Evaluation of an immunoisolation membrane formed by incorporating a polyvinyl alcohol hydrogel within a microporous filter support.

An immunoisolation membrane formed by incorporating a high water content polyvinyl alcohol (PVA) hydrogel into a microporous polyether sulfone (PES) filter has been investigated in this study. The PVA hydrogel is formed in situ within the filter pores via glutaraldehyde (GA) crosslinking under acidic conditions. The tortuous nature of the microporous filter pores securely anchors the embedded hydrogel to provide excellent structural integrity. The high void fraction of the PES filter support (>80%) and high water content of the PVA hydrogel (>85% water by weight) allow excellent solute transport rates, while an appropriate level of glutaraldehyde crosslinking supplies the required molecular size selectivity. In vitro permeability measurements made with solutes covering a wide range of molecular sizes demonstrate high transport rates for small nutrient molecules with rapidly diminishing permeabilities above a molecular weight of approximately 1,000 Dalton. Implantation experiments show that the membrane properties are not deleteriously affected by prolonged in vivo exposure or common sterilization techniques. Thus, this hybrid hydrogel/filter membrane system offers a promising approach to the immunoisolation of implanted cells.

High density insulin receptor-positive T lymphocytes from nonobese diabetic mice transfer insulitis and diabetes.

In the nonobese diabetic mouse, insulin-dependent diabetes is an autoimmune disease characterized by T cell-mediated invasion and destruction of pancreatic islet beta cells. The importance of insulin receptor (IR) expression in the pathogenesis of diabetes was examined, since it has been shown that the IR is a chemotactic receptor capable of directing cell movement in response to insulin. Using polyclonal antisera to the IR, phenotypic analysis of purified splenic T cells from diabetic mice showed that about 15% of T cells expressed high density IR (IRhigh). In addition, IRhigh T cells were already a dominant phenotype in the insulitis of young prediabetic mice. To determine the ability of IRhigh T cells to transfer diabetes, cells were sorted by flow cytometry before adoptive transfer into young (6- to 8-wk-old) nondiabetic irradiated nonobese mice. Transfer of as few as 3 x 10(6) purified IRhigh T cells alone resulted in rapid onset of insulitis and diabetes, and IRhigh-depleted T cells were essentially unable to passage either insulitis or diabetes. The adoptive transfer of disease was not due to the transfer of activated cells, since removal of IL-2R+ or transferrin R+ cells did not alter diabetes transfer. Therefore, IRhigh T cells are aggressively diabetogenic, suggesting that increased IR expression may provide a mechanism for delivering potentially autoreactive T cells to the islet, regardless of their activation state.

Detection of genomic alterations in human cervical cancer by two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis was used to comprehensively scan the whole genome of 6 cervical intraepithelial neoplasia (CIN) lesions, 7 cervical squamous cell carcinomas, 1 cervical adenosquamous cell carcinoma, and 2 cervical adenocarcinomas for multiple genetic alterations, such as DNA amplification, chromosome deletion, loss of heterozygosity, and chromosome translocation, as compared with the paired normal tissues. DNA spot analysis of the genomic 2-dimensional gels was performed by a computer color overlay system and by spot recognition software allowing for objective spot comparison and quantitation. Nine spots were found to be amplified in the cervical carcinomas while two amplified spots were detected in the CIN III lesions. Fourteen DNA spots were either reduced in their intensity or absent in cervical carcinomas as compared to their normal paired tissues. Reduction of intensity in 6 spots was observed in the 5 CIN III lesions. These genetic alterations may represent changes in cancer genes that are associated with human cervical carcinogenesis. Further characterization of these alterations may be significant to the understanding of cervical tumorigenesis and to the development of biomarkers for clinical trials in cancer chemoprevention.

DNA quantification in cervical intraepithelial neoplasia thick tissue sections by confocal laser scanning microscopy.

Image analysis of tissue biopsies for determination of DNA content as an early marker of neoplasia is hampered by the complexity of corrections necessary to dea with nuclear truncation and overlap in thin sections. The use of confocal laser scanning microscopy (CLSM) for measurement of cellular DNA content on whole cells within thick tissue sections offers the advantage of preservation of cellular architecture, capacity for 3-dimensional analysis, and absence of sectioning artifacts. We have applied this technique to pararosaniline-Feulgen stained human cervical tissues graded from normal to cervical intraepithelial neoplasia (CIN) III. For the purpose of comparison, 15 microns sections were stained and mapped so that the same cell population could be analyzed by both integrated optical density and fluorescence intensity. Distribution of DNA content from normal cervical epithelial cells 2-3 layers out from the basal cell layer measured by both methodologies showed a stable G0/G1 population with no observable S-phase or G2 cells. Cells measured from areas of increasing CIN grade showed progressively higher DNA content values that were not observable in normal tissue. Although these data are preliminary they suggest that CLSM can be used to identify aneuploid states within defined structural areas of pre-invasive neoplasia.

