TERATOLOGY 42:619-627 (1990)

A Critical Review of the Developmental Toxicity and Teratogenicity of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin: Recent Advances Toward Unde rstanding the Mechanism* LAURIE A. COUTURE, BARBARA D. ABBO'IT, LINDA S. BIRNBAUM Experimental Toxicology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27514 (L.A.C.); Developmental Toxicology Division (B.D.A.1,Environmental Toxicology Division (L.S.B.), U S . Environmental Protection Agency, Research Triangle Park, North Carolina 27711 AND

ABSTRACT A specific teratogenic response is elicited in the mouse as a result of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin). The characteristic spectrum of structural malformations induced in mice following exposure to TCDD and structurally related congeners is highly reproducible and includes both hydronephrosis and cleft palate. In addition, prenatal exposure to TCDD has been shown to induce thymic hypoplasia. These three abnormalities occur a t doses well below those producing maternal or embryo/fetal toxicity and are thus among the most sensitive indicators of dioxin toxicity. In all other laboratory species tested, TCDD causes maternal and embryo/fetal toxicity but does not induce a significant increase in the incidence of structural abnormalities even at toxic dose levels. Developmental toxicity occurs in a similar dose range across species; however, mice are particularly susceptible to development of TCDD-induced terata. Recent experiments using a n organ culture were a n attempt to address the issue of species and organ differences in sensitivity to TCDD. Human palatal shelves examined in this in vitro system were found to approximate the rat in terms of sensitivity for induction of cleft palate. Investigators have suggested that altered regulation of growth factors and their receptors may involve inappropriate proliferation and differentiation of target cells, ultimately producing TCDD-induced terata. Why the teratogenic effects of TCDD are so highly species and tissue specific, and which animal species most accurately predicts the response of the human embryolfetus, at the levels of exposure experienced by humans, still remains to be clarified. TCDD (Fig. 1)'a chemical with no known industrial use, is a ubiquitous environmental contaminant. This polychlorinated aromatic hydrocarbon is a by-product formed during the production of chlorinated phenols and their derivatives, such as phenoxy acid herbicides (Firestone et al., '72).Dioxin is also produced as a pyrolysis product during high-temperature combustion processes (Rappe et al., '83) and is formed during the chemical bleaching of pulp (EPA, '87). TCDD is one of 75 possible polychlorinated

dibenzodioxins, dibenzofurans, biphenyls, and naphthalenes. Within this class of structurally related chemicals, TCDD is the most toxic (reviewed in EPA, '85). Toxic potency has been demonstrated to be asso-

a class if structurally related polyhalogenated aromatics, including the halogenated

Environmental Protection Agency policy and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

0 1990 WILEY-LISS, INC.

Received March 15, 1990; accepted July 11, 1990 Address reprint requests to Dr. Linda S. Birnbaum, Environmental Toxicology Division, Health Effects Research Laboratory (MD-66), U S . Environmental Protection Agency, Research Triangle Park, NC 27711.

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L.A. COUTURE ET AL

Fig. 1. Chemical structure of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

ciated with the number and position of chlorine atoms, since congeners lacking chlorines in the four lateral positions, as well as those having chlorines in addition to those in the 2,3,7, and 8 positions, have been shown to be less toxic than TCDD (reviewed in EPA, '89). However, all laterally substituted congeners are capable of eliciting similar toxic effects if given at sufficient doses (McConnell and Moore, '79). Studies indicate that the 2,3,7,8-substituted congeners act via a common mechanism whereby TCDD binds to the Ah receptor and the ligand-receptor complex is translocated to the nucleus and binds to DNA, which in t u r n results in alterations in gene expression (Jones et al., '85; Poland and Glover, '80; Poland et al., '79). In addition, congeners chlorinated in the lateral positions, as compared with those structurally related chemicals lacking chlorines i n the 2,3,7, and 8 positions, have been found to accumulate preferentially in the tissues of fish, reptiles, birds, and mammals (Stalling et al., '85) and have been implicated in the human poisoning incidents in both Japan (Yusho) and Taiwan (Yu-Cheng) (Hsu et al., '84). As a result of its extreme potency, widespread distribution, persistency, and potential for bioaccumulation, dioxin is considered a health concern. TCDD induces a spectrum of toxic responses in laboratory. animals that has been well characterized and includes severe weight loss, thymic atrophy, fatty deposition in the liver, edema, fetotoxicity, and teratogenicity (McConnell et al., '78; Schwetz et al., '73). Pronounced species and strain differences in sensitivity to the toxic effects of TCDD have been observed. The acute oral LD,, of TCDD in experimental animals varies over a 5,000-fold range, with the guinea pig being most sensitive and the hamster the least sensitive (Poland and Knutson, '82). There is disparity in sensitivity to TCDD for the induction of terata and developmental toxicity as well.

