Prediction of naphthalene bioaccumulation using an adipocyte cell line model

A long-term goal of this research is to develop an in vitro model to study the metabolism, distribution, and fate of chemicals or pharmaceuticals in animals and humans. An important component of such a system is an in vitro model to study bioaccumulation of specific chemicals in adipose tissue. Due to the difficulties in maintaining primary adipocytes in culture and conducting reproducible experiments, transformed adipocyte cell lines have been used as an alternative. In this paper, several rodent preadipocyte cell lines (3T3-L1, 3T3-F442A, and TA1 cells) that differentiate into adipocytes when exposed to the appropriate stimuli are tested as an investigative tool to study naphthalene accumulation. The in vitro model is tested by comparison of its performance to that of primary adipocytes. All the experimental evidence supports the hypothesis that naphthalene accumulation is primarily dependent on the level of intracellular lipid. Furthermore, the level of naphthalene bioaccumulation is linearly correlated with the amount of triglyceride content with the slope of 37.7 +/- 0.5 microg of naphthalene/(mg of triglyceride). Indomethacin/dexamethasone/insulin are shown to be more effective in promoting preadipocyte differentiation than methylisobutylxanthine/dexamethasone/insulin. Additionally, external factors, such as the presence of albumin and serum in the medium, affect the cellular naphthalene uptake by decreasing the amount of naphthalene transported into fat cells. Among the three cell lines tested, 3T3-L1 adipocytes accumulated the highest intracellular lipid and, hence, yielded the highest level of naphthalene accumulation. Its ability to accumulate naphthalene is comparable to that of primary adipocytes. The 3T3-L1 adipocyte model is appropriate for studying the bioaccumulation of xenobiotics that are aromatic hydrocarbons.     

Degradation of polycyclic aromatic hydrocarbon compounds under various redox conditions in soil-water systems

This study evaluated the microbial degradation of naphthol, naphthalene, and acenaphthene, under aerobic, anaerobic, and denitrification conditions in soil-water systems. Chemical degradation of naphthol and naphthalene in the presence of a manganese oxide was also studied. Naphthol, naphthalene, and acenaphthene were degraded microbially under aerobic conditions from initial aqueous-phase concentrations of 9, 7, and 1 mg/liter to nondetectable levels in 3, 10, and 10 days, respectively. Under anaerobic conditions naphthol degraded to nondetectable levels in 15 days, whereas naphthalene and acenaphthene showed no significant degradation over periods of 50 and 70 days, respectively. Under denitrification conditions naphthol, naphthalene, and acenaphthene were degraded from initial aqueous-phase concentrations of 8, 7, and 0.4 mg/liter to nondetectable levels in 16, 45, and 40 days, respectively. Acclimation periods of approximately 2 days under aerobic conditions and 2 weeks under denitrification conditions were observed for both naphthalene and acenaphthene. Abiotic degradation of naphthalen and naphthol were evaluated by reaction with manganese oxide, a minor soil constituent. In the presence of a manganese oxide, naphthalene showed no abiotic degradation over a period of 9 weeks, whereas the aqueous naphthol concentration decreased from 9 mg/liter to nondetectable levels in 9 days. The results of this study show that low-molecular-weight, unsubstituted, polycyclic aromatic hydrocarbons are amenable to microbial degradation in soil-water systems under denitrification conditions.     

Degradation of polycyclic aromatic hydrocarbon compounds under various redox conditions in soil-water systems.