Tracer technique to measure in vivo chemical transport rates within an implantable cell transplantation device.

An in vivo tracer technique that uses radiolabeled insulin as the tracer molecule has been developed to assess the rate of chemical transport between the cell transplantation chamber of an implantable bioartificial device and the host's circulatory system. The device considered here employs site-directed neovascularization of a porous matrix to induce capillary growth adjacent to an immunoisolated cell implantation chamber. This device design is being investigated as a vehicle for therapeutic cell transplantation, with the advantages that it allows the cells to perform their therapeutic function without the danger of immune rejection and it avoids damaging contact of blood flow with artificial surfaces. A pharmacokinetic model of the mass transport between the implantation chamber, the vascularized matrix, and the body has been devised to allow proper analysis and understanding of the experimental tracer results. Experiments performed in this study have been principally directed at evaluation of the tracer model parameters, but results also provide a quantitative measure of the progression of capillary growth into a porous matrix. Measured plasma tracer levels demonstrate that chemical transport rates within the implanted device increase with the progression of matrix vascular ingrowth. Agreement between the fitted model curves and the corresponding measured concentrations at different levels of capillary ingrowth demonstrate that the model provides a realistic representation of the actual capillary-mediated transport phenomena occurring within the device.

Photodynamic therapy: a new modality for the treatment of cancer.

Photodynamic therapy is a promising new modality for the treatment of neoplastic disease. Currently, Photofrin is the only photosensitizer approved for the treatment of human cancers. In the search for new, chemically pure second generation photosensitizing agents which absorb in the deep red region of the visible spectrum, a novel and unique photosensitizer, CDS1, an iminium salt of copper octaethylbenzochlorin, was developed. This new photosensitizer is chemically pure, cationic, and possesses a strong (epsilon = 35000 M-1.cm-1) absorption peak at 750 nm (in dichloromethane). With copper in the aromatic cavity and a triplet lifetime which is not measurable (< 20 nsec), the photodynamic activity of CDS1 was unexpected. Preliminary in vitro and in vivo animal studies with a transplantable urothelial tumor indicate that CDS1 is an effective photosensitizing agent when used in conjunction with a broad band xenon arc light source or a low frequency, high peak power pulsed alexandrite laser.

Expression and localization of elements of the plasminogen activation system in benign breast disease and breast cancers.

The malignant potential of solid tumors is related to the ability to invade adjacent tissue and to metastasize. These properties of cancer cells depend on the synthesis of proteolytic enzymes which are able to digest adjacent connective tissue and basement membranes. We hypothesized that all elements of the plasminogen activation system might be overexpressed in malignant human breast tumors, functioning as an essential element in tumor invasion and metastasis. As determined by histopathological methods, the malignant tumors showed statistically significantly higher expression of urokinase plasminogen activator (uPA), type-1 plasminogen activator inhibitor (PAI-1), and especially urokinase plasminogen activator receptor (uPAR) than benign tissues. All those elements were present in higher amounts in the cancer cells than in the cells of benign or normal breast tissues. High exhibition of tissue plasminogen activator (tPA) found in cancer seems to be random and not related to the malignant or benign state, since benign and malignant tumors show overexpression of tissue plasminogen activator with similar frequency. When the tumors express high amounts of uPA, they express a high amount of uPAR in 50% of cases and PAI-1 in 57.3% of cases. When urokinase is expressed in low amount, the receptor is low in 28.6% and inhibitor in 21.4% of malignant breast tumors. This statistically significant consensus, 78.6% in the case of urokinase and its receptor and 78.6% in case of urokinase and its inhibitor, suggests that these activities may be the result of a unique mechanism of control, activated in the last steps of malignant transformation.

Keratin expression in chemically induced mouse lung adenomas.

