Assessment of human exposure to chemicals from Superfund sites.

Assessing human exposure to chemicals from Superfund sites requires knowledge of basic physical, chemical, and biological processes occurring in the environment and specific information about the local environment and population in the vicinity of sites of interest. Although progress is being made in both areas, there is still a tremendous amount to be done. Participants at this meeting have identified several of the areas in need of greater understanding, and they are listed below. Movement of dissolved and volatile organics, especially NAPLs, in the subsurface environment. This includes study of the partitioning of compounds between NAPLs, air, water, and soil. Partitioning of volatilized chemicals between gaseous and aerosol components of the atmosphere. This includes understanding how these components influence both wet and dry deposition. Long-term movement from sediments into biota and how these affect chronic toxicity to sediment biota. Broad validation of PBPK models describing partitioning of compounds from sediment and water into fish. Reactions of chemicals sorbed to atmospheric particles. This includes application of laboratory models to real and varied atmospheric conditions. Interactions between biotic and abiotic transformations in soil and sediment. Applicability of physiological pharmacokinetic models developed in laboratory studies of experimental animals and clinical investigations of humans to environmental chemicals, concentrations, and routes of exposure in humans. Use of human and wildlife behavioral and biomonitoring information to estimate exposure. This includes better understanding of human variability and the applicability of information gathered from particular wildlife species. To successfully address these gaps in our knowledge, much more analytical data must be collected.(ABSTRACT TRUNCATED AT 250 WORDS)

Disease-causing effects of environmental chemicals.

Both toxicologic studies and studies in environmental chemistry are important in assessing the potential adverse health effects of human exposures to hazardous environmental agents. This article discusses the toxic effects of chemical concentration at the target organ or site and how the concentration is related to the level of external exposure.

Comment on the significance of positive carcinogenicity studies using gavage as the route of exposure.

There is continuing controversy, extending into regulatory matters, over the significance to human health of positive results in carcinogenicity studies in animals using the gavage technique as the route of exposure. Our review of a nonrandom sample of 117 chemicals or chemical processes listed as known or reasonably anticipated to be carcinogenic in the National Toxicology Program's Third Annual Report on Carcinogens provides support for the validity of the gavage route in such studies. Twenty-three chemicals among the 117 substances and processes listed were positive by gavage. Twenty of these 23 chemicals were also appropriately studied by at least one other route of exposure. Thus, we were able to evaluate the extent to which positive gavage results were confirmed by another route of exposure in this sample. Nineteen (or 95%) of the twenty chemicals were positive for carcinogenicity by at least one other nongavage route in carcinogenicity bioassays. Moreover, in each of these 19 cases, positive carcinogenesis results were obtained by a nongavage route in the same species of animal where gavage administration led to the induction of cancer. All of the 23 gavage-positive chemicals induced tumors distal to the site of administration in at least one study, as did all 15 chemicals which were also positive by subcutaneous injection. We emphasize, however, the limited scope of our survey. We have not evaluated all chemicals that have tested positive by gavage and by at least one alternative route, nor have we assessed those chemicals found to be negative by the gavage route.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of prostaglandin PG-F2 alpha by freshly isolated alveolar type II cells from lungs of adult male or pregnant rabbits.

Prostaglandin F2 alpha (PG-F2 alpha) was metabolized to different products by freshly isolated alveolar type II cells from adult male or pregnant rabbits. The type II cells from the adult male rabbits metabolized PG-F2 alpha to products which co-chromatographed on HPLC with 15-keto PG-F2 alpha and 13,14-dihydro-15-keto PG-F2 alpha, metabolites of cytosolic metabolism. The cells from the pregnant rabbit metabolized the prostaglandin to several polar metabolites. The major peak co-eluted with 20-hydroxy-PG-F2 alpha, a product dependent on cytochrome P-450 metabolism. The other polar metabolites were likely secondary oxidation products, formed by both the cytosolic 15-dehydrogenase and/or the 13,14-reductase and the microsomal omega-hydroxylase. No metabolism of PG-F2 alpha was observed in fractions of alveolar macrophages or tracheal cells of either adult male or pregnant rabbits. Fractions enriched in Clara cells (40-60% purity) showed little and variable metabolism of PG-F2 alpha, qualitatively similar to that observed with type II cells. However, data was inconclusive due to the low Clara cell purity and low activity.