This study evaluated the microbial degradation of naphthol, naphthalene, and acenaphthene, under aerobic, anaerobic, and denitrification conditions in soil-water systems. Chemical degradation of naphthol and naphthalene in the presence of a manganese oxide was also studied. Naphthol, naphthalene, and acenaphthene were degraded microbially under aerobic conditions from initial aqueous-phase concentrations of 9, 7, and 1 mg/liter to nondetectable levels in 3, 10, and 10 days, respectively. Under anaerobic conditions naphthol degraded to nondetectable levels in 15 days, whereas naphthalene and acenaphthene showed no significant degradation over periods of 50 and 70 days, respectively. Under denitrification conditions naphthol, naphthalene, and acenaphthene were degraded from initial aqueous-phase concentrations of 8, 7, and 0.4 mg/liter to nondetectable levels in 16, 45, and 40 days, respectively. Acclimation periods of approximately 2 days under aerobic conditions and 2 weeks under denitrification conditions were observed for both naphthalene and acenaphthene. Abiotic degradation of naphthalen and naphthol were evaluated by reaction with manganese oxide, a minor soil constituent. In the presence of a manganese oxide, naphthalene showed no abiotic degradation over a period of 9 weeks, whereas the aqueous naphthol concentration decreased from 9 mg/liter to nondetectable levels in 9 days. The results of this study show that low-molecular-weight, unsubstituted, polycyclic aromatic hydrocarbons are amenable to microbial degradation in soil-water systems under denitrification conditions.     

Incorporation of 3T3-L1 cells to mimic bioaccumulation in a microscale cell culture analog device for toxicity studies

Deficiencies in the early ADMET (absorption, distribution, metabolism, elimination, and toxicity) information on drug candidates extract a significant economic penalty on pharmaceutical firms. We have developed a microscale cell culture analog (microCCA) device that can potentially provide better, faster, and more efficient prediction of human and animal responses to a wide range of chemicals. The system described in this paper is a simple four-chamber microCCA ("lung"-"liver"-"fat"-"other tissue") designed on the basis of a physiologically based pharmacokinetics (PBPK) model of a rat. Cultures of L2, HepG2/C3A, and differentiated 3T3-L1 adipocytes were selected to mimic the key functions of the lung, liver, and fat compartments, respectively. Here, we have demonstrated the application of the microCCA system to study bioaccumulation, distribution, and toxicity of selected compounds. Results from the bioaccumulation study reveal that hydrophobic compounds such as fluoranthene preferentially accumulated in the fat chamber. Only a small amount of fluoranthene was observed in the liver and lung chambers. In addition, the presence of the differentiated 3T3-L1 adipocytes in the microCCA device significantly reduced naphthalene and naphthoquinone-induced glutathione (GSH) depletion. These findings suggest the potential utilization of the microCCA system to assess ADMET characteristics of the compound of interest prior to animal or human trials.     

Cooxidation of naphthalene and other polycyclic aromatic hydrocarbons by the nitrifying bacterium,Nitrosomonas europaea

The soil nitrifying bacterium Nitrosomonas europaea has shown the ability to transform cometabolically naphthalene as well as other 2- and 3-ringed polycyclic aromatic hydrocarbons (PAHs) to more oxidized products. All of the observed enzymatic reactions were inhibited by acetylene, a selective inhibitor of ammonia monooxygenase (AMO). A strong inhibitory effect of naphthalene on ammonia oxidation by N. europaea was observed. Naphthalene was readily oxidized by N. europaea and 2-naphthol was detected as a major product (85%) of naphthalene oxidation. The maximum naphthol production rate was 1.65 nmole/mg protein-min in the presence of 240 M naphthalene and 10 mM NH₄ ⁺. Our results demonstrate that the oxidation between ammonia and naphthalene showed a partial competitive inhibition. The relative ratio of naphthalene and ammonia oxidation, depending on naphthalene concentrations, demonstrated that the naphthalene was oxidized 2200-fold slower than ammonia at lower concentration of naphthalene (15 M) whereas naphthalene was oxidized only 100-fold slower than ammonia oxidation. NH₄ ⁺- and N₂H₄-dependent O₂ uptake measurement demonstrated irreversible inhibitory effects of the naphthalene and subsequent oxidation products on AMO and HAO activity.     

The Chemical Constitution of Rubrofusarin

Alkaline degradation of rubrofusarin and nor-rubrofusarin were studied; nor-rubrofusarin readily underwent hydrolysis to give a tetrahydroxynaphthalenc, acetone, and acetic acid; whereas, rubrofusarin, after prolonged time of hydrolysis, yielded a β-methoxytrihydroxynaphthalene instead of the naphthol. Physical and chemical studies revealed that the naphthol is 1,3,6,8-tetrahydroxynaphthalene and it has been confirmed by the synthesis from chromotropic acid (disodium salt). Thus, evidently, rubrofusarin has a naphthalene nucleus to which a methoxyl group is attached at β-position. The formation, on the hydrolysis, of acetone and acetic acid, along with the naphthol, indicates the presence of 2-methyl-γ-pyrone structure in rubrofusarin.     

