The cytochrome P-450 monooxygenase system of rabbit bladder mucosa: enzyme components and isozyme 5-dependent metabolism of 2-aminofluorene.

The microsomal fraction prepared from the mucosa of rabbit bladder was analyzed for the presence of enzymes and activities associated with the cytochrome P-450-dependent monooxygenase system. Reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase (315 units/mg protein), reduced nicotinamide adenine dinucleotide:cytochrome b5 reductase (920 units/mg protein), cytochrome P-450 (0.22 nmol/mg protein), and cytochrome b5 (0.31 nmol/mg protein) were present in the microsomal preparation. Individual isozymes of cytochrome P-450, forms 2, 5, and 6, but not form 4, were detected by immunochemical methods. Treatment of rabbits with either phenobarbital or 2,3,7,8-tetrachlorodibenzo-p-dioxin did not alter the concentrations of these isozymes in the bladder preparation. Monooxygenase activities (pmol product/min/protein) in the bladder microsomal fraction were observed for benzphetamine N-demethylation (290), 7-ethoxyresorufin O-deethylation (29), 7-ethoxycoumarin O-deethylation (28), benzo(a)pyrene hydroxylation (10), and 2-aminofluorene hydroxylation (1400). The metabolism of 2-aminofluorene was determined by high performance liquid chromatography and scintillation counting; two products, 2-nitrosofluorene and 2,2'-azoxybisfluorene, were identified by chromatographic retention times, ultraviolet-visible spectroscopy, and mass spectrometry. Two additional metabolites were tentatively identified as N-hydroxy-2-aminofluorene and a ring-hydroxylated product. The metabolism of 2-aminofluorene was inhibited by antibodies to cytochrome P-450 form 5 or to reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase and by carbon monoxide (CO:O2, 4:1), but not by antibodies to cytochrome P-450 form 2. Acetylation of 2-aminofluorene in the presence of ethyl acetate (and deacetylation of 2-acetylaminofluorene) mediated by an enzyme sensitive to inhibition by either paraoxon or sodium fluoride was also observed.

Deacetylation to 2-aminofluorene as a major initial reaction in the microsomal metabolism of 2-acetylaminofluorene to mutagenic products in preparations from rabbit lung and liver.

The rabbit pulmonary and hepatic microsomal pathways for the metabolism of 2-acetylaminofluorene (AAF) and 2-aminofluorene (AF) to mutagenic products were investigated by means of high performance liquid chromatography and the Salmonella mutagenicity assay. Mutagenic activity approached a maximum with increasing concentrations of AAF incubated with hepatic microsomal preparations and Salmonella; with pulmonary microsomal preparations, mutagenic activity was proportional to the concentration of AAF over the range examined. The mutagenic activities of AF exhibited typical saturation kinetics with both hepatic and pulmonary microsomal preparations. Approximately 7 times more AF than N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) was formed in incubations of AAF (0.5 mM) with hepatic microsomal preparations. When AAF was incubated with pulmonary microsomal preparations, formation of AF, but not N-hydroxy-AAF, was detected. The inclusion of paraoxon in the pulmonary incubations blocked the formation of AF but did not lead to the recovery of any N-hydroxy-AAF. We conclude that the metabolism of AAF to mutagenic products in pulmonary microsomal preparations from rabbits is initiated primarily, if not entirely, by deacetylation of AAF to AF. The mutagenic activity of AAF with the pulmonary microsomal preparations is limited by the deacetylase activity which, like mutagenic activity, exhibits a linear relationship with the concentration of AAF. On the basis of the rates of formation of AF and N-hydroxy-AAF and their mutagenic activities, we estimate that about 60% of the hepatic metabolism of AAF to mutagenic products is dependent upon deacetylation of AAF and subsequent oxidation of the AF formed.

High-resolution computed tomography: Part 5. Evaluation of the temporomandibular joint.