Xenobiotic metabolism in human alveolar type II cells isolated by centrifugal elutriation and density gradient centrifugation.

Alveolar type II cells were isolated from five human lung specimens obtained during resection or lobectomy and enriched to 63-85% purity. Digestion with Sigma protease type XIV followed by centrifugal elutriation and Percoll density gradient centrifugation yielded 1.2 +/- 0.4 X 10(6) cells/g lung in the type II cell fractions. The activities of some enzymes involved in the metabolism of xenobiotics were determined in these freshly isolated type II cells and compared with activities in alveolar macrophages and fractions of unseparated cells from the same tissue samples. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity was similar in the three cell fractions from all five patients (18-29 nmol/mg protein/min). An antibody to rabbit reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase inhibited reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reduction as much as 70% in microsomal preparations of the isolated human pulmonary cells, although this same antibody barely reacted with microsomes of the human cells in a Western blot assay. Epoxide hydrolase activity was highest in the alveolar type II cells (1.08 +/- 0.17 nmol/mg protein/min). This activity was 6 times higher than in the alveolar macrophage or unseparated cell fractions. 7-Ethoxycoumarin deethylase activity, a cytochrome P-450-dependent pathway, was low or undetectable in the three cell fractions. Trace amounts of 7-ethoxyresorufin O-deethylase activity (0.5-1.5 pmol/mg protein/min) were detected in microsomes of the isolated human cells, even though a polycyclic hydrocarbon-inducible cytochrome P-450 which metabolizes 7-ethoxyresorufin (form 6 in rabbits) was not detected immunochemically.

Biotransformation of xenobiotics in individual rabbit hepatocytes. Application of microspectrofluorometry.

We have investigated several methods for determining rates of xenobiotic biotransformation in individual rabbit hepatocytes by microspectrofluorometry. Experiments designed to measure monooxygenase activity by following fluorescent product formation (i.e. 7-ethoxycoumarin deethylase or oxidation of the fluorescein derivatives ethoxyfluorescein ethyl ester and 5- and 6-ethoxycarbonyl ethoxyfluorescein ethyl ester) demonstrated that the fluorescent products were released from cells to the surrounding media. Thus, metabolically active cells probably had underestimated activities, and inactive cells could accumulate fluorescent product and appear metabolically active. We also utilized benzo(a)pyrene (BP) as a substrate in our system. By selecting appropriate wavelengths (370 nm excitation, 407 nm emission), it was possible to selectively monitor BP disappearance in single cells. In hepatocytes from rabbits, disappearance of fluorescence was observed after a 10-min incubation at 37 degrees C with 1-40 microM BP followed by a wash to remove extracellular substrate. The decrease in fluorescence followed apparent first-order kinetics. Measurement of extracellular fluorescence indicated that the substrate did not leave the hepatocytes. Furthermore, in both control and beta-naphthoflavone-treated rabbits, the observed fluorescence changes in the cells were inhibited by the presence of equimolar concentrations of ellipticine or alpha-naphthoflavone, inhibitors of BP metabolism. Storage of hepatocytes from untreated animals on ice for 3.5 hr did not affect rate constant values in cells with a normal appearance, whereas those lacking refringent edges or having numerous cytoplasmic blebs demonstrated greatly reduced (up to 88% loss) activities compared with normal, freshly isolated cells. Potential applications of this system (e.g. to other cell types) are discussed.

Mixed-function oxidase activity in enriched populations of ciliated cells freshly isolated from rabbit tracheas.

