The role of metallothionein induction and altered zinc status in maternally mediated developmental toxicity: Comparison of the effects of urethane and styrene in rats

TOXICOLOGYANDAPPLIEDPHARMACOLOGY

ll@,

450-463(1991)

The Role of Metallothionein Induction and Altered Zinc Status in Maternally Mediated Developmental Toxicity: Comparison of the Effects of Urethane and Styrene in Rats GEORGE P. DASTON,* GARY J. OVERMANN,* MARIE W. TAUBENECK,~ LOIS D. LEHMAN-MCKEEMAN,* JOHN M. ROGERS,+ AND CARL L. Kent *Miami University

Valley Laboratories, The Procter & Gamble Company, Cincinnati, Ohio 45239; tDepartment of Nutrition, of California, Davis, California 95616, and SDevelopmental Toxicology Division. Health Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Received

March

II,

1991: accepted June 18, 1991

The Role of Metallothionein Induction and Altered Zinc Status in Maternally Mediated Developmental Toxicity: Comparison of the Effects of Urethane and Styrene in Rats. DASTON. G. P.,~VERMANN, G.J., TAUBENECK. M. W.,LEHMAN-MCKEEMAN, L. D., ROGERS,J. M., AND KEEN, C. L. (199 I). Toxicol. Appl. Pharmacol. 110,450-463. We hypothesize that maternal metallothionein (MT) induction by toxic dosages of chemicals may contribute to or cause developmental toxicity by a chain of events leading to a transient but developmentally adverse decrease in Zn availability to the embryo. This hypothesis was tested by evaluating hepatic MT induction, maternal and embryonic Zn status, and developmental toxicity after exposure to urethane, a developmental toxicant, or styrene, which is not a developmental toxicant. Pregnant Sprague-Dawley rats were given 0 or 1 g/kg urethane ip, or 0 or 300 mgjkg styrene in corn oil po, on Gestation Day 11 (sperm positive = Gestation Day 0). These were maternally toxic dosages. As both treatments decreased food consumption, separate pair-fed control groups were also evaluated for effects on MT and Zn status and development. In addition, Gestation Day 11 rat embryos were exposed to urethane in vitro in order to determine whether urethane has the potential to be directly embryotoxic. Urethane treatment induced hepatic MT 1Cfold over control; styrene treatment induced MT 2.5-fold. The MT induction by styrene could be attributed to decreased food intake, as a similar level of induction was observed in a pair-fed untreated control group. However, the level of MT induction by urethane was much greater than that produced by decreased food intake alone. Hepatic Zn concentration, particularly in the cytosol, was increased in the presence of increased hepatic MT concentration. Plasma Zn concentration was significantly decreased (approximately 30%) by urethane treatment, but not by styrene or food restriction (pairfeeding). Distribution of 65Zn to the liver of urethane-treated dams was significantly greater (by 300/o),while distribution to embryonic tissues was significantly lower (by at least 50%) than in pair-fed or ad lib.-fed controls. Styrene treatment had no effect on 65Zn distribution. Urethane was developmentally toxic, causing an 18% decrease in fetal weight and a significant delay in skeletal ossification, but was not toxic to rat embryos in vitro. Styrene was not developmentally toxic. The changes observed after urethane treatment, namely substantial hepatic MT induction and altered maternal and embryonic Zn status, along with the lack of direct embryotoxicity of urethane in vitro, support the hypothesis that these maternal effectscontribute to developmental toxicity. The lack of similar changes in styrene-intoxicated dams provides one explanation for its low developmental toxicity at maternally toxic dosages. 0 1991 Academic F-MS, IX.

Developmental toxicity assessments routinely include a high dose level which produces significant maternal toxicity, typically character0041-008X/91

$3.00

Copyright 0 1991 by Academic F’res, Inc. All right.5 of reproduction in any form r.zserved.

ized by a low incidence of mortality, decreased body weight gain during pregnancy, or significant clinical manifestations of intoxication. 450

DEVELOPMENTAL

EFFECTS

OF

MATERNAL

Developmental toxicity is frequently observed only at the maternally toxic dose level, raising the question of whether the developmental effects in such instances are a result of an interaction of the toxicant with the embryo, or are a consequence of a significant alteration in maternal function. It has been suggested that the manifestation of a specific syndrome of developmental toxicity frequently observed in association with maternal toxicity is evidence that the developmental effects are caused by maternal toxicity (I&era, 1984, 1985). However, Kavlock et al. ( 1985) observed no consistent pattern or frequency of developmental effects after administration of 10 unrelated chemicals at maternally toxic dosages to pregnant mice, with the possible exception of supernumerary ribs. Furthermore, some chemicals are not developmentally toxic at or above dose levels which are maternally toxic (Chemoff et al., 1990). In evaluating how maternal toxicity may produce developmental effects, it is necessary to propose a mechanism of action which is consistent with the otherwise diverse effects of chemically unrelated toxicants, and accounts for the lack of developmental toxicity of other agents at maternally toxic dosages. One potential common manifestation of maternal toxicant exposure which may contribute to developmental toxicity is altered Zn status. A number of diverse chemical insults produce an acute phase reaction which includes the induction of metallothionein (MT), especially in the liver. MT is a low molecular weight, cysteine-rich cytosolic protein which binds divalent metals, and has a high affinity for Zn. Although divalent metals such as Cd and Zn are regarded as the classical inducers of MT, a number of organic compounds have been shown to induce this protein (for a recent review see Klaassen and Lehman-McKeeman, 1989). Although the physiological role of MT is not known, one consequence of its synthesis is the sequestration of Zn (Cousins, 1985). This sequestration of Zn can result in a decrease in plasma Zn concentration, as extrahepatic Zn