Chemically induced mouse lung tumors exhibit distinctive growth patterns, characterized by an alveolar or solid appearance, a papillary appearance, or a combination of the two. Lung tumors induced in strain A/J mice by either benzo(a)pyrene (BP) or by N-nitrosoethylurea (ENU) were examined for expression of low- and high-molecular-weight cytokeratins. Simple cytokeratins (low molecular weight) were found in all epithelial cells of the normal mouse lung and in all tumor types, whereas higher-molecular-weight cytokeratins were found only in normal bronchiolar cells and in papillary tumor cells. These data lend support to the hypothesis that chemically induced papillary lung tumors in strain A/J mice are derived from bronchiolar Clara cells.

Glyceraldehyde-3-phosphate dehydrogenase and other enzymatic activity in normal mouse lung and in lung tumors.

The histogenesis of chemically induced mouse lung adenomas is currently being debated. Tumors induced by a variety of chemicals and in a number of different strains exhibit growth patterns having a solid/alveolar appearance, a papillary appearance, or a mixture of both. Ultrastructural observations suggest that solid tumors are derived from the alveolar type II pneumocyte and that papillary tumors arise from the bronchiolar Clara cell. However, recent immunocytochemical investigations have concluded that most mouse lung tumors are derived solely from the alveolar type II cell. Enzyme histochemical methods have previously been utilized to identify Clara cells in pulmonary cell isolates and also to characterize mouse lung tumors. This report demonstrates a difference in glyceraldehyde-3-phosphate dehydrogenase (G3PD) activity in type II pneumocytes and Clara cells. Solid tumors and type II cells appear to have a similar G3PD activity, and this activity is different from that observed in papillary tumors and bronchiolar cells. These findings support morphological evidence that suggests mouse lung tumors are phenotypically different and may arise from at least two different cells of origin.

Strain A/J mouse lung adenoma growth patterns vary when induced by different carcinogens.

The histogenesis of mouse lung adenomas is currently being investigated in several laboratories. Based upon studies of a limited number of carcinogens in different mouse strains, some investigators suggest that all lung adenomas in mice are derived from alveolar type II cells, whereas others suggest a Clara cell origin for a majority of the tumors. This report differs from previous investigations in that 12 different carcinogens were evaluated for the types of lung tumor growth patterns they induced in a single mouse strain (strain A mice). The carcinogens aflatoxin B1 (AFB1), benzo(a)pyrene (BP), 1,2-dimethylhydrazine (DMH), 3-methylcholanthrene (MCA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosomethylurea (MNU) induced tumors with a predominantly solid/alveolar growth pattern, whereas N-nitrosodiethylamine (NDEA) induced predominantly papillary tumors. Most of the other carcinogens induced a higher proportion of lung tumors with the solid/alveolar growth pattern than with the papillary growth pattern; however, ratios between the 2 growth patterns varied. If, as suggested by others, solid tumors are derived from alveolar type II cells and papillary tumors from Clara cells, then carcinogens may differ with respect to their ability to transform one cell type or the other.

Relationship between in utero development of the mouse liver and tumor development following transplacental exposure to ethylnitrosourea.

Pregnant C3HeB/FeJ mice were treated with ethylnitrosourea (ENU) on one of gestation Days 10, 13, or 15 to determine if ENU treatment at different stages of gestation would result in morphological or quantitative differences in liver tumors induced in the offspring. Liver tumors were counted and measured 6 mo after treatment with ENU. Foci of cellular alteration were identified histologically and counted. Liver tumor number and foci of cellular alteration increased as a function of increasing dose and age at the time of ENU treatment. An inverse relationship between age at the time of treatment and the size of liver tumors was found. The mean tumor volume of male mice exposed on Day 10 of gestation was 123-fold larger than for spontaneous tumors observed in controls. The differences between mean liver tumor volume in mice which had been exposed to ENU on Days 10, 13, or 15 of gestation appeared to be associated with the exponential growth of the fetus during this period of gestation. Unique, large, multinodular foci of cellular alteration were found in mice treated on Day 10 of gestation. The relationship between the stage of gestation and the size of chemically induced liver tumors in these mice is similar to previous observations with transplacentally induced lung tumors in C3HeB/FeJ mice. This indicates that developmentally regulated cell proliferation occurring at the time of carcinogen exposure may affect the subsequent extent of tumor development in both the liver and lung. Therefore, cells transformed during early development may result in tumors that pose a greater biological hazard than those transformed in later development.

Metabolic studies of carnitine in a child with propionic acidemia.