A method is described for the preparation of enriched populations of ciliated cells from rabbit tracheas. Following protease digestion of tracheal lumen tissue, cells were subjected to centrifugal elutriation. This produced two cell fractions of interest: an 8 micron diameter fraction believed to be composed largely of basal cells, and a 15 micron diameter fraction containing a mixture of ciliated cells and Clara cells. Further treatment of the 15 micron cells with a dextran/polyethylene glycol/phosphate buffer system resulted in separation of a highly enriched ciliated cell fraction (84.3 +/- 2.7% ciliated cells with 6.5 +/- 1.5% Clara cells) from a fraction containing both ciliated cells (42.0 +/- 2.1%) and Clara cells (27.0 +/- 3.5%). The yield of cells in the enriched ciliated cell fraction was 0.68 +/- 0.09 X 10(6) cells/trachea. Analysis of mixed-function oxidase activity in tracheal cells showed 7-ethoxycoumarin deethylase and coumarin hydroxylase activities to be present in the 8 micron cells as well as in ciliated cells and Clara cells. Enzyme activities measured in the ciliated cells (152 +/- 66 pmol/min/mg protein or 51.2 +/- 20.5 pmol/min/10(6) cells for 7-ethoxycoumarin deethylase and 31.7 +/- 15.4 pmol/min/mg protein or 10.5 +/- 4.8 pmol/min/10(6) cells for coumarin hydroxylase) were not attributable to contamination with Clara cells.

Cytochrome P-450 monooxygenase, epoxide hydrolase and flavin monooxygenase activities in Clara cells and alveolar type II cells isolated from rabbit.

The activities of several enzymes which metabolize xenobiotics were measured and compared in freshly isolated rabbit Clara cells (50-70% purity) and alveolar type II cells (80-95% purity) or microsomal preparations from the isolated cell fractions. The presence of 1 mM nicotinamide in protease and cell isolation buffers increased significantly 7-ethoxycoumarin (7-EC) deethylase and epoxide hydrolase activities in the isolated Clara and type II cells. Isolated Clara cell fractions metabolized 7-EC to umbelliferone at a rate of 241 +/- 27 pmoles/mg prot/min (mean +/- S.E., N =5), while the 7-EC deethylation rate in type II cells was 111 +/- 15 pmoles/mg prot/min. Coumarin hydroxylation activity, however, was more than ten times greater in the Clara cells than in the type II cells on a per mg cellular protein basis. N-oxidation of N,N-dimethylaniline, catalyzed by a flavin monooxygenase, was about 2 times as great in microsomes of Clara cells as in microsomes of type II cells. Epoxide hydrolase activity with benzo(a)pyrene 4,5-oxide as substrate was about 10 times higher in Clara cells than in type II cells. Because of the greater cellular, structural and functional heterogeneity in lung, differential distribution of enzymes responsible for xenobiotic metabolism in this tissue may contribute to cell selective chemical toxicity and carcinogenesis.

Oxidation of 7-ethoxycoumarin and conjugation of umbelliferone by intact, viable epidermal cells from the hairless mouse.

Intact, viable (greater than 80%) epidermal cells were isolated from the hairless mouse. These cells metabolized 7-ethoxycoumarin (7-EC) to umbelliferone ( UMB ) (3 pmol/min/10(6) cells) and UMB to the sulfate and glucuronide conjugates (1 pmol/min/10(6) cells). The rate of oxidation in intact cells compared well with that in disrupted cells with added NADPH, but conjugation proceeded more rapidly in disrupted cells with added cofactors, due to a combination of "activation" of the UDP-glucuronosyltransferase, and to a limitation of activity by the concentration of UDP-glucuronic acid in the intact cells. Pretreatment of the animals with 5,6-benzoflavone resulted in a 5-fold increase in the rate of oxidation, and a 2-fold increase in both the rate of conjugation and the intracellular concentration of UDP-glucuronic acid. UDP-glucuronic acid concentration in isolated cells increased during incubation with glucose, and was regenerated to a steady-state concentration on incubation of cells with UMB . Pretreatment of animals with 5,6-benzoflavone decreased the percentage of metabolite conjugated (from 30% to 15%), whereas adding an inhibitor of oxidation, ellipticine, to cells isolated from pretreated animals, increased the percentage of metabolite conjugated (from 15% to 40%). Sulfation of UMB was almost undetectable, except at very low concentrations (less than 10 nM) of substrate. Thus, glucuronidation of UMB in epidermal cells may be limited by UDP-glucuronic acid availability; sulfation in the epidermis may contribute little to the conjugation of UMB ; and greater than 70% of the products of 7-EC oxidation in the skin may remain unconjugated.

