Distribution and induction of aflatoxin B1-9a-hydroxylase activity in rat liver parenchymal and non-parenchymal cells

 Chronic administration of aflatoxin B₁ (AFB₁) to rats gives rise to hepatocellular and cholangiocellular carcinomas without affecting Kupffer and endothelial cells. The enzymatic conversion of AFB₁ to AFB₁-8,9-epoxide is the critical step in the activation of the mycotoxin, while the conversion of AFB₁ to aflatoxin M₁ (AFM₁), catalyzed by the AFB₁-9a-hydroxylase, is considered to be a detoxication route for the toxin. In the present study the distribution and inducibility of AFB₁-9a-hydroxylase were analyzed in microsomes derived from freshly isolated liver parenchymal (PC) and nonparenchymal cells (i.e. Kupffer+endothelial cells, NPC). AFB₁-9a-hydroxylase activity was clearly measurable in NPC and similar to that of PC. In NPC the rate of formation of AFM₁ was higher (when incubating with 16 μM AFB₁) than or similar (with 128 μM AFB₁) to that of AFB₁-8,9-epoxide, while in PC it was significantly lower. Taken together, these results suggest that the AFB₁-9a-hydroxylase activity might be particularly important in NPC to protect these cells from AFB₁ by converting it to a significantly less mutagenic metabolite and by reducing the amount of AFB₁ available for epoxidation. Furthermore, it is shown that AFB₁-9a-hydroxylase activity is inducible by phenobarbital (only in PC), 3-methylcholanthrene, isosafrole and Aroclor 1254, thus indicating that in rat liver the conversion of AFB₁ to AFM₁ is catalyzed by members of the cytochrome 1A and 2B families. Received: 7 December 1995/Accepted: 13 February 1996     

The distribution of UDP-glucuronosyltransferases in rat liver parenchymal and nonparenchymal cells.

Activities for the glucuronidation of 1-naphthol, morphine and bilirubin as well as for the sulfation of 2-naphthol have been determined in homogenates of parenchymal, Kupffer and endothelial cells isolated from livers of untreated and Aroclor 1254-pretreated rats. In addition, Western blot analyses using different polyclonal antibodies against UDP-glucuronosyltransferases (UDP-GTs) were performed with similar preparations. All enzymes under investigation were expressed at high levels in liver parenchymal cells. The constitutive expression and inducibility of UDP-GT isozyme(s) for 1-naphthol glucuronidation was also clearly demonstrated in Kupffer and endothelial cells. Furthermore, the presence of other UDP-GT isozymes was detected in preparations from these cells. No significant sulfation of 2-naphthol was detectable in Kupffer and endothelial cell homogenates. While the glucuronidation of 1-naphthol and morphine was significantly induced in all cell types by Aroclor 1254-pretreatment of the animals, the glucuronidation of bilirubin and the sulfation of 2-naphthol remained unchanged. Since the specific activity of conjugation reactions is much lower in liver nonparenchymal cells than in liver parenchymal cells, and nonparenchymal cells contribute only about 6% to the total liver protein, protection of the cells themselves rather than contribution to the overall metabolism of xenobiotics seems to be the significant role of these xenobiotic-metabolizing enzymes in the sinusoidal lining cells.     

Xenobiotic metabolizing enzymes are not restricted to parenchymal cells in rat liver