Carnitine metabolism was studied in a 7-y-old boy with propionic acidemia due to an almost total deficiency of propionyl-CoA carboxylase. The initial diagnosis was made at 3 wk of age followed by numerous episodes of metabolic acidosis despite a low-content branch-chain amino acid diet containing supplemental biotin. Although clinically stable and in a nonacidotic state, the plasma concentration of total carnitine was normal (38.9 microM; normal = 46 +/- 10, mean +/- SD, n = 30) whereas free carnitine was decreased (5.7 microM; normal = 37 +/- 8) and short-chain acylcarnitines were increased (28.6 microM; normal = 5.7 +/- 3.5). Skeletal muscle and liver specimens obtained at open biopsy had low total and free carnitine contents and increased ratio of short-chain acylcarnitines to free carnitine. Short-chain acylcarnitine content was low in liver but increased in skeletal muscle. The liver contained fatty vacuoles, enlarged mitochondria with paracrystalline inclusions, and numerous peroxisomes whereas the skeletal muscle also had lipid vacuoles and an increase in number and size of mitochondria. A carnitine challenge test (100 mg L-carnitine/kg body wt via a gastrostomy tube) resulted in a peak plasma carnitine concentration at 120 min. With maintenance therapy of 100 mg L-carnitine/kg/day the plasma free carnitine remained relatively low, the plasma glycine concentration decreased, and urinary acylcarnitine excretion increased. This study demonstrates that the alterations in carnitine and its derivatives observed in plasma and urine reflect the same type of altered distribution in tissue and provides further data on the effects of L-carnitine therapy.

Morphometric evaluation of the renal arterial system of Dahl salt-sensitive and salt-resistant rats on a high salt diet. II. Interlobular arteries and intralobular arterioles.

The progression of small vessel renal vascular disease was studied in inbred Dahl salt-sensitive (SS/Jr) and salt-resistant rats with acute hypertension induced by a high salt diet. Corrected cross-sectional areas of wall (WAC) and lumen were measured by planimetry and histologic staining for fibrin, hyalin deposition, and elastic lamellae was performed. In SS/Jr rats on the high salt diet, the hallmarks of malignant hypertension (fibrinoid necrosis, hyperplastic and necrotizing arteritis) appeared by week 2 and were intensified after 4 weeks on the high salt diet. Renal vascular lesions from SS/Jr rats were characterized by: hyperplasia and/or hypertrophy of medial smooth muscle cells; intimal proliferation; fibrin, basophilic mucoid, and hyalin deposition within the the subendothelial space and media; variable adventitial fibrosis; and accumulation of mononuclear inflammatory cells in the adventitia and media. Interlobular arteries from both rat strains exhibited significantly increased cross-sectional areas over time for all measured parameters. Intralobular arterioles from both rat strains exhibited significantly increased cross-sectional areas over time for all measured parameters except lumen from SS/Jr rats. For SS/Jr rats, increased WAC from both arterial divisions correlated positively with systolic blood pressure, but not body weight. In salt-resistant rats, increased WAC from both arterial divisions correlated positively with body weight, but not systolic blood pressure. We concluded that the rapid increase in WAC from SS/Jr rats could not be attributed solely to the normal growth of the rat. With the development of acute hypertension in the SS/Jr rat, these results demonstrate the potential usefulness of this model to investigate the pathogenesis of similar renal vascular alterations which are observed in man.

Functional units in rainbow trout (Salmo gairdneri, Richardson) liver: II. The biliary system.

The intrahepatic biliary system was studied in the rainbow trout (Salmo gairdneri), a teleost known to form liver neoplasms after exposure to various carcinogens. Normal adults (N = 25) were examined using light microscopic, enzyme histochemical, and transmission and scanning electron microscopic methods. In light micrographs, longitudinal arrays of hepatocytes appeared as double rows incompletely divided by elongated darkly stained cells. Electron micrographs showed tubules of five to nine pyramidally shaped hepatocytes with their apices directed toward a central biliary passageway and their bases directed toward sinusoids. Sequentially, beginning with hepatocytes, biliary passageways included canaliculi, preductules, ductules, and ducts. Canaliculi were short and joined transitional passageways (preductules) formed by junctional complexes between plasma membranes of hepatocytes and small, electron-dense cells with a high nuclear to cytoplasmic ratio. Ductules, completely lined by biliary epithelial cells, occupied central regions of hepatic tubules. Relatively elongated, ductular cells were intimately associated with surrounding hepatocytes, separated from them by only a thin extracellular space devoid of a basal lamina. Epithelium of bile ducts included cuboidal through mucus-laden columnar cells, surrounded by basal lamina and, in larger ducts, by fibroblasts, smooth muscle cells, and a capillary plexus. Bile ducts and hepatic arterioles, but not venules, were distributed together. The ultrastructure of biliary epithelium, periductular, and periductal cells is presented.