7-ethoxycoumarin O-deethylation activity in viable basal and differentiated keratinocytes isolated from the skin of the hairless mouse.

Epidermal cells in suspension were prepared from the skin of hairless mice by digestion of skin strips with pronase. The viability of cells in such suspensions was routinely greater than 75%. Fractions enriched in different cell types were prepared from the original cell suspensions using metrizamide gradients and elutriation techniques. These fractions were studied histologically and enzymically. The cells of greater size both were more differentiated and had higher xenobiotic metabolizing enzyme activities. Also increasing in parallel with cell size were parameters such as protein to DNA ratios. Lowest in all respects were basal cells (size, enzyme activities, protein/DNA ratios, etc.). The present techniques seem superior to previously described methods for isolating skin cells for study of xenobiotic metabolisms and possible distribution of these metabolisms in different cell types.

Estimation of the styrene 7,8-oxide-detoxifying potential of epoxide hydrolase in glutathione-depleted, perfused rat livers.

The importance of epoxide hydrolase in preventing styrene 7,8-oxide-induced hepatoxicity was studied in isolated perfused rat livers depleted of GSH. GSH depletion was accomplished by treating the rats with diethyl maleate 45 min before surgical removal of their livers. Diethyl maleate itself caused mild hepatotoxicity that was observed histologically and measured biochemically by the release of hepatic transaminase enzymes into the circulation. GSH transferase activity was decreased in perfused livers from diethyl maleate-treated rats as shown by the persistence of circulating styrene oxide compared with that seen in experiments with livers from control animals. In the absence of GSH transferase activity, the rate of styrene oxide biotransformation by epoxide hydrolase was sufficient to prevent measurable hepatotoxicity, up to epoxide concentrations tolerated by livers from control rats. The administration of 500 mumol of styrene oxide to perfused livers from diethyl maleate-treated rats caused periportal necrosis and extensive covalent binding of styrene oxide-derived radioactivity to tissue protein. These effects, however, were no more severe than those seen in perfused livers from control animals given 500 mumol of styrene oxide. Due to high GSH transferase activity in perfused livers from untreated rats, the capacity of epoxide hydrolase to detoxify styrene oxide is difficult to measure in this system. The detoxication potential of epoxide hydrolase was clearly demonstrated in this study with GSH-depleted liver preparations.

Hepatic microsomal NADPH-cytochrome P-450 reductase from little skate, Raja erinacea. Comparison of thermolability and other molecular properties with a mammalian enzyme.

Components of little skate (an elasmobranch) and rabbit hepatic microsomal cytochrome P-450 dependent monooxygenase systems were examined for differences which might explain the decreasing xenobiotic-metabolizing activity of little skate microsomes assayed at temperatures above 30 degrees C. The proportion of saturated fatty acids in microsomal lipids and the habitat temperature are both lower in skate as compared to rabbit, which is consistent with the known adaptive pattern. The more thermolabile enzyme of the skate system in microsomal preparations is NADPH-cytochrome P-450 reductase. The optimal assay temperature for purified skate reductase (30 degrees C) is 10 degrees C lower than that for the purified rabbit reductase. The purified skate reductase differs from rabbit reductase in monomeric molecular weight, in peptides produced by partial proteolysis, in immunochemical properties, but not in flavin content.

Cytochrome P-450-dependent xenobiotic metabolizing activity in Zymbal's gland, a specialized sebaceous gland of rodents.

Homogenates of Zymbal's glands from beta-naphthoflavone-treated rats and mice have 7-ethoxycoumarin O-deethylase activity, while those from rats also have aryl hydrocarbon hydroxylase activity. Measured concentrations of cytochrome P-450 in microsomes from Zymbal's glands of beta-naphthoflavone-treated rats are not higher than those from untreated rats. Studies of inhibitors of 7-ethoxycoumarin O-deethylation and aryl hydrocarbon hydroxylation in homogenates of Zymbal's glands from beta-naphthoflavone-treated rats suggest that these enzyme activities are catalyzed by cytochrome P-450. These findings indicate that reactive metabolites of chemical carcinogens may be formed in Zymbal's gland, a target organ for chemical carcinogenesis.