The teratogenic response induced by dioxin is highly restricted, as not all species or strains are susceptible (Table 1). Our objective in writing this review is to discuss the various TCDD-induced responses in the different species, a s well a s to outline possible mechanisms that may underlie those responses. EXPERIMENTAL SUBJECTS

Mice The induction of terata is one of the most sensitive indicators of dioxin toxicity in mice, a s hydronephrosis and cleft palate are induced at doses below those resulting in either maternal or embryolfetal toxicity (Courtney and Moore, '71; Neubert et al., '73). Indices of maternal and embryo/fetal toxicity classically reported for TCDDexposed mice include increased maternal mortality, overt clinical signs of maternal toxicity, decreased maternal weight gain, increased maternal liverlbody weight ratios, increased fetal mortality, and decreased fetal weight (Courtney, '76; Courtney and Moore, '71; Neubert and Dillman, '72). In susceptible strains of mice, such as the C57BL, the teratogenic response is extremely tissue specific as only the kidney, secondary palate, and thymus show alterations. Species, strain, and tissue specificities for response to TCDD may reflect dissimilarities in embryonic exposure that occur as a result of pharmacokinetic differences, and/or variations in the intrinsic sensitivity of the developing tissues (i.e., palatal and ureteric cells respond, while other systems developing concurrently are not affected). The data suggest t h a t interspecies differences in TCDD distribution do not appear to play a role in the species differences in toxic effects observed (Neal et al., '82). Pharmacokinetic differences also fail to account for strain differences in sensitivity, as Long-Evans rats were approximately 300fold more sensitive to TCDD than were H a d

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TERATOGENICITY OF TCDD

Speciesistrain Mice C57BLl6N C57BW6N C57BLl6N

TABLE 1 . Teratogenic effects of 2,3,7,8-TCDD1 Treatment Sacrifice Maternal Embryo/fetal Daily dose' days3 day3 effects4 effects4 0,1,35 0,12,17,225 0,3,125

10,lO-13 10 11,lO-13

18

18 18

NR T LilBW t LilBW

T C P THN T C P THN T C P THN

C57BLl6N

0,35

10-13

18

t LilBW

T HN

C57BW6N

0,6,9,12,15,1S5

10,12

18

TCP tHN

C57BLi6J

0,35 (SC)

6-15

18

t LilBW; t WG T Li/BW

C57BLlM

205

10

17

t LilBW

C57BLlM

0,.5,1,2,46

6-15

18

t LilBW

NMR

0.3,3,4.5,g5

6-15

18

NR

CF-1

6-15

NR

DBA

0,0.001,0.01, 0.1,1.3' 0,35 (SC)

6-15

DBA

0,0.5,2,4@

CD-1 CD-1 Rats CD CD

Sprague-Dawley Sprague-Dawley

Wistar

T C P TKA J C P , THN;

t FW

Reference Moore e t al., '73 Weber e t al., '85 Birnbaum e t al., '85 Birnbaum et al., '86 Birnbaum e t al., '89 Courtney and Moore, '71 Haake e t al., '87

None

t C P THN; T FW TCP; / F W T FM t C P ; THN

Silkworth e t al., '89 Neubert and Dillman, '72 Smith e t al., '76

18

T LilBW

tCP; t K A

6-15

18

t CP; t HN

0,1,35 (SC)