METALLOTHIONEIN

INDUCTION

451

is not under homeostatic control (Hurley et al., 1982). Zn is essential for normal development, and either chronic or transient Zn deficiency produces abnormal development (Hurley et al., 197 1; Keen and Hurley, 1989). There is a significant flux of Zn into the embryo throughout development, and decreased maternal plasma Zn is reflected by decreased Zn accumulation by the embryo. Recently, it has been reported that maternal administration of either 6-mercaptopurine (Amemiya et al., 1989) or valproic acid (Keen et al., 1989) decreases the proportion of a pulse of 65Zn distributed to the rat embryo. At the same time, 65Zn retention by the maternal liver was increased with hepatic 65Zn being primarily associated with MT, whose synthesis had been induced by the toxicants. These observations have led to the formulation of the hypothesis that some chemicals may produce developmental toxicity by a maternally mediated chain of events. This chain is initiated by the induction of MT in the maternal liver, which increases the association of Zn with hepatic MT and thereby decreases plasma Zn concentration. The ultimate result is a transient, but developmentally adverse, embryonic Zn deficiency. This hypothesis is further supported by the fact that 6-mercaptopurine and valproic acid produce similar forms of developmental toxicity (Hirsch and Hurley, 1978; Hurd et al., 1983) despite having very different pharmacological actions and that maternal dietary Zn supplementation partially ameliorates the developmental toxicity of 6-mercaptopurine (Amemiya et al., 1986). In the present study, we tested this hypothesis further by assessing the effects of two compounds on maternal MT induction and Zn status at a maternally toxic dosage: one which is developmentally toxic at maternally toxic dosages, urethane, and another which is not, styrene. Toxic dosages of urethane are known to induce hepatic MT in the male rat (Brzeznicka et al., 1987). Urethane is developmentally toxic in rats (Hall, 1953; Takaori et al., 1966) at dosages which are toxic to the

452

DASTON

adult (Field and Lang, 1988). In this study we examined whether urethane can induce high levels of hepatic MT in the pregnant rat and whether this induction is associated with altered Zn metabolism and, ultimately, developmental toxicity. The direct development toxic potential of urethane was also examined by treating intact embryos in vitro. Conversely, if the hypothesis is correct, a toxic dosage of styrene should not induce MT or alter maternal Zn metabolism, as styrene is not developmentally toxic at maternally toxic dosages (Murray et al., 1978; Beliles et al., 1985). MATERIALS

AND

METHODS

Animals. Sprague-Dawley CD virgin female rats (Charles River Breeding Laboratories, Portage, MI, or Bantin and Kingman, Fremont. CA for the 65Zn distribution experiment) were used. Rats were housed individually in suspended stainless-steel wire cages and given ad libitum access to rat chow (Purina, St. Louis, MO) and tap water. Animal rooms were controlled for temperature (22 f 1“C), relative humidity (55 + 5%). and photoperiod ( 12 hr light/day). Breeding was accomplished by housing one female and one male (of the same strain and supplier) overnight. Successful mating was confirmed the following morning by the presence of a copulatory plug or spermatozoa in the vaginal smear. Females were considered to be at Gestation Day 0 at this time. Compound administration. Urethane (Sigma Chemical, St. Louis. MO) was dissolved in sterile 0.85% NaCl at a concentration of 100 mg/ml. Styrene (Aldrich Chemical, Milwaukee, WI) was dissolved in corn oil at a concentration of 60 mg/ml. Urethane was given by ip injection at a dose volume of 10 ml/kg (1 .O g/kg) body wt. Styrene was given by gavage at a dose volume of 5 ml/kg (300 mg/ kg). These dose levels had previously been reported to be maternally toxic. Controls for each group received an equivalent volume of the vehicle, ip or po. respectively. All dosing was carried out between 1400 and 1600 on Gestation Day 11. Animals were killed either on Gestation Day 12 (18 hr after dosing) for hepatic MT and tissue metal analyses or 65Zn distribution to maternal or embryonic tissues, or on Gestation Day 20 to assessthe developmental effects of urethane or styrene exposure. Muternal observations. Animals were weighed on Gestation Days 0, 11, and 12 for those killed on Gestation Day 12, and on Gestation Days 0, 11. 12, 14, 18, and 20 for those killed on Gestation Day 20. Food consumption was measured for the intervals of Gestation Days 11- 12, 12- 14 and 14- 18, respectively. Animals were also observed for clinical manifestations of toxicity daily after treatment.