Foreign compound metabolism by isolated skin cells from the hairless mouse.

A method for isolating mouse skin cells by enzymatic digestion with trypsin was developed. Cell populations of 33% viability could be further separated by metrizamide and Percoll gradient centrifugations into three fractions enriched in different cell types. in one fraction 80% of the cells were sebaceous, in the second fraction 50% of the cells were basal and the third fraction consisted predominantly of differentiated keratinocytes. Different cell types were characterized by electron microscopy, light microscopy, staining and enzyme activities. Measurement of benzo(a)pyrene hydroxylase, 7-ethoxycoumarin O-deethylase, UDP-glucuronosyltransferase and GSH-S-transferase activities in different cell types from control mice and mice topically treated with beta-naphthoflavone showed that different cell populations metabolized foreign compounds at different rates. The sebaceous cells were the most active xenobiotic-metabolizing cells. beta-Naphthoflavone increased relative enzyme activities of the original cell population and basal cell-enriched fraction more than that of the already highly active sebaceous cell population.

Prostaglandin synthetase and cytochrome P-450-dependent metabolism of (+/-)benzo(a)pyrene 7,8-dihydrodiol by enriched populations of rat Clara cells and alveolar type II cells.

The metabolism of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol) by prostaglandin synthetase and cytochrome P-450-dependent monooxygenases was studied using enriched fractions of Clara cells and alveolar type II cells from rat lung. Arachidonic acid-fortified fractions enriched in Clara cells and alveolar type II cells metabolized BP-7,8-diol to the 7,10/8,9-tetrol of benzo(a)pyrene and the 7/8,9,10-tetrol of benzo(a)pyrene. These tetrols are formed upon solvolysis of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha- epoxy-7,8,9,10-tetrahydrobenzo(a)-pyrene (BP diol-epoxide I). Arachidonic acid-dependent metabolism of BP-7,8-diol to BP diol-epoxide I in enriched Clara cells and alveolar type II cells was completely inhibited by indomethacin, a classical inhibitor of prostaglandin synthetase. Enriched Clara cells and alveolar type II cells also metabolized BP-7,8-diol to BP diol-epoxide I in the presence of NADPH. Amounts of BP diol-epoxide I-derived tetrols formed from BP-7,8-diol by the prostaglandin synthetase-dependent and the cytochrome P-450-dependent pathways varied significantly between the two pulmonary cell fractions examined. In fractions enriched in Clara cells, cytochrome P-450-dependent BP-7,8-diol oxidation was higher than was prostaglandin synthetase-dependent BP-7,8-diol oxidation; while in fractions of alveolar type II cells, prostaglandin synthetase-dependent BP-7,8-diol oxidation to BP diol-epoxide I predominated. Pretreatment of rats with beta-naphthoflavone resulted in a 2- to 3-fold increase in BP diol-epoxide I formation by prostaglandin synthetase and cytochrome P-450-dependent monooxygenases in both enriched Clara cells and alveolar type II cells. These increases in BP-7,8-diol oxidation to BP diol-epoxide I appear to be due to induction of the two enzymatic pathways in both pulmonary cell types. No qualitative changes in the pattern of BP-7,8-diol metabolism by either enzymatic pathway in enriched Clara cells or alveolar type II cells were observed following beta-naphthoflavone treatment. The results suggest that pulmonary prostaglandin synthetase may serve as either an additional or an alternative bioactivating enzyme to the cytochrome P-450-dependent monooxygenases for the formation of reactive chemical carcinogens in the lung.

Xenobiotic metabolism in Clara cells and alveolar type II cells isolated from lungs of rats treated with beta-naphthoflavone.