To characterize the distribution and inducibility of drug metabolizing enzymes within different hepatic cell populations, the activities of aminopyrine N-demethylase, ethoxyresorufin O-deethylase, microsomal epoxide hydrolase and cytosolic glutathione transferase were measured in liver parenchymal, Kupffer, and endothelial cells isolated from untreated rats or rats pretreated with phenobarbital, 3-methylcholanthrene, or Aroclor 1254. Enzyme activities, measurable in all cases, were 2.3- to 5.7-fold higher in parenchymal cells than in Kupffer and endothelial cells. Phenobarbital increased aminopyrine N-demethylase, microsomal epoxide hydrolase, and cytosolic glutathione transferase activities, whereas 3-methylcholanthrene enhanced ethoxyresorufin O-deethylase, epoxide hydrolase, and glutathione transferase activities in the three cell populations. Aroclor 1254 consistently induced each of the enzyme activities in parenchymal, Kupffer, and endothelial cells. Western blot analyses revealed clear differences in the expression of proteins immunologically related to cytochrome P-450 PB-1, and glutathione transferases B and X in parenchymal cells compared with the corresponding Kupffer and endothelial cells. In contrast, only minor differences between the cell types were apparent in the expression of cytochromes P-450 PB-4, P-450 MC1a, P-450 MC1b and microsomal epoxide hydrolase. These studies establish that oxidative and postoxidative drug metabolizing enzymes are not restricted to parenchymal cells: similar but distinguishable complements of these enzymes are also found in Kupffer and endothelial cells.     

Inhibitory effect of Aroclor 1254 on aflatoxin-initiated carcinogenesis in rainbow trout and mutagenesis using a Salmonella/trout hepatic activation system

Duplicate lots of 140 rainbow trout (Salmo gairdneri) were fed either control diet (CD) or 100 ppm Aroclor 1254 (a polychlorinated biphenyl—PCB) for 3 mth followed by initiation of liver carcinomas with 20 ppb dietary aflatoxin B₁ (AFB₁) for 2 wk. At 8 and 12 mth after AFB₁ treatment, fish were sampled and tumor incidence determined. Trout that were prefed PCB showed a 45% inhibition in tumor incidence at 12 mth, when compared to those prefed CD. Throughout the experiment fish were sampled to determine the time relationship of PCB bioaccumulation. A rapid uptake of PCB into total body fat was seen, with concentration of 594 ppm at the time of AFB₁ exposure. Liver benzo[a]pyrehe monooxygenase (B[a]PM) activity was also induced at the time AFB₁ exposure began. The Ames mutagenesis assay was used to determine the effects of in vitro Aroclor 1254 on AFB₁-induced mutagenesis using a trout liver preparation. A PCB dose-related inhibition was observed with a 67% inhibition at the highest dose tested (500 μg Aroclor 1254/plate). Proposed mechanisms of the inhibition of AFB₁-induced carcinogenesis/mutagenesis are discussed.     

Aflatoxin B1Epoxide,the Ultimate Carcinogenic form of Aflatoxin B1: Synthesis and Reaction with Dna

Abstract

The ultimate carcinogenic form of aflatoxin B₁, the exo-8,9-epoxide, has long evaded both isolation and synthesis. The epoxide has now been prepared in essentially quantitative yield by oxidation of aflatoxin B₁ with dimethyldioxirane. The epoxide is sufficiently stable that it can be isolated and characterized but it undergoes rapid solvolysis in hydroxylic media. Highly efficient reaction occurs with DNA to give N⁷ adducts on guanine. The oligodeoxynucleotide duplexes d(ATCGAT)₂ and d(ATGCAT)₂ react with the epoxide to give the AFB₁ adduct at guanine N⁷. Stoichiometry of adduct formation is 1 per duplex in the first case and 2 per duplex in the second. NMR studies, including measurement of nuclear Overhauser effects by one and two dimensional techniques, reveal that the aflatoxin moiety is intercalated in the duplex on the 5’ side of the point of attachment. The difference in stoichiometry observed with the two duplexes reflects the fact that with d(ATCGAT)₂ intercalation of aflatoxin between the C:G and G:C base-pairs precludes entry of a second molecule into that site, whereas intercalation occurs between T:A and G:C base-pairs in the second case so that two sites of intercalation are available. Several lines of evidence indicate that non-covalent association of aflatoxin with DNA involves intercalation and lead to the hypothesis that the transition state for reaction of aflatoxin B₁ epoxide with DNA is also intercalative.     