ET AL. Pair-fed groups. Experimental groups with restricted food intake were included in the study after it was determined that treatment with either chemical caused a significant decrease in food consumption for the day following treatment. Three groups were used: one in which access to the diet was completely deprived, matched to the urethane-exposed group which ate virtually nothing over this period; one in which only 16 g of diet was given to each animal, matched to the average amount consumed by styrene-exposed dams; and a third allowed ad libitum access to diet. These animals were not treated in any other way. Food restriction was begun in the afternoon of Gestation Day 11 and continued until the morning of Gestation Day 12. At this time, all animals except for nine in the urethane pair-fed (food deprived) group were killed for hepatic MT and tissue metal and 65Zn distribution analyses. The remaining urethane pair-fed rats were given adlibitum access to food and euthanized on Gestation Day 20 in order to assesspotential developmental effects of the short-term restriction. Analyses of hepatic MT induction and tissue Zn concentration. For this experiment animals were treated on Gestation Day 11 and were killed 18 hr later, between 0800 and 1000 on Gestation Day 12. They were anesthetized with ether and exsanguinated from the inferior vena cava into Zn-free tubes containing ammonium heparin. Blood was centrifuged to obtain plasma. The liver was removed and approximately 3 g was used to prepare a cytosolic fraction for the quantitation of MT. The remainder was weighed and frozen at -70°C for subsequent analysis of metal concentration. Lastly, embryos were dissected free of extraembryonic tissue, pooled within litters, weighed, and frozen at -70°C for subsequent metal analysis. Maternal liver cytosol was isolated by differential centrifugation. Approximately 3 g of liver was homogenized in two volumes of ice-cold 10 mM Tris-HC1 buffer (pH 7.4 at 25°C). using a glass homogenizer with a motordriven Teflon pestle. The homogenate was centrifuged at 10,OOOgfor 10 min; the supematant was then centrifuged at 100,OOOgfor 60 min. The resultant supernatant was considered to be cytosol. This was frozen at -70°C until used for MT and metal determination. MT was measured using the Cd-hemoglobin radioassay (Onosaka et al., 1978: Eaton and Toal, 1982). ‘09CdC1z (New England Nuclear, Boston, MA), with a radiochemical purity of 99% and a specific activity of 125 mCi/mg was used in the assay. MT data are expressed as micrograms MT/g liver, assuming a metal-binding capacity of 7 g-atoms/mol MT and a molecular weight for MT of 7000 Da. Tissue concentrations of Zn, Cu. and Fe were determined by flame atomic absorption spectrophotometry (Instrumentation Laboratories Model 55 1, Wilmington, MA). Tissues were wet ashed in 12 N nitric acid. Standards for each element were made from certified atomic absorption standards (Fisher, Fair Lawn, NJ) by addition of known volumes of standards to 0.1 N nitric acid. Recovery

DEVELOPMENTAL

EFFECTS OF MATERNAL

of trace elements by this method is 98-102% (Clegg et al., 198 1). Metal concentrations are expressed as micromoles per gram wet tissue. Assessment of 6’Zn distribution. For this experiment animals were treated on Gestation Day 11 as described above. Following dosing, all dams were fed a semipurified egg white protein-based diet containing 25 pg Zn/g, by analysis (Keen et al., 1989). Control groups were included which were pair-fed to styrene- and urethane-treated groups. and an ad lib.-fed group. Eight hours after treatment, all dams were dosed orally with 32 PCi 6SZn (New England Nuclear) in a 25% w/v slurry of a 0.5 rg Zn/g diet in 0.85% saline (Keen et al., 1989). Dams were killed 18 hr after chemical treatment by COr asphyxiation and exsanguinated via cardiac puncture. Maternal and embryonic tissues were excised, weighed and frozen in liquid nitrogen. Tissues were analyzed for 65Zn content by counting (Beckman Instruments Model 8500, Fullerton, CA). The counting efficiency was 20%. Data are expressed as percentage in each tissue of the total recovered counts. Developmental assessments.For this experiment, dams from each treatment group were killed on Gestation Day 20 in order to evaluate the developmental effectsof styrene, urethane, or dietary restriction (pair-feeding) regimens administered on Gestation Day 11. Rats were killed by CO* asphyxiation. Laparotomies were performed, gravid uteri were excised and weighed, and the number of live and dead fetuses and resorptions was recorded. Fetuses were removed and weighed as a litter; therefore, fetal weights reported are means of litter averages. Fetuses were examined for external abnormalities. Approximately half of each litter was preserved in Bouin’s fixative and subsequently examined for soft tissue development (Wilson, 1965) the other half was eviscerated, fixed in acetone, cleared in KOH, and stained with Ahzarin red S for skeletal examination (Dawson. 1926). Whole embr.vo culture. Intact rat embryos in culture were exposed to urethane in order to determine whether it had direct effectson embryonic development. Embryos were explanted from their dams on Gestation Day 10 and cultured for 48 hr, according to established methods (New, 1978). The culture medium consisted of 56% heat-inactivated male rat serum in Waymouth’s 751 medium (GIBCO, Grand Island, NY). Urethane was added to the culture medium on Gestation Day 11. The concentrations of urethane used were 2250 pg/ml (the peak systemic concentration if 1 g/kg urethane distributed to the extracellular fluid compartment), 1500 pg/ml (the peak systemic concentration if 1 g/kg urethane distributed to total body water), and 1000, 500, 250, and 0 *g/ml. The number of embryos used at each concentration ranged from 8-25. Empirical measurements in mice indicate that peak blood concentration after a I g/kg ip injection of urethane is approximately 1250 &ml (O’Flaherty and Sichak, 1983). Embryos were assessedfor viability, growth, development, and presence of abnormalities 20 hr after the addition of urethane. Embryos and visceral yolk sacs (the principal

METALLOTHIONEIN

INDUCTION

453

extraembryonic membrane) were frozen at -70°C for subsequent determination of protein (Lowry et al., 195 1) and DNA (Boer, 1975) content. Statistics. Comparisons between each treatment group and the appropriate control for continuous variables were made using Student’s t test (Sokal and Rohlf, 1969) or ANOVA (SAS, 1987). Discrete data and data expressed as frequencies were analyzed using Fisher’s exact test (Sokal and Rohlf, 1969). Whole embryo culture data were analyzed for concentration-response trends by general linear model procedure and differences between individual concentration groups and control by ANOVA (SAS, 1987). All data are expressed as mean f standard error of the mean.