6-15

17

LilBW; 1TylBW None

0,25,50,100, 200,400'

6-15

17

Courtney and Moore, '71 Silkworth et al., '89 Courtney and Moore, '71 Courtney, '76

0,0.55 (SC)

6-15

20

None

T KA

0,0.125,0.5,25

2 wk7

20

.1 WG; t prel

IFW, TFM

T LiiBW

t C P ; TKA TCP; THN;

T FM

0,0.125,0.5,2' 0.03,0.125, 0.5,2,S6

0-2 6,15

0,0.125,0.25, 0.5,1,2, 4,8,16'

5-14

20 21

21

postimplantation loss J WG I WG; clinical toxicity Toxicity

1FW

Courtney and Moore, '71 Giavini e t al., '83

t FM;

Giavini e t al., '82a Sparschu e t al., '71

hemorrhage TFM; I F W t Edema;

Khera & Ruddick, '73

T resorptions; t edema; T GI

t GI hemorrhage

Hamsters Golden Syrian

0,1.5,3,6,1S5

7,9

15

t LiiBW

t FM; J. thymus size; t HN; t renal Congestion

Rabbits New Zealand

0,0.1,0.25,0.5,16

6-15

28

I WG; clinical toxicity

Monkeys Rhesus

0,50,500 ppt

7-9 mo7

-

t mortality; clinical toxicity

Chickens White leghorn eggs

0.009-77.5 pmollegg

0

14

-

T FM; T resorptions;

Olson and McGarrigle, '90

Giavini e t al.. '82b

extra ribs

J fertility; t abortions

Allen e t al., '79

cardiovascular Cheung e t al., '81 malformations

'CP, cleft palate; FM, fetal mortality; FW, fetal body weight; HN, hydronephrosis; KA, kidney anomaly; LilBW, liver-to-body weight ratio; NR, not reported; SC, subcutaneous (exposure);Ty/BW, thymus-to-body weight ratio; WG, weight gain. 'Oral exposure unless otherwise noted. 3All days adjusted to reflect plug day = gestation day 0. 4Effects reported are only those that were statistically significant. 'Dose: pgikg 'Dose: pgkglday. 7Exposure prior to mating.

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L.A. COUTURE ET AL.

Wistar rats, in spite of similar overall distribution patterns between these two strains (Phojanvirta et al., '90). In addition, data on the distribution of TCDD to the mouse embryoifetus indicates that pharmacokinetic differences do not appear to be responsible for tissue differences in sensitivity to TCDD. Of the dose reaching the embryo/ fetus, equal levels of TCDD, when measured on a pgimg basis, distribute between the fetal body and head (Abbott et al., '89). As has been reported for adult animals, in the embryo/fetus the liver is the major tissue depot (Nau and Bass, '81). Less than 0.0005% of the maternally administered dose reaches the embryonic palatal shelves or urinary tract, resulting in 1.5 pg TCDDimg in the palatal shelves and 1 pg TCDDimg in the kidneys 3 days postexposure to 30 pg TCDDikg (Abbott et al., 1989). In spite of the extremely low levels of TCDD detected in the target tissues of mice, the levels are sufficient for induction of both hydronephrosis and cleft palate. The data support the view that species and target organ specificities are related to differences in intrinsic sensitivity of the developing organ systems. As a result of the extreme persistence (Gasiewicz et al., '83) and potency of TCDD (reviewed in EPA, '851, i t is likely that all developing organ systems are exposed while undergoing morphogenesis. Yet the kidney, hard palate, and thymus are selectively affected. Hydronephrosis is induced in the absence of palatal clefting; thus, the urinary tract is more sensitive to TCDD than is the secondary palate (Birnbaum et al., '89; Couture et al., '90; Moore et al., '71). In addition, while palatal sensitivity to TCDD increases with gestational age at days 6-12 in the C57BLl 6N mouse, the urinary tract appears to be equally sensitive throughout the major period of organogenesis (Couture e t al., '90). Therefore, the renal system remains susceptible for induction of structural anomalies over a n extended period of time. The data also indicate that the urinary tract remains sensitive to perturbation by TCDD postnatally, as hydronephrosis is induced in neonates subsequent to lactational exposure to this chemical (Couture-Haws et al., '90b; Moore et al., '73). Another sensitive indicator of TCDD-induced developmental toxicity is the thymic effects. In a recent study by Fine et al. ('891, TCDD was found to induce inhibition of lymphopoiesis, which in t u r n

