old rat pups, and DEHP was present in the neonatal liver, suggesting the presence of DEHP in breast milk (Pat-mar et al., 1985). Because of the likelihood that a lipid soluble chemical like DEHP would be transferred through breast milk in large amounts, DEHP and its major monoester metabolite, mono(2-ethylhexyl) phthalate (MEHP), were quantitated in the milk and plasma of lactating rats dosed with DEHP and the effects on the hepatic peroxisomal enzyme activities in the pups were determined. In addition, the effects of DEHP on milk composition and milk production were examined. To assess milk production, mammary gland weight was determined, as were DNA and RNA contents, which indicate the cellularity and protein synthetic activity of the mammary gland, respectively. A high dose level was chosen to provide optimal conditions for the transfer of DEHP through the milk without causing severe toxicity in the dams. MATERIALS

AND

METHODS

Materials Di(Z-ethylhexyl-l-‘4C) phthalate ([“‘CIDEHP), 98% pure, was obtained from Pathfinder Laboratories, Inc.

AND SCHWETZ (St. Louis, MO). Unlabeled di(2-ethylhexyl) phthalate was obtained through the Radian Corp. (Morrisville, NC) from Aldrich Chemical Co. (Milwaukee, WI) (Lot No. 040-3-DL, batch 1) or from Hatco Chemical Co. (Fords, NJ) (Lot No. H8 10626, batch 2) and was >99% pure by gas chromatography and infrared spectroscopy. Dosing solutions of 400 m&ml in Mazola corn oil were prepared before each experiment, and the concentrations were verified to be +5% of the target concentration on a Perkin-Elmer Sigma 2000 gas chromatograph equipped with a 6 ft X 2-mm OV17 on Supelcoport 80/100 glass column (Supelco, Bellefonte, PA). The chromatographic standard MEHP was obtained from Fairfield Chemical Co., Inc. (Blythewood, SC). The HPLC internal standard di-n-octyl phthalate was custom synthesized by the Midwest Research Institute(Kansas City, MO), and the internal standard mono-n-octyl phthalate was custom synthesized by the Research Triangle Institute (Research Triangle Park, NC). All biochemical reagents were obtained from Sigma Chemical Co. (St. Louis, MO). All solvents used were HPLC grade or better. All glassware used for collection, storage, and extraction of plasma and milk for DEHP analyses was rinsed with n-hexane (HPLC grade or better) before use. Contamination of blank milk samples with DEHP (4.7 ag/ ml) could not be eliminated, whereas blank plasma contained
TRANSFER

OF DEHP INTO RAT MILK

the mothers’ cages to allow milk to accumulate in the glands. Six + one hours after dosing the rats were injected ip with oxy-tocin (1 IU) and anesthetized with 100 mg/ kg ketamine and 32 mg/kg xylazine. Milk (l-2 ml) was collected manually from all teats, and blood, liver, and the six abdominal-inguinal mammary glands were removed. The mammary glands were cut and blotted to remove any visible milk which could affect the gland weight. Mammary glands were frozen at -70°C for later analysis of DNA and RNA.