RESULTS Maternal toxicity. Both urethane, at 1 g/kg ip, and styrene, at 300 mg/kg po, were maternally toxic when administered on Gestation Day 11. Both produced a significant body weight loss and decrease in food consumption for the day following treatment (Fig. 1). In addition, urethane treatment produced an anesthesia which persisted for approximately 18 hr. The body weight decrement which occurred during this 18-hr period persisted over the remainder of gestation in the urethanetreated dams; however, weight loss was transient as the net body weight change in this group for Gestation Days 12-20 was not different from controls (data not shown). In order to control for potential MT-inducing or developmental effects related to decreased food intake, additional dams were offered either 16 g of food or no food over the 18-hr period between 1400 of Gestation Day 11 and 0800 of Gestation Day 12. This approximated the average amount of food consumed over this period by styrene- and urethane-treated rats, respectively. These were compared to a concurrently run ad Zib.fed control group. The body weight loss over the period for the styrene pair-fed group was 10 f 2 g, comparable to the loss of 6 f 1 g in the styrene-treated group. The weight decrease in the urethane pair-fed and urethanetreated groups was also comparable, 25 f 1 vs 28 f 8 g, respectively. A group of urethane

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FIG. 1. Indicators of maternal toxicity after treatment with styrene (300 mg/kg po in corn oil) or urethane ( 1 g/kg ip in saline) on Gestation Day 11. Numbers in parentheses are the number of animals in each group. Asterisks indicate a significant difference from the appropriate control group. Asboth agents causeda significant reduction in food consumption for the day following treatment (see top) additional experimental groups which were pair-fed to match the mean dietary consumptions of the treated groups were added, along with an untreated, ad lib.-fed control group (see bottom). CO, corn oil (PO) control; STY, styrene (300 mg/kg po in corn oil); PF-S, untreated control pair-fed to styrene-treated group; INJ, saline (ip) control; URE, urethane (1 g/kg ip in saline); PF-U, untreated control pair-fed to urethane-treated group; ALF, ad lib.-fed untreated control. All values are mean + SEM.

pair-fed animals was maintained until Gestation Day 20 in order to make fetal observations; the remaining urethane pair-fed, styrene pair-fed and ad lib.-fed animals were killed on Gestation Day 12 for MT and tissue metal determination. The urethane pair-fed

animals maintained after Gestation Day 12 quickly regained the weight that they had lost, gaining 40 f 4 g over the next 2 days of gestation, significantly more than the saline injected controls over that period of time.

DEVELOPMENTAL

EFFECTS OF MATERNAL

It should be noted that the data in Fig. 1 represent all animals in the study except for those used for the 65Zn distribution experiment, as these animals were purchased from a different supplier. However, the effects of styrene and urethane on food consumption and maternal weight in this latter group were comparable to those observed for the rest of the animals on study. Developmental toxicity. The results of the fetal examinations are presented in Table 1. No developmental toxicity was observed in litters of styrene-treated dams. Urethane, on the other hand, was developmentally toxic. Average fetal weight was 20% lower than the saline-injected control, a significant decrease. Although no frank malformations were observed, skeletal development was markedly delayed, as there was a significantly lower number of ossified sternebrae in fetuses of urethane-treated dams compared to controls. Food deprivation (pair-feeding) on Gestation Days 11 and 12 produced a slight decrease

METALLOTHIONEIN

455

INDUCTION

in Gestation Day 20 fetal weight. Average fetal weight in this group was 3.29 + 0.08 g, significantly different from the saline-injected control (3.64 f 0.10 g), but not from the corn oil-gavaged control (3.48 +- 0.15 g). No increase in the incidence of visceral abnormalities or delays in skeletal ossification were observed in the food deprived group. Live embryo number and embryo weight were determined in litters of dams killed on Gestation Day 12 (Table 2). As two separate experimental blocks were conducted on Gestation Day 12 animals, the results from each is reported. Embryo weight was decreased after urethane treatment in both experiments. This was statistically significant (p < 0.05 vs the pair-fed control and p < 0.01 vs the ad lib.fed control) for the 65Zn distribution experiment, and was close to statistical significance (p < 0.1 but >0.05) for the Zn concentration experiment. Styrene had no effect on embryo weight. There were no differences between groups in the number of live embryos/litter.

TABLE I DEVELOPMENTAL

EF’F-ECTS OF STYRENE,

URETHANE,

Corn oil control No. of litters No. of implantations No. of live fetuses No. of dead fetuses No. of resorptions % resorbed/litter Fetal weight (g) Soft tissue analysis No. fetusesexamined No. abnormal’ Sex ratio (F/M, %) Skeletal analysis No. fetusesexamined No. with decreased ossificationd Avg. no. stemebrae

1

14.1 + 1.5 13.7 f 1.4 O&O 0.4 + 0.2 3?2 3.48 f 0.15

OR Foot

DEPRIVATION

ON GESTATION

DAY

11

Styrene

Injected control

Urethane

8 14.5 + 1.9 13.9 + 2.1 Ok0 0.6 f 0.3 754 3.69 f 0.13

8 17.5 +- 0.5 16.8 -c 0.7 o+o 0.8 + 0.3 5&2 3.64 + 0.10

16.6 + 0.5 15.3 iT 0.7 o+o 1.3 f 0.4 8*2 2.98 k 0.10””

9 14.7 + 1.1 14.0 + 1.0 O&O 0.7 f 0.3 4+2 3.29 + 0.08”

57

69 2 58142

80 0 48152

64 3 48152

65

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62 3 4.9 k 0.3

50 3 58142

4915 1

46 3 5.0 + 0.3

53 2 5.5 + 0.2

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Food deprived

o Significantly different from injected control, p < 0.05. b Significantly different from pair-fed (food deprived) control, p < 0.05. ’ Abnormalities were undescended testes and enlarged renal pelvis. d Fetuses with skull bones less than half ossified, completely unossified pubic bones, metatarsals, metacarpals, or sternum.