Stimulation and inhibition of lipolysis in isolated rat adipocytes: evidence for age-related changes in responses to forskolin and PGE1

The ability of a number of hormones to activate cellular responses in a variety of cells declines with age. The mechanisms responsible for these alterations are complex and incompletely understood. Rat adipocytes have served as an important model to study blunted responses to stimulatory hormones which function by activating cAMP accumulation. We have previously found that the blunted lipolytic response of adipocytes from older rats to the beta adrenergic receptor agonist isoproterenol appeared to be due to a lessened ability of isoproterenol to activate cAMP accumulation. Further, the blunted response to isoproterenol was apparently caused by an accentuated inhibition of lipolysis, mediated by adenosine receptors activated by endogenously released adenosine. The present studies were designed to test and extend those conclusions. We have utilized forskolin to augment the cAMP accumulation that occurs in the presence of isoproterenol. Isoproterenol-activated lipolysis was greater in adipocytes from 2 month old rats compared with those from 12 month old rats (603 +/- 32 vs 450 +/- 29 nmol/10(5) cells/hr, P less than 0.01). However, in the presence of forskolin (10(-6) M), there was no significant difference in the response to isoproterenol between the two groups (646 +/- 23 vs 615 +/- 29 nmoles/10(5) cells/hr). As we had seen previously, the adenosine receptor agonist phenylisopropyladenosine more effectively inhibited lipolysis in the adipocytes from older rats. We now also find that PGE1 more efficaciously inhibits lipolysis in the cells from older rats. These data confirm that diminished cAMP accumulation in adipocytes from older rats appears to be a rate-limiting alteration in the regulation of lipolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

A microfluidic lung-on-a-chip based on biomimetic hydrogel membrane

Lung-on-chips have showed great promise as a tool to recapitulate the respiratory system for investigation of lung diseases in the past decade. However, the commonly applied artificial elastic membrane (e.g., polydimethylsiloxane, PDMS) in the chip failed to mimic the alveolar basal membrane in the composition and mechanical properties. Here we replaced the PDMS film by a thin, biocompatible, soft, and stretchable membrane based on F127-DA hydrogel that well approached to the composition and stiffness of extracellular matrix in human alveoli for construction of lung-on-a-chip. This chip well reconstructed the mechanical microenvironments in alveoli so that the epithelial/endothelial functions were highly expressed with a well established alveolar-capillary barrier. In opposite to the unexpectedly accelerated fibrotic process on the PDMS-based lung-on-a-chip, HPAEpiCs on hydrogel-based chip only presented fibrosis under nonphysiologically high strain, well reflecting the features of pulmonary fibrosis in vivo. This physiologically relevant lung-on-a-chip would be an ideal model in investigation of lung diseases and for development of antifibrosis drugs.     

Isoenzymes and histochemistry of non-specific esterases in normal and cancerous skin

Synopsis Non-specific esterases in normal and carcinomatous skin of the mouse have been investigated electrophoretically and histochemically. Three esterase bands were obtained on electrophoresis from homogenates of normal skin; homogenates of carcinomas showed an accumulation of esterase-Ia and esterase-Ib.^* However, using several ester substrates, substrate-specific patterns were demonstrated in the electrophoresis separations and histochemically in tissue sections. On the electrophoresis separations, -naphthyl acetate, -naphthyl acetate, 6-bromo-2-naphthyl acetate, naphthol AS acetate, naphthol AS-D acetate and naphthol AS-LC acetate gave rise to similar patterns, but with -naphthyl propionate as subsmate, more esterase-Ib was indicated and with 5-bromo-indoxyl acetate a distinctive preponderance. Peripheral or uniformly distributed staining was found histochemically in tumour epithelium using -naphthyl acetate, -naphthyl propionate and -naphthyl acetate, whereas with the substrates of naphthol AS acetate, naphthol AS-D acetate and indoxyl acetate an intermediate pattern of staining related to keratinization was obtained.     