Analyses Plasma lipids. Blood was collected by heart puncture, using heparinized syringes, and stored on ice until centrifugation to obtain plasma. Plasma was stored at 4°C overnight and analyzed by an Encore automated serum analyzer (Baker Instruments Corporation, Allentown, PA) for cholesterol and triglyceride concentrations. Total cholesterol was determined by the enzymatic method of Allain et al. (1974), and triglyceride was determined by the method of Megraw et al. ( 1979). Peroxisomal enzyme activities. Livers were removed, weighed, placed in cold 0.25 M sucrose, and then homogenized in 3 vol of0.25 tu sucrose. Palmitoyl CoA oxidase activity was determined on fresh homogenates by a modification of the fluorometric assay of Walusimbi-Kisitu and Harrison (1983). The reaction mixture contained 1 pM scopoletin, 25 pM palmitoyl &enzyme A, 50 PM FAD, 75 a(22.5 units) horseradish peroxidase, Type VI, 0.75 mg bovine serum albumin, 0.01% Triton X-100, and 30 mM potassium phosphate buffer, pH 7.4, in a total volume of 3.0 ml. The loss of scopoletin fluorescence was monitored at 46 1 nm in a Perkin-Elmer 650-40 tluorescence spectrophotometer. Camitine acetyltransferase activity was determined in freeze-thawed liver homogenates by the method of Gray et al. (1982). The reaction mixture contained 2.5 mM EDTA, 0.3125 mM 5,5’dithiobis-(2-nitrobenzoic acid), 0.25 mM acetyl CoA, homogenate (50- 150 pg protein), and 50 mM Tris-HCl buffer, pH 8.0, in a total volume of 1.O ml. The reaction was initiated by adding 3.125 mM m=carnitine and monitored at 412 nm using a PerkinElmer Lambda 5 UV/VIS spectrophotometer. Protein concentrations were determined by the method of Bradford ( 1976) using the Bio-Rad protein assay reagent and bovine serum albumin as standard. Milk composition. Total milk solids were determined by drying 100 ~1 of milk to constant weight at 95’C. Milk protein was determined using the Bio-Rad protein assay reagent. Total lipid was determined by the method of Bligh and Dyer (1959). Milk lactose was determined enzymatic&y by the method of Kun and Wallenfels (1974).

317

Mammarygland DNA andRNA. DNA and RNA were isolated from mammary glands by Method I of Kombrust et al. (1982). DNA was quantified by the diphenylamine assay of Burton ( 1956) and RNA was quantified by the orcinol method of Ceriotti ( 1955), using a PerkinElmer Lambda 5 UV/VIS spectrophotometer. Quantitation of DEHP and MEHP. Determination of DEHP and MEHP in plasma and milk was performed by sequential extraction of DEHP and MEHP by modifications of the procedures of Teirlynck and Rosseel ( 1985) and Pollack et al. ( 1985) and analysis by HPLC. Internal standards di-n-octyl phthalate and mono-n-octyl phthalate (20 pg in acetonitrile) were added to 1.O-ml samples of plasma or milk and 2.0 ml of acetonitrile was added. The mixtures were extracted with 3.0 ml of hexane containing 1.5 g% of silicic acid by vortexing for 1 min. After centrifugation for 5 min at lOOOg, the top hexane layer was removed and the lower phase was reextracted with 3.0 ml of hexane. The hexane layers were combined and evaporated under N2 before addition of 1.O ml of HPLC mobile phase. The milk extracts contained a lipid residue after evaporation which did not dissolve in the mobile phase. The mean extraction recoveries for di-n-octyl phthalate from plasma and milk were 85 and 29%, respectively. DEHP standard curves were linear from 0.5 to 100 &ml with an intraassay coefficient of variation of 10.3% (assessedwith six 1.0 &ml standards). The minimum quantifiable level of DEHP was 0.5 &ml. For determination of MEHP, the remaining lower phase was acidified with 75 ~1 of 2 N HCl and then extracted with 3.0 ml of methylene chloride by gentle rotation on a tube rotator for 5 min. After centrifugation for 10 min at 12OOg. the lower phase was removed. Large emulsions formed in the milk extracts between the top and bottom layers but the clear lower layer could be removed carefully. The upper phase was then reextracted with 3.0 ml of methylene chloride and the methylene chloride extracts were combined. The extracts were evaporated under Nr before addition of 1.0 ml of HPLC mobile phase and were centrifuged slightly to precipitate any protein particulate. Mean extraction recoveries for mono-n-oetyl phthalate from plasma and milk were 95 and 8 l%, respectively. MEHP standard curves were linear from 0.5 to 100 &ml with an intraassay coefficient of variation of 2.7% (amessed with ten 1.0 &ml standards). The minimum quantifiable level of MEHP was 0.5 j&ml. For method validation, 1.0 ml of plasma was spiked with 2.0-20 &ml DEHP and MEHP, and milk was spiked with 50-300 &ml DEHP and 5-75 &ml MEHP. Samples were extracted and the internal standards were added at concentrations of 20 &ml just prior to analysis. Correlation coefficients for DEHP and MEHP in plasma were 0.9986 and 0.9964, respectively, and calculation of the F statistic showed linearity. Co-

