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.

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.

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.

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.

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.

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)

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.

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.

Relationship between induction of aryl hydrocarbon hydroxylase and de novo synthesis of cytochrome P-448 (P1-450) in mice.

Phenobarbital, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), benzpyrene, 3-methylcholanthrene (3-MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were administered i.p. for 1 or 3 days to genetically "responsive" (C57BL/6J) and genetically "non-responsive" (DBA/2J) mice. 3-MC or benzpyrene stimulated aryl hydrocarbon hydroxylase (AHH) activity in C57BL/6J (B6) mice but not in DBA/2J (D2) mice. TCDD induced AHH activity in both B6 and D2 mice. Time-course studies showed that in the first 12 h after a single injection of 3-MC to B6 mice there was no shift in the reduced cytochrome P-450-CO complex absorption spectra from 450 to 448 nm, although AHH activity increased 4-5 times over (above) that of the control group. The relationship between induction of AHH activity by polycyclic hydrocarbons in B6 mice and the concomitant synthesis of cytochrome P-448 is discussed.

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.

Metabolism of benzo(a)pyrene and benzo(a)pyrene 4,5-oxide in rabbit lung.

For several years our laboratory has been investigating the biotransformation of various environmental pollutants by lung. Studies have been performed with pulmonary subcellular fractions, purified monooxygenase and glutathione transferase enzymes, and preparations having intact cellular structure including the isolated perfused lung and cell fractions enriched in alveolar macrophages, Clara cells and alveolar type II cells. Collectively, these investigations have identified several metabolic factors which may contribute to the pulmonary toxicity mediated by certain polycyclic aromatic hydrocarbons (PAH). First, although lung has low overall cytochrome P-450-dependent monooxygenase activity for many substrates, relative to liver, this activity is localized in only a few cell types and specific activity in certain cell types, such as the non-ciliated bronchiolar epithelial (Clara) cell, can be high. Second, oxidative metabolites of benzo(a)pyrene tend to accumulate in pulmonary tissue due, at least in part, to the low ability of lung (relative to liver) to conjugate and detoxify phenolic, dihydrodiol and epoxide metabolites. Thus, products such as benzo(a)pyrene 7,8-dihydrodiol are available for further cytochrome P-450-dependent oxidation to ultimate carcinogens and cytotoxins. Moreover, the lung is efficient in removing benzo(a)pyrene 4,5-oxide and presumably other oxidized PAH metabolites, from the bloodstream. Consequently, the uptake of relatively stable electrophilic metabolites released by the liver may also contribute to pulmonary toxicity.