Constitutive and inducible hydroxylase activities involved in the degradation of naphthalene by Cunninghamella elegans

The non-ligninolytic fungus Cunninghamella elegans was investigated for its ability to produce naphthalene hydroxylase (NAH) and naphthol hydroxylase (NOH) activities under various conditions. When the organism was cultivated on a rich growth medium, the mycelia exhibited significant constitutive NAH activity in the late exponential growth phase, but not in the early-exponential-growth-phase. On incubating the early-exponential-growth-phase mycelia with naphthalene, NAH activity was increased five-fold; however, this increase did not occur in the presence of the protein synthesis inhibitor cycloheximide. Since incubation of the late-phase mycelia with naphthalene did not lead to a higher degradation rate of naphthalene, mycelia in this physiological state have apparently lost the ability to induce synthesis of the enzyme exhibiting NAH activity. This is not due to an overall inability to perform de novo protein synthesis, since NOH activity, non-constitutive at all growth phases, could be induced by incubating late-phase mycelia with naphthalene. Whether inducible and constitutive NAH activity originate from one and the same enzyme remains to be elucidated. It is suggested that naphthalene oxidizing enzyme(s) may also oxidize pyrene, but not anthracene or benzo[a]pyrene, although the latter are degradable by C. elegans.     

A dual component analysis explains the distinctive kinetics of cAMP accumulation in brown adipocytes

The mechanism behind the distinctive non-Michaelis-Menten, bell-shaped kinetics of cAMP accumulation in brown adipocytes (which underlies the similar kinetics of UCP1 and beta(1)-adrenoreceptor gene expression) was investigated. A theoretical dual component analysis indicated that the observed dose-response curves could be constructed as the resultant of a stimulatory and an inhibitory component. Experimentally, inhibition of the alpha(1)-component of the norepinephrine response revealed the underlying existence of a much larger stimulatory beta(3)-component which displayed monophasic Michaelis-Menten kinetics. The inhibitory alpha(1)-component (which was also monophasic but had a 2-fold higher EC(50)) was mediated via an increase in [Ca(2+)](i); the protein kinase C pathway was not involved. The [Ca(2+)](i) increase which resulted in massive inhibition of cAMP accumulation was very low: <100 nM. The [Ca(2+)](i) signal stimulated a calmodulin-controlled phosphodiesterase, possibly PDE-1. The acquirement of this specific interaction pattern between beta- and alpha(1)-adrenergic stimulation was thus part of the differentiation program of the brown adipocytes. It was concluded that an array of synergistic or inhibitory alpha(1)/beta interactions occur in the adrenergic regulation of this cell type which is unique in its dependence upon adrenergic stimulation for cellular proliferation, differentiation, and metabolic function.     

Trans10, cis12-conjugated linoleic acid prevents triacylglycerol accumulation in adipocytes by acting as a PPARgamma modulator

A group of polyunsaturated fatty acids called conjugated linoleic acids (CLAs) are found in ruminant products, where the most common isomers are cis9, trans11 (c 9,t11) and trans10, cis12 (t10,c12) CLA. A crude mixture of these isomers has been shown in animal studies to alter body composition by a reduction in body fat mass as well as an increase in lean body mass, with the t10,c12 isomer having the most pronounced effect. The objective of this study was to establish the molecular mechanisms by which t10,c12 CLA affects lipid accumulation in adipocytes. We have shown that t10,c12 CLA prevents lipid accumulation in human and mouse adipocytes at concentrations as low as 5 microM and 25 microM, respectively. t10,c12 CLA fails to activate peroxisome proliferator-activated receptor gamma (PPARgamma) but selectively inhibits thiazolidinedione-induced PPARgamma activation in 3T3-L1 adipocytes. Treatment of mature adipocytes with t10,c12 CLA alone or in combination with Darglitazone down-regulates the mRNA expression of PPARgamma as well as its target genes, fatty acid binding protein (aP2) and liver X receptor alpha (LXRalpha). Taken together, our results suggest that the trans10, cis12 CLA isomer prevents lipid accumulation in adipocytes by acting as a PPARgamma modulator.     