318

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efficients of variation for DEHP in plasma were 1.63% at 20 &ml and 4.92% at 2 &ml, and for MEHP in plasma coefficients of variation were 6.93% at 20 &ml and 3.10% at 2 &ml. Correlation coefficients for DEHP and MEHP in milk were 0.9984 and 0.9997, respectively, and calculation of the F statistic showed linearity. Coefficients of variation for DEHP in milk were 3.80% at 300 &ml and 1.62% at 50 r&ml, and for MEHP in milk coefficients of variation were 1.50% at 75 pgrglml and 6.46% at 5 &ml. An HPLC method for analysis of DEHP and MEHP was developed by the Radian Corp. which consisted of separation on a 30-cm PBondapak Cls column (Waters Assoc., Milford, MA) using a Perkin-Elmer Series 4 LC system. The flow rate was 1.0 ml/min, and the eluant was monitored at 254 nm with a Perkin-Elmer LC-85B detector. For analysis of DEHP extracts, the mobile phase consisted of 90% acetonitrile/lO% water-both containing 1% H,P04. DEHP and di-n-octyl phthalate eluted at 13.4 and 15.6 min. For MEHP analyses, the mobile phase consisted of 60% acetonitrile/40% water with 1% HsPO,,. MEHP and mono-n-octyl phthalate eluted at 11.6 and 13.1 min, respectively. Data were collected and analyzed by an IBM System 9000 computer. Distribution of [14C]DEHP in milk fractions. 0.15 &i of [ 14C]DEHP plus 200 pg of DEHP were added in 8 ~1 of acetonitrile to 2 ml of milk and allowed to equilibrate with occasional mixing on ice for 1 hr. The milk was centrifuged at 100,OOOgfor 1 hr in a swinging bucket rotor. The radioactivity was then determined in the three phases: top fat globule layer, infranatant whey layer, and casein pellet. Statistics. Statistical analyses were performed by an unpaired t test which was modified for unequal variances when necessary. The nonparametric Mann-Whitney test was used when populations did not have a normal distribution. Statistical significance was assumed at p < 0.05. When control values in two experiments were not significantly different in an unpaired t test, the data for individual dose groups were combined.

RESULTS Experiment

I

Five daily doses of DEHP (2 g/kg) caused significant decreases in body weight in lactating rats (Table 1) and their suckling pups (Table 2) when administered on Days 2-6,6- 10, or 14- 18 of lactation. Food consumption by the 14- to 18-day lactating DEHP-treated rats was significantly reduced throughout the dosing period, and during the last 24 hr, food

AND SCHWETZ

consumption was reduced by 62% (5 1.5 + 1.3 gin controls vs 19.5 + 2.0 gin DEHP-treated rats, mean + SE, p < 0.05). Rats which were pair fed on Days 14- 18 of lactation and their pups also had reduced body weights and their weights were not significantly different from the body weights of the DEHP-treated rats (Tables 1 and 2). Relative liver weight was increased by DEHP treatment in the lactating dams (Table l), but not in the suckling pups (Table 2) at all three stages of lactation. Decreased food consumption by pair-fed dams caused a decrease in relative liver weight in lactating dams and in the suckling pups. Although food consumption was decreased, water consumption was not affected in the DEHP-treated rats (70 ? 4 ml in controls vs 61 +- 9 ml in DEHP-treated rats during the last 24 hr, mean f SE). However, pair-fed rats drank 46% less water (38 f 3 ml, p < 0.05). The reason for the lack of decrease in water consumption in the DEHP-treated rats is unknown. The hepatic peroxisomal enzyme activities, palmitoyl CoA oxidase and carnitine acetyltransferase, were increased by five- to eightfold in DEHP-treated dams at all three stages of lactation (Table 1). Twofold increases in these enzyme activities were also observed in the pups suckling the DEHPtreated dams (Table 2). The increases in enzyme activities in the dams could not be due to the decreased food consumption since there was little or no increase in these activities in the pair-fed rats. In the pups suckling the pair-fed dams, there was a small but significant increase in palmitoyl CoA oxidase activity but no increase in carnitine acetyltransferase activity. However, the palmitoyl CoA oxidase activity in the pups of the DEHP-treated dams was significantly greater than that of the pair-fed pups. Hypolipidemia was observed in the DEHP-treated lactating rats at all three stages of lactation (Table 3). Plasma cholesterol and triglyceride concentrations were decreased by 30-50%. A small decrease in plasma choles-