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TABLE

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2

E~CTS OF STYRENE, URETHANE, OR FOOD RESTRICTION (PAIR-FEEDING) ON GESTATION DAY 11 ON LIVE EMBRYO NUMBER AND WEIGHT ON GESTATION DAY 12 Treatment Tissue mineral concentration experiment Corn oil control Styrene Injected control Urethane Ad lib. fed Food restricted (pair-fed to styrene) Food deprived (pair-fed to urethane) 65Zn distribution experiment Corn oil control (pair-fed to styrene) Styrene Injected control (ad lib. fed) Injected control (pair-fed to urethane) Urethane

No. pregnant dams 9 10 7 7 4 4 4 6 6 6 6 6

Live embryos/litter

Avg. embryo weight (md

12.7 iz 1.6 14.7 Ifr 1.0 15.3 + 0.6 14.7 * 1.4 13.8 k 1.3 13.3 + 0.6 14.0 * 0.7

25.9 25.8 23.7 22.1 25.4 26.7 26.9

14.5 + 13.3 f 13.8 f 14.3 f 12.2 +

28.3 f 30.1 f 31.0 f 26.5 f 20.7 +

0.8 1.0 1.0 1.0 0.9

+ 1.4 f 1.8 + 1.1 + 0.6“ f 0.4 + 1.6 + 1.2 0.8 2.1 1.9 1.7’ 1.8’

a Different from injected control, 0.05 < p < 0.1. b Significantly different from ad lib.-fed control, p < 0.05. ’ Significantly different from ad lib.- and pair-fed controls, p < 0.05.

Hepatic MT induction. The MT concentration in maternal livers was determined on Gestation Day 12, 18 hr after treatment. Administration of 1 g/kg urethane produced a 1Cfold induction of hepatic MT (Fig. 2). Styrene exposure also increased hepatic MT, but this induction was of a much smaller magnitude. The hepatic MT concentration of styrene-treated dams was 2.5-fold higher than in vehicle controls (Fig. 2). Interestingly, corn oil gavage appeared to slightly increase hepatic MT concentration in the pregnant rat, as these animals had a MT level approximately twice as high as the saline-injected controls (22.8 + 2.7 vs 10.5 f 2.5 pg/g liver, respectively). To control for the decreased food intake following urethane or styrene treatment, groups of untreated animals were fed amounts of food equivalent to those consumed by styrene- and urethane-treated rats on Gestation Days 11 and 12, and hepatic MT concentration was measured. An ad lib.-fed control group was concurrently evaluated. Both food restricted groups had higher hepatic MT levels than the ad lib.-fed group (Fig. 2). Hepatic MT

concentration in the ad lib.-fed group was equivalent to that for the saline-injected control for the urethane-treated group, 11 .O ? 1.1 vs 10.5 + 2.5 pg/g liver, respectively. The group pair-matched to the styrene-treated group had a 2.2-fold increase in hepatic MT compared to the ad lib control, about the same magnitude of increase as that produced by styrene treatment when compared to its vehicle control. The group pair fed to urethanetreated rats (which ate virtually nothing over the day following treatment) had an approximately 6.5-fold increase in hepatic MT concentration over the ad lib.-fed control (Fig. 2). Although this increase was substantial, it is less than half of the 1Cfold increase caused by urethane treatment. The difference in MT induction between urethane-treated and pairfed groups was significant (p < 0.0 1). It should be noted in making statistical comparisons that these two groups were run in different experimental blocks with different control groups (vehicle injected for urethane, untreated for the pair-fed group). However, as both control groups had virtually identical he-

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FIG. 2. Hepatic metallothionein concentrations on Gestation Day 12, 18 hr after treatment. Legend is the same as for Fig. 1. Numbers in parentheses are the number of animals in each group. Styrene and urethane treatment caused significant increases in hepatic MT as compared to vehicle controls. Both food restriction (pair-feeding) regimens caused significant increases in hepatic MT as compared to the ad lib.-fed control.

patic MT levels, we believe that the comparison is appropriate. Zn concentrations of tissues. The concentration of Zn was measured in maternal liver homogenate and liver cytosol, plasma, and embryos 18 hr after styrene or urethane treatment. Zn concentrations were increased in maternal liver and liver cytosol by styrene, urethane, and both of the pair-feeding regimens compared to controls. The magnitude of change was highest in the urethane-treated dams, in which liver cytosolic Zn concentration was almost doubled over control. The increase in liver cytosolic Zn was more modest in styrene-treated dams, approximately 25% higher than control (Fig. 3). Plasma Zn concentrations were significantly decreased by urethane treatment (Fig. 3). Plasma Zn level was unaffected by styrene or either of the food restriction regimens. Embryonic Zn concen-

tration was unaffected by any of the treatment regimens. Maternal and embryonic distribution of”‘Zn. Tissue distribution of orally administered Zn was significantly different among the experimental groups. Zn retention in the livers of urethane-treated dams was a mean of 38.4 f 3.0% of the total ‘j5Zn recovered, significantly greater than the 29.0 + 0.9% retained in the livers of pair-fed controls, or the 22.9 & 0.7% retained in the livers of ad lib.-fed controls (Fig. 4). Conversely, the percentage of @Zn retained in embryos and extraembryonic tissue (extraembryonic membranes and fluid, decidua) was significantly less than in the other groups (Fig. 4). Distribution of 65Zn was also decreased in other organs of the urethane-treated animals, including spleen, heart, lung, thymus, pancreas, bone, muscle, brain, skin, ovaries and uterus, as compared to both pair-fed and ad lib.-fed

458

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FIG. 3. Zn concentrations in maternal liver cytosol, plasma, and embryos on Gestation Day 12, 18 hr after treatment. (The effects on liver homogenate Zn concentration were qualitatively comparable to that for liver cytosol, and therefore are not shown here.) Legend is the same as for Fig. 1. The number of animals in each group is the same as for Fig. 2. Asterisks indicate significant differences from the appropriate vehicle control for styrene or urethane, or from the ad lib.-fed group for the pair-fed groups. It should be noted that the pair-fed groups represent a separate experimental block, as data on food consumption after styrene or urethane treatment had to be collected prior to starting this block. This may explain the slight variability in tissue Zn concentrations between experimental blocks.

control groups (data not shown). Styrene had no effect on the distribution of 65Zn to maternal or embryonic tissues (Fig. 4).