Changes in the binding of "fast"-form alpha 2-macroglobulin to 3T3-L1 cells after differentiation to adipocytes

Human alpha 2-macroglobulin (alpha 2M)-CH3NH2 specifically binds to 3T3-L1 fibroblasts and adipocytes with an apparent Kd of 0.3 nM at 4 degrees C. Binding to fibroblasts follows first-order kinetics only for the first 20-30 min of reaction, k1 = 160 microM-1 h-1, and then proceeds in a non-first-order reaction that takes 28 h to reach steady state. Receptor activity is 120 fmol of alpha 2M-CH3NH2/mg of cell protein or 60 000 molecules/cell. Binding is nondissociable. In contrast, binding to adipocytes follows first-order kinetics, k1 = 720 microM-1 h-1, and reaches steady state in 6-8 h. Receptor activity is 35 fmol of alpha 2M-CH3NH2/mg of cell protein or 60 000 molecules/cell. Binding is reversible with a k2 of 0.4 h-1. Control studies with 3T3-C2 cells, which do not differentiate after hormone treatment, indicate that these differences are not due to hormone treatment alone. Binding to both fibroblasts and adipocytes is specific for "fast"-form alpha 2M but not for native alpha 2M. Inhibition studies with neoglycoproteins demonstrate that binding does not occur via any of the known carbohydrate receptors. Some cross-reactivity with antithrombin III-trypsin complexes is demonstrated. Both fibroblasts and adipocytes take up and degrade alpha 2M-CH3NH2 at 37 degrees C. For both cell types, the concentration of alpha 2M-CH3NH2 needed for half-maximal uptake is 65 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anaerobic degradation of naphthalene and 2-methylnaphthalene by strains of marine sulfate-reducing bacteria

The anaerobic biodegradation of naphthalene, an aromatic hydrocarbon in tar and petroleum, has been repeatedly observed in environments but scarcely in pure cultures. To further explore the relationships and physiology of anaerobic naphthalene-degrading microorganisms, sulfate-reducing bacteria (SRB) were enriched from a Mediterranean sediment with added naphthalene. Two strains (NaphS3, NaphS6) with oval cells were isolated which showed naphthalene-dependent sulfate reduction. According to 16S rRNA gene sequences, both strains were Deltaproteobacteria and closely related to each other and to a previously described naphthalene-degrading sulfate-reducing strain (NaphS2) from a North Sea habitat. Other close relatives were SRB able to degrade alkylbenzenes, and phylotypes enriched anaerobically with benzene. If in adaptation experiments the three naphthalene-grown strains were exposed to 2-methylnaphthalene, this compound was utilized after a pronounced lag phase, indicating that naphthalene did not induce the capacity for 2-methylnaphthalene degradation. Comparative denaturing gel electrophoresis of cells grown with naphthalene or 2-methylnaphthalene revealed a striking protein band which was only present upon growth with the latter substrate. Peptide sequences from this band perfectly matched those of a protein predicted from genomic libraries of the strains. Sequence similarity (50% identity) of the predicted protein to the large subunit of the toluene-activating enzyme (benzylsuccinate synthase) from other anaerobic bacteria indicated that the detected protein is part of an analogous 2-methylnaphthalene-activating enzyme. The absence of this protein in naphthalene-grown cells together with the adaptation experiments as well as isotopic metabolite differentiation upon growth with a mixture of d(8)-naphthalene and unlabelled 2-methylnaphthalene suggest that the marine strains do not metabolize naphthalene by initial methylation via 2-methylnaphthalene, a previously suggested mechanism. The inability to utilize 1-naphthol or 2-naphthol also excludes these compounds as free intermediates. Results leave open the possibility of naphthalene carboxylation, another previously suggested activation mechanism.     

Accumulation of polychlorinated biphenyls in adipocytes: selective targeting to lipid droplets and role of caveolin-1

Background

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that preferentially accumulate in lipid-rich tissues of contaminated organisms. Although the adipose tissue constitutes a major intern reservoir of PCBs and recent epidemiological studies associate PCBs to the development of obesity and its related disorders, little is known about the mechanisms involved in their uptake by the adipose tissue and their intracellular localization in fat cells.