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319

OF DEHP INTO RAT MILK TABLE 1

EFFECTSOF DEHP ON LACTATING RATS’ B&Y

Relative liver weight (g/ 100 g body wt)

Palmitoyl CoA oxidase (nmol/min/mg)

Carnitine acetyltransferase (nmol/min/mg)

Days of lactation

Dose wow

weight (g)

2-6

Control DEHP

271 +7 224 + 5*

5.05+0.12 5.87 +0.13*

6.63 f 0.4 I 40.8 2 3.3*

9.72 + 0.68 53.1 t 3.4*

6-10

Control DEHP

295 -t 3 243 i 5*

5.03 k 0.08 5.49 2 0.13*

5.22 + 0.58 37.5 * 2.5*

7.16 + 0.58 58.0 f 4.8*

14-18

Control DEHP Pair fed

288 + 6 24lk

5.21 kO.10 5.56 f 0.07*,** 4.03 + 0.12*

7.20 -+ 0.43 37.3 ?I 1.7*x** 7.13 rkO.89

8.29 f 0.43 42.2 f 1.9*.** 10.4 t 0.7*

5*

235 + 6*

’ Rats were dosed orally with DEHP (2 g/kg) or corn oil once daily on the days of lactation indicated. Pair fed rats were given the same amount of food as was consumed by the DEHP-treated rats during the previous 24 hr. Twentyfour hours after the fifth dose, the rats were killed and the livers were removed. Values are the means f SE for seven or eight rats. * Significantly different from control; p i 0.05. ** Significantly different from pair fed; p < 0.05.

terol and no change in plasma triglycerides was observed in pair-fed rats. Experiment

II

To determine the effects of DEHP on milk composition and lactational capacity, and to

determine the concentrations of DEHP and MEHP in the milk, milk and mammary glands were collected from DEHP-treated rats. Since five daily doses of DEHP apparently compromised the quality of lactation in Experiment I as indicated by the reduced body weights of the pups, it was expected that

TABLE 2 EFFECTSOF MATERNAL DEHP DOSING ON SUCKLING RAT PUPS’ Days of lactation

Dose group

Body b weight cd

Relative liver weight (g/lOOgbodywt)

Palmitoyl CoA oxidase (nmol/min/mg)

Camitine acetyltransferase (nmol/min/mg)

2-6

Control DEHP

13.7 f 0.4 10.1 f 0.2*

2.98 ? 0.06 2.93 + 0.05

3.35 t 0.16 7.22 k 0.35*

7.60 +- 0.45 13.6& l.l*

6-10

Control DEHP

21.4+ 1.0 17.1 *0.7*

2.73 f 0.04 2.60 f 0.06

2.47 -t 0.2 1 4.68 + 0.26*

8.43 + 0.47 16.0 + 1.6*

14-18

Control DEHP Pair fed

34.9 2 1.0 30.0 +- 0.8* 31.6 f l.l*

3.17+-0.06 3.10 + 0.04** 2.84 f 0.04*

2.14 + 0.20 5.75 t 0.36*,** 3.40 f 0.20*

7.01 IO.43 15.1 + 1.o*.** 6.62 +- 0.31

a Lactating rats were given daily oral doses of DEHP (2 g/kg) or corn oil on the days of lactation indicated. Two pups from each litter were killed 24 hr after the fifth dose and livers were removed and assayed individually. The average for each litter was then used to calculate the means + SE for seven or eight litters. b Body weight is the average of 10 pups per litter for seven or eight litters. * Significantly different from control; p < 0.05. ** Significantly different from pair fed; p < 0.05.