Effect of gestational development of lung tumor size and morphology in the mouse.

Pregnant C3HeB/FeJ mice were treated with ethylnitrosourea (ENU) on one of gestation Days 10, 13, or 15 to determine if ENU treatment at different stages of gestation would result in qualitative or quantitative differences in lung tumors induced in the offspring. Lung tumors were counted and measured 6 mo after treatment with ENU. Offspring of mice treated with ENU on Day 10 of gestation had a small increase in lung tumors while those treated on gestation Day 13 or 15 had significantly more tumors than controls and 6- to 8-fold more tumors than the treated mothers. An inverse relationship between age at the time of treatment and lung tumor size was found. The mean lung tumor volume of mice exposed on Day 10 of gestation was 167-fold larger than that of mice exposed to ENU as adults. The difference between mean lung tumor volume in mice which had been exposed to ENU on Day 10, 13, or 15 of gestation appeared to be associated with the exponential growth of the fetus during this period of gestation. Lung tumors induced on Days 10 and 13 of gestation were irregular in contour and were multinodular. Sixty-five to 85% of the lung tumors in offspring treated during gestation versus 20% in mice treated as adults had a papillary morphology. These differences in tumor size and morphology indicate that cells transformed during early development may pose a greater biological hazard than cells transformed in older animals.

Morphometric evaluation of the renal arterial system of Dahl salt-sensitive and salt-resistant rats on a high salt diet. I. Interlobar and arcuate arteries.

Structural changes in intrarenal arteries of inbred Dahl salt-sensitive and salt-resistant rats with acute hypertension were studied morphometrically. After a week on a normal salt diet (1% NaCl), animals were placed on a high salt diet (8% NaCl) for 4 weeks. Systolic blood pressure (BP) and body weights (BW) were recorded, and six salt-sensitive and six salt-resistant animals were sacrificed weekly for a total of five sampling periods. Corrected cross-sectional (C/S) areas of adventitia, media (MAC), intima, wall (WAC), and lumen (LAC) were measured by planimetry. Although significant increases (p less than 0.01) in both BW and systolic BP were observed over time in both strains, salt-sensitive rats became hypertensive (systolic BP greater than 150 mm Hg) by week 2 on a high salt diet, while salt-resistant rats remained normotensive. In interlobar arteries, significant increases over time were observed for the WAC, MAC, and LAC in salt-resistant rats and in the WAC, adventitia, MAC, and LAC for salt-sensitive rats. Significant increases over time were observed for the WAC, adventitia, MAC, and LAC in arcuate arteries from salt-sensitive rats only. Increased C/S areas observed over time in both strains were observed by week 3 on the high salt diet, after the elevated systolic BP. Analysis of covariance indicated that increased C/S areas observed over time in salt-sensitive rats paralleled elevated systolic BP but did not follow an increase in BW. On the other hand, in salt-resistant rats, increased C/S areas observed over time correlated with BW but not systolic BP. The documented rapid development of vascular changes in salt-sensitive rats in conjunction with the development of acute hypertension demonstrates the potential usefulness of this model for investigating the pathogenesis of hypertensive renal vascular alterations.

Resident sinusoidal macrophages in the liver of the brown bullhead (Ictalurus nebulosus): an ultrastructural, functional and cytochemical study.