Methodology/Principal Findings

We have examined the intracellular distribution of PCBs in mouse cultured adipocytes and tested the potential involvement of caveolin-1, an abundant adipocyte membrane protein, in the uptake of these compounds by fat cells. We show that 2,4,4′-trichlorobiphenyl (PCB-28), 2,3′,4,4′,5-pentachlorobiphenyl (PCB-118) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB-153) congeners rapidly and extensively accumulate in 3T3-L1 or mouse embryonic fibroblast (MEF) derived cultured adipocytes. The dynamics of accumulation differed between the 3 congeners tested. By subcellular fractionation of primary adipocytes, we demonstrate that these pollutants were almost exclusively recovered within the lipid droplet fraction and practically not associated to cell membranes. The absence of caveolin-1 expression in primary adipocytes from cav-1 deficient mice did not modify lipid droplet selective targeting of PCBs. In cav-1 KO MEF differentiated adipocytes, PCB accumulation was decreased, which correlated with reduced cell triglyceride content. Conversely, adenoviral mediated cav-1 overexpressing in 3T3-L1 cells, which had no impact on total cell lipid content, did not change PCB accumulation.

Conclusion/Significance

Our data indicate that caveolin-1 per se is not required for selective PCB accumulation, but rather point out a primary dependence on adipocyte triglyceride content. If the crucial role of lipid droplets in energy homeostasis is considered, the almost exclusive accumulation of PCBs in these organelles warrants future attention as the impairment of their function could be linked to the worldwide obesity epidemic.     

Stinging Nettle (Urtica dioica L.) Attenuates FFA Induced Ceramide Accumulation in 3T3-L1 Adipocytes in an Adiponectin Dependent Manner

Objective

Excess dietary lipids result in the accumulation of lipid metabolites including ceramides that can attenuate insulin signaling. There is evidence that a botanical extract of Urtica dioica L. (stinging nettle) improves insulin action, yet the precise mechanism(s) are not known. Hence, we examined the effects of Urtica dioica L. (UT) on adipocytes.

Research Design

We investigated the effects of an ethanolic extract of UT on free fatty acid (palmitic acid) induced inhibition of insulin-stimulated Akt serine phosphorylation and modulation of ceramidase expression in 3T3-L1 adipocytes. Adipocytes were exposed to excess FFAs in the presence or absence of UT. Effects on adiponectin expression, ceramidase expression, ceramidase activity, ceramide accumulation and insulin signaling were determined.

Results

As expected, FFAs reduced adiponectin expression and increased the expression of ceramidase enzymes but not their activity. FFA also induced the accumulation of ceramides and reduced insulin-stimulated phosphorylation of Akt in adipocytes. The effects of FFA were partially reversed by UT. UT enhanced adiponectin expression and ceramidase activity in the presence of excess FFAs. UT abated ceramide accumulation and increased insulin sensitivity via enhanced Akt phosphorylation. A siRNA knockdown of adiponectin expression prevented UT from exerting positive effects on ceramidase activity but not Akt phosphorylation.

Conclusions

In adipocytes, the ability of UT to antagonize the negative effects of FFA by modulating ceramidase activity and ceramide accumulation is dependent on the presence of adiponectin. However, the ability of UT to enhance Akt phosphorylation is independent of adiponectin expression. These studies demonstrate direct effects of UT on adipocytes and suggest this botanical extract is metabolically beneficial.     

Metabolism of covalent receptor-insulin complexes by 3T3-L1 adipocytes. Synthesis and use of photosensitive insulin analogs to study insulin receptor metabolism in cell culture