resulted in thymic atrophy and suppression of cell-mediated immunity. The teratogenic sensitivity of specific strains of mice has been shown to segregate with the Ah receptor (Poland and Glover, '80). This receptor has been detected in the developing palate of responsive strains of mice (Pratt et al., '84b). In a series of blastocyst transfer experiments using both responsive and nonresponsive strains of mice, d'Argy et al. ('84) demonstrated that TCDD interacted directly with embryonic cells to interfere with development. Hence, fetal genotype appears to be a n important determinant of teratogenic outcome. It is clear, however, that sensitivity is a relative term. Recent studies by Silkworth et al. ('89) demonstrated that the "nonresponsive" DBA/2 mouse strain, which has a defective Ah receptor, does develop hydronephrosis and cleft palate after prenatal exposure to TCDD, but requires doses that are two to four times higher than needed in the responsive C57BL/6 strain. Thus, responsiveness in a given species or strain may be a function of dose. The finding that the spectrum of dioxin-induced toxicity is a function of dose and is independent of the allele a t the Ah locus has also been observed in adult animals, as the dose necessary for induction of dioxin toxicity in congenic mice homozygous for the d allele was 8-24x greater than for the wild-type animals carrying the b/b gene (Birnbaum et al., '90). The mechanism by which TCDD induces hydronephrosis has been examined in C57BL/6N mice and has been found to be a consequence of increased proliferation of the ureteric epithelial cells (Abbott et al., '87). Hyperplasia of the epithelial lining of the ureter results in occlusion of the lumen and restriction of urine outflow. The end result is the development of hydroureter and hydronephrosis. The hyperplastic response induced in the ureter was found to correlate with increased ureteric epithelial expression of EGF receptors and high levels of 3HTdR incorporation (Abbott and Birnbaum, '90a). The induction of cleft palate was also examined a t the cellular and molecular levels in C57BL/6N mice. After exposure to TCDD, palatal shelves of normal size came into contact but failed to fuse as a result of altered differentiation of the medial epithelial cells (Abbott and Birnbaum, '89; Pratt et al., '84a). When palatal shelves were

TERATOGENICITY OF TCDD

placed in organ culture and subsequently exposed to TCDD, the response induced in vitro was identical to t h a t detected after in vivo exposure (Abbott e t al., '89). As was seen for ureteric epithelial cells, TCDD altered expression of EGF receptors in palatal medial epithelial cells and of specific growth factors (i.e., EGF, TGF-a, TGF-P1, and TGF-(32) (Abbott and Birnbaum, '90b). EGF and TGF-P1 disrupted normal medial cell differentiation and proliferation after exposure of palatal shelves to exogenous growth factors in vitro, further implicating growth factors in the genesis of TCDD teratogenicity (Abbott and Birnbaum, '90b; Abbott and Pratt, '87). The various terata induced in the mouse are not limited to TCDD but have been observed after exposure to other structurally related polyhalogenated aromatic hydrocarbons. Several of the polyhalogenated dibenzofurans and biphenyls, as well a s hexabromonaphthalene and 3,3',4,4'-tetrachloroazoxybenzene, induce the same spectrum of developmentally toxic and teratogenic effects as TCDD and are thought to act by a common mechanism (Birnbaum e t al., '87a,b; Hassoun et al., '84; Marks et al., '81; Miller and Birnbaum, '86; Weber et al., '85). However, as is the case for the acute toxicity of this class of compounds, teratogenic potency of the structurally related congeners is less than that of TCDD. Coadministration of several different chemicals and TCDD results in a pattern of effects that is both compound and dose specific and may provide insight into understanding the genesis of terata. The teratogenicity of dioxin was enhanced when 2,3,4,5,3',4'-hexachlorobiphenyl, retinoic acid, hydrocortisone, or thyroid hormones were coadministered with TCDD (Birnbaum et al., '85, '86, '89; Lamb et al., '86). At the cellular and molecular levels, coadministration of TCDD and retinoic acid resulted in a n even greater reduction in expression of growth factors than was seen with a teratogenic dose of TCDD alone (Abbott and Birnbaum, '90b). Thus, the interaction of these different compounds appears to involve regulation of growth factors. However, in contrast to the additive or synergistic effects reported, Haake et al. ('87) found that the incidence of palatal clefting decreased when TCDD was given in combination with Aroclor 1254. Their results suggested that Aroclor 1254 was a partial