Simplified Synthesis and Stability Assessment of Aflatoxin B1-Lysine and Aflatoxin G1-Lysine

Aflatoxins B₁ (AFB₁) and G₁ (AFG₁) are carcinogenic mycotoxins that contaminate crops such as maize and groundnuts worldwide. The broadly accepted method to assess chronic human aflatoxin exposure is by quantifying the amount of aflatoxin adducted to human serum albumin. This has been reported using ELISA, HPLC, or LC-MS/MS to measure the amount of AFB₁-lysine released after proteolysis of serum albumin. LC-MS/MS is the most accurate method but requires both isotopically labelled and unlabelled AFB₁-lysine standards, which are not commercially available. In this work, we report a simplified synthetic route to produce unlabelled, deuterated and ¹³C₆ ¹⁵N₂ labelled aflatoxin B₁-lysine and for the first-time aflatoxin G₁-lysine. Additionally, we report on the stability of these compounds during storage. This simplified synthetic approach will make the production of these important standards more feasible for laboratories performing aflatoxin exposure studies.     

Agonistic properties of alniditan,sumatriptan and dihydroergotamine on human 5-HT1B and 5-HT1D receptors expressed in various mammalian cell lines

1. Alniditan, a novel migraine abortive agent, is a potent 5-HT_1B/5-HT_1D receptor agonist of nM affinity. We compared the agonistic properties of alniditan, sumatriptan and dihydroergotamine on the cloned human 5-HT_1B receptor expressed at 200 fmol mg⁻¹ protein (B_max) in non-induced L929sA cells, at 740 fmol mg⁻¹ protein in HEK 293 and at 2300 fmol mg⁻¹ protein in mIFNβ-induced L929sA cells, and on the human cloned 5-HT_1D receptor expressed in C6 glioma cells (B_max 780 fmol mg⁻¹ protein).
2. Sodium butyrate treatment increased the expression level of human (h)5-HT_1B receptors in HEK 293 cells and h5-HT_1D receptors in C6 glioma cells approximately 3 fold, the binding affinities of [³H]-5-HT and [³H]-alniditan were unaffected.
3. Agonistic properties were evaluated based on inhibition of cyclic AMP accumulation in the cells after stimulation of adenylyl cyclase by forskolin or isoproterenol. Alniditan, sumatriptan and dihydroergotamine were full agonists at the h5-HT_1B receptor (IC₅₀ values were 1.7, 20 and 2 nM, respectively in HEK 293 cells) and h5-HT_1D receptors (IC₅₀ values of 1.3, 2.6 and 2.2 nM, respectively). At the h5-HT_1B receptor the agonist potency of the compounds slightly increased with higher receptor density. The opposite was seen for antagonists (ocaperidone, risperidone and ritanserin).
4. This comparative study demonstrated that alniditan was 10 times more potent than sumatriptan at the h5-HT_1B receptor, and twice as potent at the h5-HT_1D receptor. Dihydroergotamine was more potent an agonist at the h5-HT_1B receptor when expressed at high and low level in L929sA cells (but not in HEK 293 cells), and was less potent at the h5-HT_1D receptor.

     

Synthesis and Pharmacokinetics of a New Liver-Specific Carrier,Glycosylated Carboxymethyl-Dextran,and Its Application to Drug Targeting

To develop a new carrier system for hepatic targeting, carboxymethyl-dextran (CMD) was modified with galactose and mannose residues (Gal-CMD, Man-CMD), and their disposition characteristics were studied in mice using ¹⁴C-labeled dextran. At a dose of 1 mg/kg, i.v.-injected Gal-CMD and Man-CMD rapidly accumulated in the liver parenchymal and nonparenchymal cells, respectively, because of their preferential uptake via carbohydrate receptors in these cells. Pharmacokinetic analysis revealed that their uptake rates were sufficiently large for selective drug targeting. Targeting of cytosine -D-arabinoside (araC) was studied using Gal-CMD as a specific carrier to the hepatocytes. From the conjugate of araC with Gal-CMD, araC was released with a half-life of 36 hr in phosphate buffer (pH 7.4) and 23 hr in plasma. An in vivo biodistribution study demonstrated a disposition profile of the conjugated araC similar to that of the carrier, and selective delivery to hepatocytes of up to 80% of the dose was achieved. These findings suggest that glycosylated CMDs are carriers with a high affinity to liver parenchymal or nonparenchymal cells without any affinity to other tissues.     