Developmental eflects of urethane in vitro. Intact rat embryos were exposed to urethane from Gestation Days 11 to 12 in order to de-

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FIG. 4. Distribution of 65Zn to maternal liver and embryonic and extraembryonic tissues on Gestation Day 12. CO, corn oil (PO) pair-fed to styrene-treated group; STY, styrene (300 mg/kg po in corn oil); INJ, saline (ip), ad lib fed control; INJP, saline (ip), pair fed to urethane-treated; URE, urethane (1 g/kg ip in saline); EEM/F, extraembryonic membranes and fluids. The data are expressed as the percentage of total body cpm in each tissue. There were six animals in each group. A single asterisk indicates significant difference from adlib.-fed control; a double asterisk indicates a significant difference from adlib.and pair-fed controls. (In this experiment, treated and pair-fed groups were run concurrently.) The urethane-treated group retained significantly more 65Zn in their livers than either its pair-fed or adlib.-fed control group. The pair-fed control group for urethane also retained more 65Zn in their livers than did the adlib.-fedgroup. Styrene treatment did not cause a significant increase in hepatic 65Zn distribution. Urethane treatment also caused a significant decrease in 65Zn distribution to embryos and extraembryonic tissues as compared to either control group.

termine whether urethane had any direct embryotoxic effects during this period. Concentrations of urethane up to 2250 fig/ml were used. Embryo viability was 100% at all concentrations. Urethane had no effect on growth and development of embryos or extraembryonic membranes as measured by size, somite number, or protein or DNA content. No abnormalities were observed. Thus, urethane was not developmentally toxic to Gestation Days 11 and 12 rat embryos in vitro.

DISCUSSION Under the conditions of this study, both styrene and urethane were maternally toxic, causing a decrease in food consumption and body weight following treatment. Urethane was also developmentally toxic, causing a decrease in fetal weight at term and delayed skeletal ossification. Styrene was not develop mentally toxic in this study, a result which is consistent with published work (Murray et al.,

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1978; Beliles et al., 1985). Urethane treatment produced a marked induction in hepatic MT synthesis, an effect attributable both to maternal anorexia and to the compound itself. This effect was associated with an increase in hepatic cytosolic Zn concentration and in Zn distribution to the liver, and a decrease in circulating Zn concentration. Styrene treatment and partial dietary restriction also caused an increase in hepatic MT concentration; however, this was much less marked than the induction caused by urethane, and was not associated with any measurable change in plasma Zn levels. These results are consistent with the hypothesis that substantial induction of hepatic MT causes a redistribution of Zn into the liver, decreasing circulating Zn. This latter effect may lead to developmental toxicity by depriving the embryo of sufficient Zn for normal development, and we have determined that Zn distribution to the embryo was decreased in urethane-treated rats. That urethane was not directly toxic to embryos in vitro, despite being developmentally toxic in vivo, is consistent with the hypothesis that the developmental toxicity of this agent is due, at least in part, to alterations in maternal Zn metabolism. The developmental toxicity observed after urethane treatment was limited to decreased fetal weight and skeletal ossification, indicative of growth retardation and/or developmental delay. Takaori et al. (1966) also reported decreased fetal weight after urethane administration at this point in gestation. Although urethane has been reported to produce frank malformations in the rat, these were produced by exposure to the compound earlier in organogenesis (Hall, 1953; Takaori et al., 1966). Decreased fetal weight and developmental delay are also observed after transient maternal Zn deficiency (Hurley et al., 197 1; Keen and Hurley, 1989). Urethane treatment altered the pattern of distribution of orally administered 65Zn, causing a marked increase in the fraction of Zn retained by the liver, and decreased the fraction of Zn accumulated by other tissues, in-

ET

AL.

eluding embryonic, extraembryonic, and reproductive tissues. A similar pattern of 65Zn distribution has been observed in previous studies 24 hr after dosing with 6-mercaptopurine (Amemiya et al., 1989) or valproic acid (Keen et al., 1989). Both of these compounds induced maternal hepatic MT, and both were developmentally toxic. Although embryonic Zn accumulation was significantly decreased, Zn concentration of embryos was not decreased after urethane treatment. This result appears to be inconsistent with the hypothesis that the developmental effects of urethane are caused by embryonal Zn deficiency. However, concentration of Zn in the embryo may not be as appropriate an early indicator of embryonal Zn status as Zn accumulation. Whereas 6-mercaptopurine (Amemiya et al., 1989), valproic acid (Keen et al., 1989), and urethane (this study) all caused a significant decrease in the distribution of 65Zn to embryos, none decreased embryonic Zn concentration. There are at least two possible explanations for the lack of effect on embryonic Zn concentration in these studies. First, it is possible that the time point selected for measurement was insufficiently long after treatment to permit detection of a decrease in embryonic Zn concentration. Second, and more likely, it is possible that decreased availability of Zn slows embryonic development proportionately; thus, development would be affected, but embryonal concentration of Zn would not change. Consistent with this idea is the observation that Gestation Day 12 embryos from urethane-treated dams were substantially smaller than controls. This result is consistent with the biological effects of Zn deficiency in that a number of enzymes essential for cell growth and proliferation are Zn-dependent (Keen and Hurley, 1989). Thus, Zn accumulation is probably a better measure of embryonic Zn status than whole embryo Zn concentration. Urethane was not toxic to Gestation Day 11-12 embryos in vitro, despite the fact that exposure was quite high. Concentrations of 1500 and 2250 pg/ml were used, as these rep-