623

antagonist. However, recent studies in our laboratory have shown that the partial antagonism occurs only over a very narrow range of extremely high doses of 2,4,5, 2' ,4' 3'-hexachlorobiphenyl (unpublished observations). Such findings are of great importance in terms of evaluating human risk, as most individuals are exposed to mixtures of environmental chemicals.

Rats In laboratory species other than mice, TCDD appears to act as a developmental toxicant but induces structural abnormalities only at doses that are maternally and/or embryoifetally toxic. Induction of cleft palate has been reported in the rat after administration of high doses of TCDD and other structurally related dioxin congeners, but these doses, when both maternal and embryoifetal toxicity occurred (as was evident from the overt toxicity), produced a significant decrease in maternal weight gain, increase in fetal mortality, and decrease in mean fetal weight (Couture e t al., '89; Olson and McGarrigle, '89; Schwetz et al., '73). As cleft palate was induced only at doses that were extremely toxic to the dam, i t is possible that maternal toxicity had a n etiologic role in the induction of terata. However, recent findings indicated that cultured rat palatal shelves responded to TCDD with the same alterations in medial cell differentiation, survival, and proliferation as had been seen for both in vitro- and in vivo-exposed mouse palates (Abbott and Birnbaum, '9Oc). In this culture system, r a t palates were found to be 200 times less sensitive to TCDD than were the mouse palatal shelves, as measured by alterations in medial epithelial cell differentiation. Thus, the differences in palatal response observed among species may reflect inherent variations in sensitivity. Other investigators have not reported a significantly increased incidence of cleft palate in the rat after in vivo exposure to TCDD (Courtney and Moore, '71; Giavini e t al., '82a; Giavini et al., '83; Khera and Ruddick, '73; Sparschu et al., '71). Additional effects detected in the rat included kidney malformations, subcutaneous edema, andlor gastrointestinal hemorrhage (Courtney and Moore, '71; Giavini et al., '82a, '83; Khera and Ruddick, '73; Schwetz et al., '73; Sparschu et al., '71). The biological significance of hydronephrosis, descriptively termed dilated renal pelvis, induced

624

L.A. COUTURE ET AL.

in the rat following exposure to TCDD and related congeners is questionable, as review of the literature revealed that rigorous statistical analyses to determine whether hydronephrotic incidence was significantly elevated above controls were most often lacking (Courtney and Moore, '71; Giavini et al., '83). Sparschu et al. ('71) stated that the occurrence of dilated renal pelvis was unrelated to compound or dose, while Giavini et al. ('82a) reported induction of hydronephrosis in the rat but stated that the incidence was not increased relative to controls.

ations in growth or water content, but instead represented a direct effect (Cheung et al., '81).