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TABLE 3 HY~OLIPIDEMIC EFFECTSOF DEHP IN LACTATING RA& Plasma cholesterol (mg/lOO ml)

Plasma triglyceride (mg/lOO ml)

Days of lactation

Dose group

2-6

Control DEHP

94 + 3 44*3*

73 + 3 33*2*

6-10

Control DEHP

93k5 52 + 3*

57+6 42+4

14-18

Control DEHP Pair fed

100*5 46 + 4*~** 86 + 4*

46 f 3 27 k 3**** 49+4

“Rats were treated as described in the legend to Table 1. Values are the means + SE for seven or eight rats. * Significantly different from control; p < 0.05. ** Significantly different from pair fed; p < 0.05.

milk production would be significantly reduced, thus making milk collection difficult. Therefore, in this experiment only three daily doses of DEHP (2 g/kg) were given. The third week of lactation was chosen because it is easier to collect milk later in lactation and because the pair feeding in Experiment I was done during this week. Peak milk concentrations were expected approximately 6 hr after the dose since plasma time course experiments in adult male rats have shown that peak concentrations of DEHP in plasma occur at 3-4 hr (Pollack et al., 1985; Teirlynck and Belpaire, 1985). As in Experiment I, DEHP dosing on Days 15-I 7 of lactation caused increases in the peroxisomal enzyme activities palmitoyl CoA oxidase and carnitine acetyltransferase (3- to 4-fold) but had no significant effect on body weight in the dams (Table 4). The pups suckling the DEHP-treated dams showed a 2.3-fold increase in palmitoyl CoA oxidase activity and a small (1.6-fold) but statistically nonsignificant increase in carnitine acetyltransferase activity with no change in body weight. No significant increases in peroxisomal enzyme activities were observed in the

AND SCHWETZ

pair-fed lactating rats or in their suckling PUPS. In DEHP-treated rats, total milk solids, lipid, and protein were increased relative to ad-lib.-fed controls (Fig. l), whereas milk lactose was significantly decreased. Decreased food consumption, as indicated by the pairfed rats, could account for the increased protein and decreased lactose concentrations, but not the increases in solids and lipid. Mammary gland weight, both absolute (Table 5) and relative to body weight (not shown), was significantly reduced in DEHPtreated rats compared to both control and pair-fed rats. The total DNA in the excised glands was not significantly different in any of the treatment groups, indicating that mammary cell number was not affected by DEHP treatment. Expressed relative to body weight (not shown), the DNA content was not affected by DEHP compared with controls. The total RNA content of the gland (Table 5), as well as the RNA content relative to body weight (not shown), was significantly reduced in the DEHP-treated rats compared with controls, and the RNA/DNA ratio was also reduced in both DEHP-treated and pairfed rats (Table 5), indicating reduced synthetic activity in the mammary cells. Since peroxisomal enzyme activities were increased in the pups suckling the DEHPtreated dams, the presence of DEHP and/or MEHP in the milk was expected. Milk collected 6 hr after the third dose of DEHP contained 2 16 clglml DEHP and 25 &ml MEHP (Table 6). Although the milk levels of DEHP were much higher than the levels of MEHP, the plasma contained virtually no DEHP and substantial amounts of MEHP. This gave a very high milk/plasma ratio for DEHP and a low milk/plasma ratio for MEHP. DEHP was not detectable in control rat plasma, but approximately 5 pg/ml of DEHP was found in the control milk. This was most likely due to contamination of the milk during collection and not to its presence