DEVELOPMENTAL

EFFECTS

OF

MATERNAL

resent the maximum possible peak serum concentrations achievable if 1 g/kg urethane distributed into total body water or extracellular water, respectively. (It is most likely that urethane distributes to total body water, as 1 g/kg urethane administered ip to mice resulted in a peak blood level of approximately 1250 pg/ml (O’Flaherty and Sichak, 1983)). Thus, the concentrations of urethane to which embryos were exposed in vitro probably represent an exaggeration of in utero levels of urethane after a 1 g/kg dosage. Furthermore, as a considerable amount of even a large dose of urethane is eliminated over a day’s time in rats, the duration of exposure to high levels of urethane was also exaggerated in vitro. It should be noted that the in vitro embryo culture did not include an exogenous metabolizing system. This is of concern, as the carcinogenicity of urethane, and probably other aspects of its toxicity, are dependent on its metabolism (Lawson and Pound, 1973). However, urethane is not metabolized by mixed function oxidases such as those which comprise the metabolizing systems for in vitro preparations; in fact, its metabolism by rat liver microsomes in vitro is not appreciably higher than by rat plasma (Nomeir et al., 1989). This indicates that metabolism is nonspecific. Thus, although no exogenous metabolizing system was included in the rat embryo cultures, it is likely that the embryo itself, or the male rat serum which comprised 50% of the culture medium, would have been capable of metabolizing urethane. Thus, this culture system is relevant for the study of the direct effects of urethane on embryonic development. That urethane was not directly toxic to embryos provides further support for the contention that this chemical produced developmental toxicity in vivo via an indirect, maternally mediated mechanism. The induction of MT in this study was substantially greater in pregnant female SpragueDawley rats than has been reported for males. A 1 g/kg dosage of urethane resulted in a mean hepatic MT concentration of about 65 pg/g liver in male rats (Brzeznicka et al., 1987) as

METALLOTHIONEIN

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compared to a mean of 143 pg/g in our pregnant females. Hepatic MT levels in male and pregnant female controls were comparable in the two studies. The pregnant rat was also much more sensitive to the MT-inducing effects of dietary restriction than are male rats (Bremner and Davies, 1975). MT induction by physiological stressors appears to be secondary in part to increased circulating levels of adrenal corticosteroids, which are proximate inducers of MT synthesis (Failla and Cousins, 1978; I&in et al., 1981). It is possible that the increased metabolic demands associated with pregnancy are sufficient to augment the degree of stress, and consequently the amount of adrenal corticosteroid release, to a level which is MT-inducing. In any case, it is clear that significant decreases in food intake can result in hepatic MT induction in the pregnant rat. Although this inducing stimulus alone was apparently insufficient to affect circulating Zn levels in the rat, it may be sufficiently synergistic with even weak chemical inducers of MT to significantly decrease circulating Zn. Decreased food consumption is a common observation in developmental toxicity studies and may, at least in some instances, contribute to developmental toxicity by inducing maternal hepatic MT to an extent that maternal Zn metabolism is affected. In summary, treatment of midgestation rats with a single, maternally toxic dosage of urethane produced a significant (14-fold) induction of hepatic MT synthesis, with a significant decrease in plasma Zn concentration and in embryonic Zn accumulation. This effect was associated with developmental toxicity, as assessed in term fetuses. Urethane was not directly toxic to intact midgestation rat embryos maintained in vitro. These results are consistent with the hypothesis that the developmental effects are a consequence of maternal toxicity, through the chain of events beginning with maternal hepatic MT induction, increased retention of Zn in maternal liver, decreased plasma Zn concentration, and, ultimately, decreased distribution of Zn to the embryo. This transient Zn deficiency may be

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developmentally adverse. Styrene, administered to midgestation rats at a maternally toxic dosage, produced a relatively small (2.5-fold) induction of hepatic MT, attributable entirely to decreased food consumption. There was no concomitant change in plasma Zn concentration or embryonic Zn accumulation, nor was styrene developmentally toxic. These results are also consistent with the hypothesis. Taken together, the results obtained in the current study strongly support the idea that changes in maternal Zn metabolism induced by toxic insult may be a common factor contributing to the developmental toxicity of a number of agents. Identification of maternal insults which affect Zn metabolism may provide considerable insight into the biochemical effects underlying the teratogenicity of an otherwise diverse class of toxicants.

ET AL. induced maternal toxicity on embryo-fetal development in the rat. Teratology 42,651-658. CLEGG, M. S., KEEN, C. L., LONNERDAL, B., AND HURLEY, L. S. (198 1). Influence of ashing techniques on the analysis of trace elements in animal tissue. I. Wet a&rig. Biol.

Trace Element

Res. 3, 107-I

15.

COUSINS,R. J. (1985). Absorption, transport and hepatic metabolism of copper and zinc: Special reference to metallothionein and ceruloplasmin. Physiol. Rev. 65, 238-309.

DAWSON,A. B. ( 1926). Note on the staining of the skeleton of cleared specimens with Alizarin red S. Stain Technol. 1, 123.

EATON, D. L., AND TOAL, B. P. (1982). Evaluation of Cd/ hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues. Toxicol. Appl. Pharmacol. 66, 134- 142. FAILLA, M. L.. AND COUSINS, R. J. (1978). Zinc accumulation and metabolism in primary cultures of adult rat liver cells. Regulation by glucocorticoids. Biochim. Biophys.

Acta 543,293-304.

K. J., AND LANG, C. M. (1988). Hazards of urethane (ethyl carbamate): A review of the literature. Lab. Anim.

FIELD,

22,255-262.

ACKNOWLEDGMENTS

HALL, E. K. (1953). Developmental anomalies in the eye of the rat after various experimental procedures. Anat. Rec. 116, 383-393.