Ultrastructural, functional, and cytochemical characteristics of resident sinusoidal macrophages (RSM) in brown bullhead (Ictalurus nebulosus) liver were examined. Following perfusion fixation of the hepatic vascular bed, light micrographs revealed RSM that possessed multiple elongate cytoplasmic processes and frequently contained erythrocytes in various stages of degradation. Following brief perfusion fixation, light microscope examination of vibratome sections of bullhead liver reacted for peroxidase revealed intensely positive RSM. By transmission electron microscopy, peroxidase activity was localized to the nuclear envelope and cytoplasmic granules of RSM and in endothelial and perisinusoidal fat-storing cells. In cryostat sections of fresh-frozen liver, glucose-6-phosphate dehydrogenase (G-6-PDH) was uniformly distributed over hepatocytes, whereas intensely positive punctate staining for G-6-PDH was localized over RSM. To test for phagocytosis by RSM, latex beads (0.81 micron) were injected into a tributary of the hepatic portal vein 2 min prior to perfusion fixation. Latex beads appeared either singly or in dense aggregates within RSM. Ultrastructurally, RSM were characterized by an irregularly shaped, eccentrically located nucleus, electron-dense vacuoles, small patches of granular endoplasmic reticulum, a well-developed Golgi apparatus, elongated mitochondria, desmosomes or desmosome-like densities that served as a source of attachment to endothelial cells, and a centriole with radiating microtubules. Invaginations of the plasma membrane (vermiform processes) characteristic of mammalian Kupffer cells were not observed in bullhead RSM. The results indicated a resident cell population of sinusoidal macrophages in the bullhead liver with properties that partially resembled mammalian Kupffer cells. These results are important for the identification of the normal resident cells in the bullhead liver.

Morphologic and histochemical analysis of the newt (Notophthalmus viridescens) liver.

Architectural arrangement, ultrastructure, and selected histochemical properties of the newt (Notophthalmus viridescens) liver were examined. Although hematopoietic tissue (1-4 cells thick) invested the liver, direct vascular communication between this tissue and hepatic parenchyma was not observed. The liver was intensely positive when stained with Oil-red-O and periodic acid-Schiff reagent and connective tissue was limited to large vascular channels and the capsule. A distinctive polarity was observed in the hepatic vascular system when lobes were viewed in cross section. Dorsally, portal venules accompanied arterioles and branches of the biliary system, while tributaries of hepatic veins were observed ventrally. Following perfusion fixation, hepatocytes appeared as sheets of cells 1-5 cells thick; however, lobules as defined in adult mammalian liver were absent. Hepatocytes contained abundant smooth endoplasmic reticulum, mitochondria, electron-dense lysosomes, patches of granular endoplasmic reticulum, and lipid droplets. Continuous endothelial cells lined sinusoids and exhibited fenestrae organized into structures similar to sieve plates observed in mammalian liver. Variable numbers of melanin-containing macrophages and subendothelial macrophages were observed; however, Kupffer cells and lipid containing perisinusoidal fat-storing cells were not seen. Patterns of reaction product for glucose-6-phosphatase (G-6-Pase), glucose-6-phosphate dehydrogenase (G-6-PDH), and succinic dehydrogenase (SDH) were localized in the newt liver. All enzymes exhibited a uniform distribution pattern; however, small punctate regions of intensely positive G-6-PDH cells were noted within hepatic parenchyma. Cells comprising the hematopoietic tissue were intensely positive for G-6-Pase, G-6-PHD, and negative for SDH.

Ethylnitrosourea-induced transplacental carcinogenesis in the mouse: tumor response, DNA binding, and adduct formation.

We have confirmed previous results which suggest that transplacental exposure of fetal mice to carcinogens does not cause an increase in tumor incidence as they mature unless treatment occurs after midorganogenesis. In C3HeB/FeJ mice we found a negligible increase in tumor incidence and multiplicity following transplacental exposure to the direct-acting carcinogen ethylnitrosourea (ENU) on gestation day 10, but significant increases in lung and liver tumor incidence following exposure on days 13 or 15 or in adults. To explore the possibility that this observed difference is due to differences in the biodistribution of the carcinogen or its interaction with cellular macromolecules, the level of covalent binding between ENU and fetal and maternal DNA following an i.p. injection of a dose of 50 mg/kg of tritium-labeled ENU was measured 30 min after its injection into pregnant females on days 10, 13, and 15 of gestation. The DNA from fetal and maternal lung, liver, and brain was isolated and the amount of covalent binding estimated from the dpm/mg DNA recovered. Samples of DNA were hydrolyzed and chromatographed to determine that the bound tritium was associated with ENU-DNA adducts and not as a product of DNA synthesis. The level of binding of ENU to fetal DNA was equivalent at all gestation days studied but was significantly less than maternal tissues. Binding to the DNA of maternal liver was 4-fold greater than to fetal DNA while maternal lung and brain DNA were bound at intermediate levels. We conclude that the lack of carcinogenic response to ENU documented here, in fetal mice exposed early in gestation (day 10), is not due to differences in ENU binding to fetal DNA during development.