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EFFECTS

OF

DEHP

INTO

TABLE

4

RAT

321

MILK

OF THREE DAILY DOSESOF DEHP ON LACTATING RATS AND THEIR SUCKLING PUPS* Dose group

Body weight(g)

Control

341+9

DEHP

322 f 1 I 320 rfr 9

Pair fed Palmitoyl CoA oxidase (nmol/min/mg)

Control DEHP

Pair fed Camitine acetyltransferase (nmol/min/mg)

Dams

Control DEHP

Pair fed

pups 38.9 k 1.7 31.5 -c 1.6

40.5 + 1.4

4.63 + 0.28 18.09 + 1.16*,** 3.57 -c 0.59

2.11 + 0.32 5.08 f 0.41*.** 2.34 f 0.38

4.9 i + 0.47 16.70 + I .77*-** 6.34 ? 0.50

5.49 f 0.66 8.95 f 1.49 6.47 X!I0.63

a Lactating dams were given three daily oral doses of DEHP (2 g/kg) or corn oil on Days 15- 17 of lactation. Six hours after the third dose the dams were killed and livers were removed. Two pups from each litter were killed 3-4 hr after the third dose and the livers were removed for determinations of enzyme activities. Body weight for the pups is the average of 10 pups per litter. Values are the means ? SE for nine rats and litters. * Significantly different from control; p < 0.05. ** Significantly different from pair fed; p < 0.05.

in the rats. MEHP was not detectable in the milk or plasma of control rats. After addition of [14C]DEHP to milk in vitro, 94.4 f 0.6% (mean + SE, n = 8) of the radioactivity was associated with the fat globule layer, 4.0 ~fr0.3% was in the whey, and 1.6 f 0.4% was in the casein pellet. Although small increases in peroxisomal enzyme activities were observed in the pups suckling the DEHP-treated dams, DEHP and MEHP were not detected in the plasma of the pups at the time of analysis (Table 6). Since the pups were only with their mothers for a short time after the last dose, there may not have been sufficient time for intestinal absorption of DEHP and MEHP in the pups. DISCUSSION DEHP administration during lactation at the high doses employed in this study caused a marked decrease in growth in the suckling rat pups which was apparently entirely due to decreased food consumption by the dams. However, changes in hepatic peroxisomal en-

zyme activities in the pups suggested the transfer of DEHP through the milk during each of the first 3 weeks of lactation. The large amounts of DEHP found in the milk in the second set of experiments were expected, due to the high lipid solubility of DEHP (octanol/ water partition coefficient 87,000: 1; Howard et al., 1985), and are consistent with the data of Parmar et ai. (1985) which showed decreases in mixed function oxidase activities in rat pups suckling dams treated with 2 g/kg of DEHP throughout lactation. Considering the concentrations of DEHP and MEHP present in the milk, and assuming that a 35-g pup consumes 4 g of milk per day, doses of 25 mg/ kg of DEHP and 3 mg/kg of MEHP would have been administered to the pups if the milk concentrations remained the same at all times. Based on our previous work, a dose of 25 mg/kg of DEHP for 5 days would probably be sufficient to cause two-fold increases in peroxisomal enzyme activities at this age (Dostal et al., 1987). The contribution of the MEHP in the milk is unknown since the responses of suckling pups to MEHP have not been reported.

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PROTEIN

400 f f \” 350 E” 300 c LIPID

D

P

C D LACTOSE

200

40

$150 \ B 100

30

P

$ 20 I l

c

D

P

1. Effects of DEHP on milk composition. Lactating rats were given three daily oral doses of DEHP (2 g/ kg) or corn oil on Days 15- 17 of lactation. Milk was collected 6 hr after the third dose. Bars represent the means + SE for eight or nine rats. C, Control, ad-lib. fed; D, DEHP treated; P, pair fed. *Significantly different from controls; p < 0.05. $Significantly different from pair fed; FIG.

p<

0.05.