This work was supported in part by U.S. EPA Cooperative Agreement CR-8 167 13 and by NIH Grant HD01743.

REFERENCES AMEMIYA, K., HURLEY, L. S., AND KEEN, C. L. (1989). Effect of 6-mercatopurine on 65Zn distribution in the pregnant rat. Teratology 39, 387-393. AMEMIYA, K., KEEN, C. L., AND HURLEY, L. S. (1986). 6-Mercatopurine induced alterations in mineral metabolism and teratogenesis in the rat. Teratology 34, 32 l334.

BELILES,R. P., BUTALA, J. H., STACK, C. R.. AND MAKRIS, S. (1985). Chronic toxicity and three-generation reproduction study of styrene monomer in the drinking water of rats. Fundam. Appl. Toxicol. 5,855-868. BOER, G. J. ( 1975). A simplified microassay of DNA and RNA using ethidium bromide. Anal. Biochem. 65,225231. BREMNER, I., AND DAVIES, N. T. (1975). The induction of metallothionein in rat liver by zinc injection and restriction of food intake. Biochem. J. 149, 733-738. BRZEZNICKA, E. A., LEHMAN, L. D., AND KLAASSEN, C. D. (1987). Induction of hepatic metallothionein following administration of urethane. Toxicol. Appl. Pharmacol.

81,457-463.

CHERNOFF,N., SETZER, R. W., MILLER, D. M., ROSEN, M. B., AND ROGERS,J. M. (1990). Effects of chemically

HIRSCH, K. S.. AND HURLEY, L. S. (1978). Relationship of dietary zinc to 6-mercaptopurine teratogenesis and DNA metabolism in the rat. Teratology 17, 303-3 14. HURD, R. W.. WILDER, B. J., AND VAN RINSVELT, H. A. (1983). Valproate, birth defects,and zinc. Luncet 1, 18 1. HURLEY, L. S., GORDON, P., KEEN, C. L., AND MERKHOF'ER, L. (1982). Circadian variation in rat plasma zinc and rapid effect of dietary zinc deficiency. Proc. Sot. Exp. Biol. Med.

170, 48-52.

HURLEY, L. S., GOWAN, J., AND SWENERTON,H. (1971). Teratogenic effectsof short-term and transitory zinc deficiency in rats. Teratology 4, 199-204. KARIN, M., SLATER, E. P.. AND HERSCHMAN, H. R. ( 198 1). Regulation of metallothionein synthesisin HeLa cells by heavy metals and glucocorticoids. J. Cell. Physiol. 106, 63-74.

KAVLOCK, R. J., CHERNOFF, N.. AND ROGERS, E. H. (1985). The effect of acute maternal toxicity on fetal development in the mouse. Teratogen. Carcinog. Mutagen. 5, 3-13. KEEN, C. L., AND HURLEY, L. S. (1989). Zinc and reproduction: Effects of deficiency on fetal and postnatal development. In Zinc and Human Biology (C. F. Mills, Ed.), pp. 183-220. Springer Verlag, New York. KEEN, C. L., PETERS,J. M., AND HURLEY, L. S. (1989). The effect of valproic acid on 65Zn distribution in the pregnant rat. J. Nutr. 119, 607-6 11. KHERA. K. S. (1984). Maternal toxicity-A possible factor in fetal malformations in mice. Teratology 29, 41 l416.

DEVELOPMENTAL

EFFECTS OF MATERNAL

KHERA, K. S. (1985). Maternal toxicity: A possible etiological factor in embryo-fetal deaths and fetal malformations of rodent-rabbit species. Teratology 31, 129153. KLAASSEN, C. D.. AND LEHMAN-MCKEEMAN, L. D. (1989). Induction of metallothionein. J. Am. CON.Toxicol. 8, 1315-1321. LAWSON, T. A., AND POUND, A. W. (1973). The interaction of carbon- 14-labelled alkyl carbonates, labelled in the alkyl and carbonyl position, with DNA in vivo. Chem. Biol. Interact. 6, 99-105. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (195 1). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 165-175. MURRAY, F. J., JOHN, J. A., BALMER, M. F., AND SCHWETZ, B. A. (1978). Teratologic evaluation of styrene given to rats and rabbits by inhalation or by gavage. Toxicology 11, 335-343. NEW, D. A. T. (1978). Whole embryo culture and the study of mammalian embryos during organogenesis. Biol. Rev. 53, 81-122. NOMEIR, A. A., IOANNOU, Y. M., SANDERS,J. M., AND MATTHEWS, H. B. (1989). Comparative metabolism and

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INDUCTION

463

disposition of ethyl carbamate (urethane) in male Fischer 344 rats and male B6C3Fl mice. Toxicol. Appl. Pharmacol. 97,203-2 15. O’FLAHERTY, E. J., AND SICHAK, S. P. (1983). The kinetics of urethane elimination in the mouse. Toxicol. Appl. Pharmacol. 68,354-358. ONOSAKA, S., TANAKA, K., DOI, M., AND OKAHARA, K. (1978). A simplified procedure for determination of metallothionein in animal tissues. Eisei Kagaku 24. 128-131.

SAS (1987). SAS/STAT Guide .for Personal Computers. SAS Institute, Cary, NC. SOKAL, R. R., AND ROHLF, F. J. (1969). Biometry. Freeman, San Francisco. TAKAORI, S., TANABE, K., AND SHIMAMOTO, K. (1966). Developmental abnormalities of skeletal systeminduced by ethylurethan in the rat. Jpn. J. Pharmacol. 16, 6373.

WILSON, J. G. (1965). Methods for administering agents and detecting malformations in experimental animals. In Teratology-Principles and Techniques (J. G. Wilson and J. Warkany, Eds.), pp. 262-277. Univ. Chicago Press, Chicago.