An investigation of several pathways for xenobiotic metabolism in rat lung cells was carried out using enriched fractions of alveolar type II cells (80% purity) and Clara cells (50% purity) which had been prepared from either untreated (control) rats or animals which had been treated with beta-naphthoflavone. Monooxygenase activities (7-ethoxycoumarin deethylase; aryl hydrocarbon [benzo(a)pyrene] hydroxylase) and activities of conjugating enzymes (glutathione transferase; glucuronosyl transferase) were found to be much higher in fractions enriched in Clara cells than in either the crude cell digest or in fractions enriched in type II cells. This was also found to be true for epoxide hydrolase activity. beta-Naphthoflavone treatment of animals was found to increase the monooxygenase and glucuronosyl transferase activities in all cell fractions, but no effect was seen on either glutathione transferase or epoxide hydrolase activity, each of which was extremely low in type II cells.

Benzo(a)pyrene hydroxylase activity in enriched populations of Clara cells and alveolar type II cells from control and beta-naphthoflavone-pretreated rats.

We have developed a method for isolation and purification of specific types of lung cells from rats. Alveolar type II cells were purified from 18% in the initial cell digest to 30% after centrifugal elutriation and finally to over 80% following density gradient centrifugation. Clara cells were enriched from 0.8% in the cell digest to 40 to 60% by use of centrifugal elutriation. In control animals, aryl hydrocarbon hydroxylase activity was found to increase in parallel with Clara cell purity but was almost undetectable in the type II cells. Following pretreatment of rats with beta-naphthoflavone, aryl hydrocarbon hydroxylase activity increased both in Clara cells and particularly, in alveolar type II cells. This methodology provides a means for investigation of the effects of toxic and carcinogenic compounds on different populations of lung cells.

Thyroid hormone-induced changes in the hepatic monooxygenase system, heme oxygenase activity and epoxide hydrolase activity in adult male, female and immature rats.

In 8-day-old rat pups, pretreatment with a single injection of L-triiodothyronine or L-thyroxine decreased hepatic cytochrome P-450 content, aminopyrine N-demethylase activity and epoxide hydrolase activity but increased hepatic microsomal cytochrome c reductase, 7-ethoxyresorufin O-deethylase and heme oxygenase activities without significantly altering UDP-glucuronosyltransferase activity (towards o-aminophenol) or the microsomal yield. In adult rats of either sex such single injections of L-triiodothyronine failed to significantly alter these enzyme activities. However, multiple injections evoked changes similar to those observed in the pups, in all these enzyme activities, except that 7-ethoxyresorufin O-deethylase activity was slightly decreased rather than increased. These findings demonstrate that: (1) The hepatic monooxygenase system in the rat pup is more responsive to thyroid hormones than that in adult. (2) Thyroid hormones can decrease rat liver cytochrome P-450 content and its dependent monooxygenase activity independently of sexual maturity. (3) Thyroid hormones also decrease hepatic epoxide hydrolase activity in both pups and adults. Thus, hyperthyroidism could render the rat pup more susceptible to hepatotoxicity from electrophilic epoxides which utilize microsomal epoxide hydrolase as the major detoxication pathway.

In vitro metabolic activation of the pulmonary toxin, 4-ipomeanol, in nonciliated bronchiolar epithelial (Clara) and alveolar type II cells isolated from rabbit lung.

The metabolism and covalent binding of 4-ipomeanol (IPO), a pulmonary toxin, were investigated in pulmonary cells isolated from rabbit. 3H-labeled IPO was incubated with freshly, isolated, intact alveolar type II cells (83% purity), nonciliated bronchiolar epithelial (Clara) cells (77% purity) and alveolar macrophages (greater than 90% purity). Covalent binding of radioactive material to type II and Clara cells was observed by autoradiography and by a biochemical method. IPO binding to cells was almost totally prevented by 1 mM piperonyl butoxide, an inhibitor of the cytochrome P-450-dependent metabolism of IPO. No covalent binding was observed with alveolar macrophages in the presence or absence of piperonyl butoxide. The maximal rates of enzyme-mediated covalent binding of IPO to protein were greater in the Clara cells (135 pmol/10(6) cells/min) than in the type II cells (13 pmol/10(6) cells/min). Incubation of either sonicated Clara or type II cell fractions with [3H]IPO, glutathione and NADPH (20 min, 37 degrees C) resulted in the formation of two distinct radiolabeled glutathione conjugates.