High-resolution computed tomography (CT) is evolving as the most valuable radiographic study when detailed information about the status of the temporomandibular joint is needed. High-resolution CT is faster, less invasive, and in most cases much more accurate than other imaging modalities used in the past. Erosive, hypertrophic, and ankylosing arthropathies are illustrated, as are condylar dislocations secondary to acute trauma, chronic trauma, and neoplasm. A detailed discussion of the common problem of the anterior displaced meniscus is undertaken.

Water systems to report drinking water quality to all customers: how can health professionals prepare for the questions that these reports will generate?

The ultimate safety of drinking water depends upon protection of source waters and construction and maintenance of reliable drinking water treatment and distribution systems. These objectives require public support. Physicians can encourage their patients to call their water suppliers and advocate for investment in effective treatment systems and support zoning that will protect water supply watersheds and wellheads. The Consumer Confidence Reports are meant to inform consumers about their drinking water supply. Consumers should use the reports to verify that their drinking water meets all health standards and to understand some of the potential threats to their drinking water quality. Physicians may use the reports as an opportunity to discuss the many types of environmental exposures and ways to reduce these exposures. As a crucial component of the public health community, this is your opportunity to encourage your patients to become more aware of their environment and its impact on their health.

Stimulation of ribonucleic acid synthesis by chloramphenicol in a rel + aminoacyl-transfer ribonucleic acid synthetase mutant of Escherichia coli.

Escherichia coli strain 9D3 possesses a highly temperature-sensitive valyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.9). Since 9D3 is a rel(+) strain, it cannot carry out net RNA synthesis at high temperature. A 100-mug amount of chloramphenicol (CAP) per ml added in the absence of valine cannot stimulate RNA synthesis. Either 300 mug of CAP or 100 mug of CAP plus 50 mug of valine per ml, however, promotes nearly maximal RNA synthesis. These results can be understood as follows. (i) Valyl-tRNA is required for net RNA synthesis, (ii) the synthetase lesion is incomplete, (iii) the rate of mutant acylation of tRNA(val) at high temperature is valine-dependent, and (iv) the CAP concentration determines the rate of residual protein synthesis. Data are also presented which demonstrate that the rate of net RNA synthesis can greatly increase long after the addition of CAP, if the amount of valyl-tRNA increases.

Reconciling science and policy in setting federal drinking water standards--four states' perspectives.

After almost 20 years of experience with implementing the Safe Drinking Water Act and eight with Amendments to the Act, the individual states within the United States have gained valuable experience while trying to reconcile the legal mandates provided by the statutes with the science underlying them. This paper presents four different topics illustrating the problems of reconciling these two issues in the regulation of toxic chemicals in drinking waters. It presents these from the perspectives of the states of Massachusetts, Rhode Island, Connecticut, and New Jersey and offers suggestions for improved program efficiency based on considerations of comparative human health risks. The approach and schedule for controlling toxic chemicals used through 1994 are first examined and a recommendation is made for more flexibility in the rate at which chemicals are regulated. Recent U.S. EPA proposals to more stringently control radon in drinking waters are presented in the context of all sources of radon exposures, illustrating the intersection of science, laws, and economic consequences of regulatory initiatives. Inhalation and dermal exposures as a result of using chemically contaminated drinking waters are then discussed with the suggestion of the possible underprotectiveness of some present standards. Finally, the difficulty faced by the states and federal government in the control of naturally occurring arsenic exposures through drinking water is also presented and an argument is made for more local flexibility in the application of health-based standards.

Epidermal growth factor (EGF) promotes chemomigration of a human prostate tumor cell line, and EGF immunoreactive proteins are present at sites of metastasis in the stroma of lymph nodes and medullary bone.