Of particular interest was the finding that DEHP was not detectable in the dams’ plasma at the time of very high milk concentrations. Several factors could contribute to the efficient extraction of DEHP from the plasma into the milk. DEHP becomes associ-

AND SCHWETZ

ated with lipoproteins in the plasma (Albro and Corbett, 1978) and these are the source of a large proportion of the milk lipid (Scow et al., 1972, 1976). Uptake of lipoproteins by the mammary gland for milk synthesis could result in uptake of DEHP, and since plasma lipids are decreased in the DEHP-treated dams (Table 3), an even greater proportion of the plasma lipoprotein could be taken up. The fact that >90% of [14C]DEHP added in vitro becomes associated with lipid, rather than becoming bound to protein, indicates that once DEHP is taken up by the gland, it diffuses into the fat and is excreted in the fat globules. Preliminary results indicated a similar distribution of [14C]DEHP in human milk (not shown). The low milk/plasma ratio for MEHP is consistent with the weakly acidic nature of MEHP and its lower lipid solubility. Furthermore, MEHP is not associated with lipoproteins but readily equilibrates between being free in solution and bound to albumin (Albro and Corbett, 1978). The effects of DEHP on the suckling pups were not limited to those produced by the metabolites in the milk, but were also due to changes in the amount of milk produced and the composition of that milk. Since our previous study showed that doses of DEHP which caused twofold increases in peroxisomal enzyme activities did not cause a decrease in

TABLE 5 DEHP EFFECTSONMAMMARYGLANDSOFLACTATINGRATS' Dose group

Mammary gland weight (9)

DNAb (mg)

RNAb (mg)

RNA/DNA

Control DEHP Pair fed

16.2 + 1.1 11.2 + 0.4**** 13.6 f 0.6

42.3 _+2.7 38.3 + 2.5 44.1 + 1.6

150+ 13 114rt 10* 12418

3.51 LO.13 2.97 f 0.13* 2.8OkO.17”

0 Rats were treated as described in the legend for Table 4. The abdominal-inguinal mammary glands were removed after milk was collected 6 + 1 hr after the third dose and were assayed in duplicate. Values are the means +- SE for nine rats. b The milligrams of DNA and RNA are the total nucleic acids in the six abdominal-inguinal glands. * Significantly different from control; p < 0.05. ** Significantly different from pair fed; p -c 0.05.

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OF DEHP INTO RAT MILK

TABLE 6 CONCENTRATIONSOF DEHP AND MEHP INPLASMA AND MILK OF LACTATING RATS AND THEIR SUCKLING

Pupsa Control

DEHP treated

DEHP analysis Dams Plasma (&ml)

(0.5 (9)

Milk (&ml) Milk/plasma

4.7 2 0.9 (7) -

<0.5 (4) 1.2 rt 0.1 (5) 216r23 (7) >200

Pups Plasma (&ml)

CO.5 (9)

-co.5 (9)

MEHP analysis Dams Plasma (&ml) Milk (&ml) Milk/plasma Pups Plasma (beg/ml)

co.5 (9) co.5 (5) 1.s (2) -

76 + 12 (9) 25+6 (8)

CO.5 (9)

CO.5 (8) 1.1(l)

0.33

’ Rats were treated as described in the legend for Table 4. Blood and milk were collected from dams 6 hr after the third dose and blood was collected from two pups per litter 3-4 hr after the third dose. Values are the means + SE or the average for the number of rats or litters in parentheses. ~0.5 indicates that the value was less than the detection limit of 0.5 pg/ml.

body weight (Dostal et al., 1987), the decrease in body weight of the pups in the present study probably resulted from decreased milk consumption. The decrease in mammary gland weight suggests that the capacity for milk production would be reduced, but the lack of a significant change in total DNA in the gland or in DNA relative to body weight (not shown) indicates that the numbers of cells were not altered. The decreases in total RNA in the glands, the RNA/ 100 g body wt, and the RNA/DNA ratio indicate that the synthetic activity of the cells was compromised since these parameters are closely asso-