Cigarette smoke exposure up-regulates endothelin receptor B in human pulmonary artery endothelial cells: molecular and functional consequences

BACKGROUND AND PURPOSE

Pulmonary arteries from smokers and chronic obstructive pulmonary disease patients show abnormal endothelium-dependent vascular reactivity. We studied the effect of cigarette smoke extract (CSE) on endothelin receptor B (ET_B) expression in human pulmonary artery endothelial cells (HPAECs) and its role in endothelial dysfunction.

EXPERIMENTAL APPROACH

ET_B receptor expression was measured by real time RT-PCR, Western blot and immunofluorescence. Cell contraction, intracellular Ca²⁺, F/G-actin, RhoA activity, myosin light chain phosphorylation, ET, NO, thromboxane (Tx)A₂ and reactive oxygen species (ROS) were measured by traction microscopy, fluorescence microscopy, phalloidin fluorescence, colorimetric assay, Western blot, elisa and DCFDA fluorescence respectively.

KEY RESULTS

Cigarette smoke extract dose-dependently increased ET_B receptor expression in HPAECs after 24 h incubation. CSE-induced ET_B expression was attenuated by bosentan, the ET_B receptor antagonist BQ788, the Rho kinase antagonist Y27632 and the antioxidant N-acetylcysteine. A monoclonal antibody to ET-1 prevented CSE-induced ET_B receptor overexpression. Twenty-four hour exposure to ET-1 dose-dependently increased ET_B receptor expression, mimicking the effect of CSE. CSE-induced ET_B receptor overexpression caused greater cell contraction; increased intracellular Ca²⁺; increased F/G-actin and RhoA activity; increased myosin light chain phosphorylation; augmented TxA₂ and ROS production; and decreased NO after acute ET-1 (10 nM). These effects were attenuated by bosentan, BQ788, Y27632 and N-acetylcysteine.

CONCLUSIONS AND IMPLICATION

Cigarette smoke extract induced ET_B receptor overexpression by a feed forward mechanism mediated partly by ET release, promoting HPAEC dysfunction and attenuated by ET_B receptor blockade, Rho kinase and ROS inhibition. These results provide support for the use of bosentan in CS-related endothelial dysfunction.     

Butylated hydroxytoluene chemoprevention of aflatoxicosis – Effects on aflatoxin B1 bioavailability, hepatic DNA adduct formation, and biliary excretion

The extreme sensitivity of turkeys to aflatoxin B₁ (AFB₁) is associated with efficient hepatic cytochrome P-450 (P450)-mediated bioactivation, and deficient glutathione S-transferase (GST) mediated detoxification. Butylated hydroxytoluene (BHT) protects against AFB₁ toxicity in turkeys through mechanisms that include competitive inhibition of P450-mediated AFB₁ bioactivation. To test whether dietary BHT alters hepatic AFB₁–DNA adduct formation, excretion, and bioavailability of AFB₁ in vivo, turkeys were given diets with BHT (4000 ppm) for 10 days, given a single oral dose of [³H]-AFB₁ (0.05 μg/g; 0.02 μCi/g), then sampled at intervals up to 24 h. Radiolabel in serum, red blood cells, liver, and breast meat was frequently lower in BHT-treated compared to control. Hepatic AFB₁–DNA adducts in BHT-treated turkeys were significantly lower at 12 and 24 h. BHT-fed birds had significant higher bile efflux, though biliary radiolabel excretion was not different from control. The amount of aflatoxin M₁ (AFM₁) excreted in the bile was lower than in control, but BHT had no effect on the biliary excretion of AFB₁, aflatoxin Q₁ or glucuronide and sulfate conjugates. Thus, the chemopreventive properties of BHT may also occur through a reduction in AFB₁ bioavailability in addition to inhibition of bioactivation.     