Humans Studies in human populations have failed to identify a n association between TCDD exposure and evidence of developmental toxicity or teratogenicity (Kimbrough et al., '84). The lack of a n association may be the result of the inadequacy and inconclusive nature of the available data. To date, there is no evidence of a n association between male exposure and either teratogenic or developmentally toxic effects in animal studOther Animal Species ies (Lamb et al., '80). Yet, most epidemioIn the rabbit, a s in the rat, TCDD induced logical studies have involved occupationally both maternal and embryo/fetal toxicity but exposed males. In addition, occupational exwas not teratogenic (Giavini et al., '82b). posures frequently involve multiple comThe most significant anomaly detected in pounds and are thus often complex. As a the rabbit in this study was the increased result, it has been difficult to conclusively incidence of extra ribs. However, supernu- identify the chemicals responsible for the merary ribs have been found to be associ- adverse effects observed. In a recent examated with chemically induced maternal ination of children born of Yucheng mothstress (Khera, '84, '87). Therefore, TCDD ers, Rogan et al. ('88) found that these appears to act as a developmental toxicant children, exposed both in utero and lacin the rabbit. Preliminary studies in the tationally to polychlorinated biphenyls and hamster indicate that TCDD induced renal furans, exhibited abnormalities of the nails, congestion and hydronephrosis, but only at hair, teeth, and gums, as well as hyperpigdoses that were both maternally and fetally mentation of the skin and growth delays. In toxic (Olson and McGarrigle, '90). Cleft pal- addition, exposed children displayed deficits ate was also induced in the hamster, but the in cognitive development and behavioral dose required resulted in greater than 50% abnormalities. Rogan et al. ('88) concluded fetal mortality (Olson and McGarrigle, '90). that their findings were consistent with dysDecreased fertility and a n increased inci- plasia of the ectodermal tissue. Hyperpigdence of spontaneous abortions and still- mentation of the skin, gingivae, and nails births have been observed in subhuman pri- have also been detected in children born of mates exposed to maternally toxic doses of Yusho mothers (Kuratsune, 1989). These TCDD (Allen et al., '79; Barsotti et al., '79; data suggest that the structurally related Schantz et al., '79). Thus in the monkey, a s polyhalogenated aromatics may act alone, in the rat, hamster, and rabbit, TCDD ap- or in combination, a s developmental toxipears to be a potent developmental toxicant, cants in humans. but not a teratogen. The effects of TCDD on Most recently, the organ culture system the chicken embryo were of interest, as used to examine and compare mouse and rat TCDD-induced toxicity had been reported in responses to TCDD was applied to human adult chickens (Firestone, '73). Cheung e t embryonic palatal shelves in early stages of al. ('81) reported a dose-related increase in palatogenesis (Abbott and Birnbaum, '90d). the incidence of cardiovascular malforma- In this system, the medial epithelial cells of tions in chick embryos exposed to TCDD. the human palatal shelves responded to These malformations included ventricular TCDD with altered medial cell survival, septa1 defects, conotruncal malformations, proliferation, and differentiation in a manand aortic arch defects. However, there was ner identical to that detected for both rat no increase in the incidence of hydropericar- and mouse medial cells. The sensitivity of dium, which was in contrast to what had the human shelves most closely resembled been found in adult chickens. The authors that of the rat, as the human palatal shelves concluded that the cardiovascular malfor- were approximately 200-fold less sensitive mations did not occur secondary to alter- than the mouse. This suggests that expo-

625

TERATOGENICITY OF TCDD

sure to high levels of TCDD would need to occur in order to alter palatal development in the human. Correlation of the LD,, data with in vitro and in vivo EC,, values for specific teratogenic responses in the various laboratory animals and humans may facilitate prediction of human responses in vivo. FUTURE DIRECTIONS