323

ciated with the synthesis of milk and have been shown to correlate well with suckling intensity and litter weight gain (Tucker, 1966). However, the decreases in RNA and the RNA/DNA ratio were similar in the pair-fed rats, so the changes may be a result of the decreased food consumption in the DEHPtreated rats. In addition to its effects on milk production, DEHP also caused changes in milk composition. Water content was decreased, as indicated by the increase in solids, which may account for the increased concentration of lipid in the milk. Although water consumption was not affected, renal concentrating and diluting ability was impaired in female rats after 17 weeks on a diet containing 2% DEHP (Gray ef al., 1977), so urinary water excretion could have been affected in the lactating rats in the present study. The increase in protein appears to be due to reduced food consumption as indicated by the pairfed rats. The significant decreases in lactose concentration in the milk of both the DEHPtreated and pair-fed rats is consistent with the fact that lactose synthesis in the mammary gland is tightly regulated by food supply (Wilde and Kuhn. 1979) and lactose is the major regulator of the quantity of milk secreted (Giacoia and Catz, 1986). Characteristic of DEHP toxicity, liver enlargement and increases in the peroxisomal enzymes palmitoyl CoA oxidase and carnitine acetyltransferase were observed in the lactating rats. Under normal conditions, liver weight is increased during lactation to provide the synthetic capacity needed for the production of the tremendous quantities of milk (40 ml/day: Williamson et al., 1984). As shown by the pair-fed rats, decreased food intake causes a decrease in relative liver weight, probably by impairing lactation. Therefore, the hepatomegalic effect of DEHP is moderated by the decrease in liver weight caused by the decreased food intake. Compared with the adult male rats in our previous study (Dostal et al., 1987) the increases in the per-

324

DOSTAL.

WEAVER,

oxisomal enzyme activities in the lactating rats were smaller (5 to 8-fold compared with 13- to 1j-fold increases in 42-day-old rats). This is consistent with reports showing that female rats are less sensitive to the peroxisome proliferating effects of DEHP than are males (Osumi and Hashimoto, 1978), but might also suggest that lactating rats are less sensitive than nonlactating females. This would not be surprising in light of the altered metabolic activity of many hepatic enzymes during lactation (Williamson, 1980). Another characteristic effect of DEHP which was observed in the lactating dams is hypolipidemia, as measured by decreases in both cholesterol and triglyceride. The hypocholesterolemic effect of DEHP was larger in the lactating rats than in 6-week-old males in our previous study (Dostal et al., 1987). Since cholesterol synthesis is increased in liver and intestine during lactation (Feingold and Moser, 1985), impairment of lactation could have contributed to the greater hypocholesterolemic effect. Triglyceride levels might also be affected by the transport of large amounts of triglyceride into the mammary gland for milk lipid synthesis (80% of human milk lipid is derived from plasma triglycerides; Neville et al., 1983). In conclusion, large amounts of DEHP and smaller, yet significant, amounts of MEHP are transported through the milk of lactating rats when high doses of DEHP are given. After multiple doses, increases in hepatic peroxisomal enzyme activities are observed in the suckling pups. The very high milk/plasma ratio for DEHP in the present study suggests that a very efficient extraction mechanism exists for DEHP which may be applicable to other lipid-soluble environmental chemicals. Elucidation of the causes of this high milk/ plasma ratio may give a better understanding of the factors that influence mammary excretion of xenobiotics. ACKNOWLEDGMENTS The authors express their thanks to Mr. Walter L. Jenkins and Mr. Eric Haskins for technical assistance; to Mr.

AND SCHWETZ Kirk Wilbourne and Mr. Mitchell Howell of Radian Corporation (Morrisville, NC); and to Mr. Brad Collins, Dr. Bill Jameson, and Dr. Tom Goehl of NIEHS for valuable analytical advice and assistance. We are grateful to Dr. Ronald Melnick for providing the automated serum analyzer and the fluorescence spectrophotometer. We also thank Louise Oyster for typing the manuscript.

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