To facilitate labeling cell surface insulin receptors and analyzing their metabolism by 3T3-L1 adipocytes, a characterization of both the interaction of photosensitive insulin analogs with 3T3-L1 adipocytes and the conditions for photocross-linking these derivatives to the insulin receptor are described. The synthesis and purification of two photoaffinity analogs of insulin are presented. Both B29-lysine- and A1-glycine-substituted N-(2-nitro-4-azidophenyl)glycyl insulin compete with 125I-insulin for binding to 3T3-L1 adipocytes, and the B29-derivative retains a biological activity similar to that for native insulin. An apparatus developed for these studies permits photolysis of cells in monolayer culture using the visible region of the lamp emission spectrum. Activation of the photoderivative by this apparatus occurs with a half-life of approximately 15 s and permits rapid photolabeling of a single species of receptor of 300,000 Da. The conditions for photolabeling permit a measurement of the turnover of covalent receptor-insulin complexes by 3T3-L1 adipocytes in monolayer culture. Degradation of this complex occurs as an apparent first order process with a half-life of 7 h. A comparison with previous studies (Reed, B. C., Ronnett, G. V., Clements, P. R., and Lane, M. D. (1981) J. Biol. Chem 256, 3917-3925; Ronnett, G. V., Knutson, V. P., and Lane, M. D. (1982) J. Biol. Chem. 257, 4285-4291) indicates that in a "down-regulated" state, 3T3-L1 adipocytes degrade covalent receptor-hormone complexes with kinetics similar to those for the degradation of dissociable receptor-hormone complexes.     

Assay and partial purification of epoxide hydrase from rat liver microsomes

Solubilized cytochrome P-450 monooxygenase and epoxide hydrase activities from rat liver microsomes have been separated by column chromatography. The highly active epoxide hydrase fraction is still contaminated with cytochrome P-450, which has very low monooxygenase activity. The highly purified cytochrome P-450 fraction possesses high monooxygenase activity and is essentially devoid of epoxide hydrase activity. Purification factors for the epoxide hydrase through four purification steps are similar with [³H]styrene oxide, [³H]naphthalene oxide, [³H]cyclohexene oxide, and benzene oxide as substrates. Failure of benzene oxide to inhibit hydration of styrene or naphthalene oxide in the most purified preparations in indicative of the presence of at least two hydrases. These purified cytochrome monooxygenase and hydrase preparations represent valuable tools for the study of the intermediacy of arene oxides in drug metabolism. Thus, with naphthalene, only naphthol is formed with the monooxygenase, while both naphthol and the dihydrodiol are formed in the presence of monooxygenase and hydrase. A convenient radiochemical synthesis of [³H]naphthalene 1,2-oxide and assays for the measurement of the hydration of [³H]naphthalene oxide and benzene oxide, based on differential extractions and high-pressure liquid chromatography, respectively, are described.     

Fluorescence quenching studies of nitrated polycyclic aromatic hydrocarbons

The analysis of nitrated polycyclic aromatic hydrocarbons (NPAHs) is of great importance because of the mutagenicity and possible carcinogenic activity of these compounds, which are distributed widely in the environment. Nitro‐substituents in aromatic compounds are known to quench fluorescence and NPAHs have no intrinsic fluorescence, but they can be determined using their quenching effects on other fluorophores. The quenching effects of several important NPAHs on 1,2,3,4‐ tetrahydro‐1‐naphthol,5,6,7,8‐tetrahydro‐1‐naphthol,4‐(2‐hydroxy‐4‐sulfo‐1‐naphthylazo)‐2‐naphthalene carboxylic acid and 7‐amino‐4‐methyl coumarin have been studied. The singlet emission of these fluorophores is efficiently quenched by all the NPAHs, the quenching following the Stern–Volmer relationship. Quenching constants and the limits of detection and linear ranges of the quenchers have been determined in each case: the limits of detection are ca 1 µm . Copyright © 2010 John Wiley & Son, Ltd.     

红树林厌氧环境对多环芳烃类有毒物的降解预测

红树林是连接陆地和海洋的重要生态系统,由于潮汐活动,氧化还原条件表现出明显的昼夜间的交替,这一生态体系中不但有大量的动植物种类,同时还有数量极高的不同种类的细菌,包括好氧和厌氧类型,厌养的硫酸(盐)还原菌已证实在降解多环芳烃有机物方面有其独特的生化优势,但从红树林中分离出的此类纯细菌还很少,在降解方面,已初步确定萘的厌氧降解途径异于好氧细菌,厌氧降解时的一系列代谢中间产物也有明显的专一性,羰基化反应是开始的一个重要步骤,而后的每步生化反应还有待进一步验证。从现有的结果可以看出,红树林中厌养的硫酸还原菌应在降解多环芳烃有机物中起到非常重要的作用。