Prostate tumor cells preferentially metastasize to bony sites and lymph nodes at a frequency in excess of that which would be predicted by random tumor cell dissemination. In order to determine whether chemoattractants in these organs promote organ-specific metastasis, we utilized human cell lines derived from and/or related to these organs as sources of potential chemoattractants. Secretory proteins derived from the cell lines MG-63 (osteosarcoma), SK-ES-1 (Ewing's sarcoma), and KG-1 (leukemia) stimulated chemomigration of the TSU-pr1 prostate tumor cells in a dose-dependent manner in Boyden chambers. In addition, secretory proteins from a human prostatic stromal cell line (hPS) and from the TSU-Pr1 prostate tumor cell line were also able to stimulate chemomigration of the TSU-pr1 cells through Boyden chambers. Since lymph nodes and bony sites represent organs of hematopoietic/lymphoid proliferation and activation, we undertook identification of specific cytokines present at these sites which may promote the chemomigration of prostate tumor cells. In this context, the cytokines interleukin-1 alpha, interleukin-2, interleukin-6, tumor necrosis factor-beta, transforming growth factor-beta, interferon alpha 2-a, and granulocyte-macrophage colony-stimulating factor did not stimulate chemomigration of the TSU-pr1 prostate tumor cell line. In contrast, the cytokine epidermal growth factor (EGF) stimulated chemomigration of the TSU-pr1 prostate tumor cells through the Boyden chambers in a dose-dependent manner. Western blot analysis of secretory proteins from the cell lines KG-1, SK-ES-1, MG-63, hPS, and TSU-pr1 identified EGF-immunoreactive proteins in all cases. In addition, EGF immunoreactivity was localized to the stroma of the human prostate, the osteogenic stroma of pelvic medullary bone, and the stroma within the capsule and trabeculae of pelvic lymph nodes. Hence, these results demonstrate that the cytokine EGF promotes the chemomigration of the TSU-pr1 prostate tumor cell line, and that EGF within the stroma of pelvic lymph nodes and medullary bone may act as a chemoattractant for prostate tumor cells, thereby facilitating the preferential formation of metastatic foci within these organs.

The radiographic diagnosis of pelvic lipomatosis.

We describe a patient who initially presented with asymptomatic hydronephrosis. He underwent extensive radiologic evaluation which led to the diagnosis of pelvic lipomatosis. The possible etiology, workup, and treatment options of this unusual entity are discussed.

Problems in assessing the risks of mixtures of contaminants in drinking water.

In conducting risk assessments on drinking water contaminants, the U.S. Environmental Protection Agency (EPA) attempts to evaluate all available toxicity data to develop Health Advisory (HA) and Maximum Contaminant Level Goal (MCLG) values. The EPA often has grappled with the issues surrounding the toxicity of chemical mixtures, including radioactive contaminants, nitrate/nitrite, and trihalomethanes (THMs). In evaluating the toxicity of chemical mixtures, the EPA's immediate concern is whether the individual HA values and MCLGs are protecting public health when multiple contaminants are present in drinking water. Potential toxic interactions between drinking water contaminants are difficult to predict because experimental studies are generally performed only at high doses relative to environmental levels. Although the contamination of drinking water involves mixtures of contaminants, drinking water regulations are generally based on an assessment of the risks of individual contaminants. This paper discusses three issues of major concern to the EPA: the synergistic effects of solvent mixtures, vehicle effects in laboratory studies, and setting standards for essential trace nutrients where the absorption and/or toxicity are affected by an individual's nutritional status or other dietary components.

Species-dependent expression and induction of homologues of rabbit cytochrome P-450 isozyme 5 in liver and lung.