Studies of the cellular and molecular effects of TCDD will advance our understanding of the mechanism of TCDD activity and of the etiology of chemically induced terata. The teratogenic effects of TCDD in C57BL/ 6 N embryos may involve altered regulation of growth factors and their receptors. The precise regulatory level a t which the effect occurs has not been revealed, and experiments are needed to examine whether TCDD exerts its effects on regulation of transcription of the messenger RNA (mRNA) or a t some later post-transcriptional level. Studies of TCDD activation of cytochrome P450IA1 gene transcription reveal that the dioxin-Ah receptor complex interacts with dioxin-responsive enhancers (DRE) upstream of the gene (Denison et al., '88). DRE-gene complexes in the genome may play a n important role in determining responsiveness to TCDD. Variations in the DNA sequence of the responsive element between species, as well as between strains of the same species, could result in altered binding affinity of the dioxin-receptor complex and consequently modulate the responsiveness of the cells to TCDD. This could account for the wide range of speciesistrain sensitivity that has been reported. It would be interesting to know whether genes regulating growth factor activity have associated DREs. The application of molecular biological techniques in mechanistic toxicology studies should expand our understanding of this compound. While advances have been made toward elucidating the basic mechanism(s1 responsible for the induction of terata after exposure to TCDD, little is known about alterations in functional integrity associated with structural defects. Assessment of alterations in function is needed to evaluate the biological significance of chemically induced malformations. Experimental questions that need to be addressed include determination of alterations in function associated with both the thymic hypoplasia and hydronephrosis, as well as assessment

of the persistence of such malformations. Investigators have demonstrated a n association between alterations in renal function and hydronephrosis in the rat (Kavlock and Gray, '82). It may be of interest to assess segments of the human population in whom exposure to TCDD and/or related compounds is known to have occurred, for alterations in both immune and renal function. Establishment of a correlation between alterations in structure and function would support use of measures of functional integrity as a noninvasive means of examining dioxin-exposed human populations for potential chemically induced effects. LITERATURE CITED Abbott, B.D., and L.S. Birnbaum (1989) TCDD alters medial epithelial cell differentiation during palatogenesis. Toxicol. Appl. Pharmacol., 99:276-286. Abbott, B.D., and L.S. Birnbaum (1990a) Effects of TCDD on embryonic ureteric epithelial EGF receptor expression and cell proliferation. Teratology, 41 t7184. Abbott, B.D., and L.S. Birnbaum (1990b) TCDDinduced altered expression of growth factors may have a role in producing cleft palate and enhancing the incidence of clefts after coadministration of retinoic acid and TCDD. Toxicol. Appl. Pharmacol. (submitted). Abbott, B.D., and L.S. Birnbaum (1990~) Rat embryonic palatal shelves respond to TCDD in organ culture. Toxicol. Appl. Pharmacol., I03 t441-451. Abbott, B.D., and L.S. Birnbaum (1990d) TCDD exposure of human palatal shelves in organ culture alters the differentiation of medial epithelial cells. Teratology (in press). Abbott, B.D., and R.M. Pratt (1987) Retinoids and epidermal growth factor alter embryonic mouse palatal epithelial and mesenchymal cell differentiation in organ culture. J . Craniofac. Genet. Dev. Biol., 7t219240. Abbott, B.D., J.J. Diliberto, and L.S. Birnbaum (1990) TCDD alters embryonic palatal medial epithelial cell differentiation in vitro. Toxicol. Appl. Pharmacol., 100r119-131. Abbott, B.D., L.S. Birnbaum, and R.M. Pratt (1987) TCDD-induced hyperplasia of the ureteral epithelium produces hydronephrosis in murine species. TeratolOgy, 35:329-334. Allen, J.R., D.A. Barsotti, L.K. Lambrecht, and J.P. Van Miller (1979)Reproductive effects of halogenated aromatic hydrocarbons on nonhuman primates. Ann. NY Acad. Sci., 320.419-425. Barsotti, D.A., L.J. Abrahamson, and J.R. Allen (1979) Hormonal alterations in female rhesus monkeys fed a diet containing 2,3,7,8-tetrachloro-p-dioxin. Bull. Environ. Contam. Toxicol., 21:463-469. Birnbaum, L.S., M.W. Harris, L.M. Stocking, A.M. Clark, and R.E. Morrissey (1989) Retinoic Acid and 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) selectively enhance teratogenesis in C57BLi6N mice. Toxicol. Appl. Pharmacol., 98t487-500. Birnbaum, L.S., H. Weber, M.W. Harris, J.C. Lamb, and J.D. McKinney (1985) Toxic interaction of specific polychlorinated biphenyls and 2,3,7&tetrachloro-

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