The presence of homologues of rabbit cytochrome P-450 isozyme 5 in pulmonary and hepatic microsomal preparations from guinea pig, mouse, monkey, hamster, and rat was examined by immunoblotting and inhibition of metabolism of 2-aminofluorene with antibodies to isozyme 5. Homologues to isozyme 5 were detected in pulmonary preparations from all five species. However, only hepatic preparations from hamster, in addition to those from rabbit, contained detectable levels of this isozyme. With the exception of induction by phenobarbital in rabbit liver, treatment of animals with phenobarbital or tetrachlorodibenzo-p-dioxin did not increase hepatic or pulmonary content of isozyme 5 homologues or the amount of 2-aminofluorene metabolism inhibited by antibodies to isozyme 5. Metabolism of 2-aminofluorene was measured both colorimetrically (formation of a reduced iron chelate from the N-hydroxyfluorene metabolite) and radiochemically (separation of 3H-metabolites by high performance liquid chromatography and quantitation by scintillation counting). A turnover number of 48 nmol of product X min-1 X nmol of enzyme-1 for isozyme 5-catalyzed metabolism of 2-aminofluorene was determined with incubations containing isozyme 5 purified from rabbit lung. A similar turnover number was calculated from the rabbit hepatic microsomal activity inhibited by antibodies to isozyme 5 and the microsomal isozyme 5 content measured by immunoquantitation. In other species, amounts of metabolism inhibited by antibodies to isozyme 5 agreed qualitatively with relative staining intensities on immunoblots. In all species except the hamster, rates of total and isozyme 5-catalyzed metabolism of 2-aminofluorene were greater with pulmonary than with hepatic microsomal preparations from untreated animals.

Refolding and characterization of recombinant human soluble CTLA-4 expressed in Escherichia coli.

CD28 and CTLA-4 are homologous cell surface proteins expressed by T cells. CD28 is constitutively expressed by most T cells, whereas CTLA-4 is expressed by activated T cells. Both proteins are ligands for the costimulatory molecules CD80 and CD86 expressed by activated B cells, macrophages, and dendritic cells. A fusion protein comprising the CTLA-4 extracellular domain joined to a human immunoglobulin heavy chain constant region (CTLA4Ig) binds CD80 and CD-86 with high affinity and inhibits CD80/CD86-dependent immune responses in vitro and in vivo. Attempts at producing the CTLA-4 extracellular domain as an unfused protein have met with limited success. Here we describe the expression and purification of the CTLA-4 extracellular domain as a nonfused protein in Escherichia coli. The 12.5-kDa CTLA-4 extracellular domain was insoluble when expressed in E. coli and required denaturation, reduction, and refolding steps to become soluble and assume its proper conformation. The protein refolded into a mixture of monomers, disulfide-linked dimers, and higher order disulfide-linked aggregates. sCTLA-4 dimers were the predominant refold form when air was used as the oxidizing agent during the refold procedure. Purified sCTLA-4 dimers were 10- to 50-fold more potent than sCTLA-4 monomers at inhibiting T cell activation using a CD80-dependent in vitro bioassay.

Multifunctional regulation of the biological effects of TNF-alpha by the soluble type I and type II TNF receptors.

Two soluble receptors of tumour necrosis factor were evaluated for development as potential therapeutic agents for inflammatory disease. The recombinant human soluble Type I and Type II TNF receptors, rsTNF-RI and rsTNF-RII, were expressed at high levels in E. coli, refolded, and chromatographically purified to homogeneity. The potencies of both recombinant soluble receptors were similar to their naturally occurring soluble receptors. In in vitro cytotoxicity and competitive binding assays, both recombinant soluble receptors functioned to inhibit the biological effects of rhTNF-alpha although rsTNF-RI was a 5 to 30 fold more potent inhibitor of rhTNF-alpha than was rsTNF-RII or a truncated form of the soluble receptor, TNF-RII delta. In in vivo experiments in mice, rsTNF-RI was a better inhibitor than rsTNF-RII delta of rhTNF-alpha-stimulated changes in the percentages of circulating lymphocytes and neutrophils, influx of neutrophils into the peritoneal cavity, and serum IL-6 induction. At molar ratios of 0.1:1 and 0.01:1 (rsTNF-R:rhTNF-alpha), using the rsTNF-I or rsTNF-II delta, there was a trend towards enhancement of the induction of IL-6. However, higher ratios of either rsTNF-RI or rsTNF-RII delta significantly inhibited the rhTNF-alpha-stimulated increase in serum IL-6 levels. In a murine model of cytokine-induced shock, either rsTNF-RI or rsTNF-RII delta provided protection against the lethality of shock induced by a synergistic combination of rhTNF-alpha and rhIL-1 beta. Based on the results of these experiments, the rsTNF-RI was chosen as the better candidate for development as an anti-inflammatory agent.