Exposure to Aroclor‐1254 impairs spindle assembly during mouse oocyte maturation

Polychlorinated biphenyls (PCBs), as typical environmental estrogen disruptors, are a structurally‐related group of halogenated aromatic hydrocarbons that are composed of 209 isomers and present as a mixture in the environment. PCBs congener with different numbers and positions of chlorine atoms substituted on the biphenyl moiety. Aroclor‐1254 is a mixture of more than 60 PCB congeners. Previous studies have provided the evidence that PCBs have severe negative effects on reproductive functions, but the effects of PCBs on spindle assembly during mouse oocyte maturation in vitro have not been reported. In the present study, female ICR mouse immature oocytes were cultured in M2 medium with 1 and 10 μg mL^?1 Aroclor‐1254 separately in vitro. The percentage of germinal vesicle breakdown (GVBD) and the first polar body extrusion were recorded. The results showed no significant difference in the percentage of GVBD or the first polar body extrusion between control oocytes and Aroclor‐1254‐treated oocytes. Further studies showed that the normal localization of γ‐tubulin and Aurora‐A kinase was interfered and α‐tubulin assembling into spindle was affected when mouse oocytes were exposed to Aroclor‐1254. The length of spindle from 10 μg mL^?1 Aroclor‐1254‐treated oocytes was longer than that from control oocytes, and the spindle area in the Aroclor‐1254‐treated groups were decreased. Furthermore, the percentage of DNA damage in cumulus cells revealed an increase after exposed to Aroclor‐1254. These results will provide the important reference for the prevention of reproductive disorders caused by PCBs. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1652–1662, 2016.     

Endothelium-derived bradykinin is responsible for the increase in calcium produced by angiotensin-converting enzyme inhibitors in human endothelial cells

Summary The effects of angiotensin-converting enzyme (ACE) inhibitors on intracellular calcium concentration ([Ca²⁺]_i) were examined under resting conditions and after stimulation with bradykinin in cultured human umbilical vein endothelial cells. The ACE inhibitors ramiprilat and enalaprilat (0.3 M) enhanced the increase in [Ca²⁺]_i elicited by bradykinin (3 nM) and also caused an increase in resting [Ca²⁺]_i when given alone. This increase in resting [Ca²⁺]_i was long-lasting and accompanied by an increased formation of nitric oxide, as assessed by a N^G-nitro-l-arginine-sensitive cyclic GMP accumulation in the cells. Both increases in resting [Ca²⁺]_i and nitric oxide production by ACE inhibitors were inhibited by preincubation of the cells with the B₂-receptor antagonist Hoe 140. These data indicate that ACE inhibitors are able to unmask a release of bradykinin from cultured human endothelial cells. This endothelium-derived bradykinin can exert an autocrine function by stimulating endothelial B₂-receptors with a subsequent increase in [Ca²⁺]_i and nitric oxide formation. Send offprint requests to R. Busse at the above address     

Protective role of Lycopene against Aroclor 1254-induced changes on GLUT4 in the skeletal muscles of adult male rat

Aroclor 1254 is the commercial mixture of highly toxic environmental pollutant, polychlorinated biphenyls (PCBs). Being immensely durable, it is extensively used and widely distributed. Studies show that Aroclor 1254 causes a variety of adverse health effects through free radical generation. The present investigation was designed to check the effect of Aroclor 1254 on the glucose transporter protein, GLUT4, which plays a key role in glucose homeostasis. The protective role of lycopene against the adverse effect of Aroclor 1254 was also tested. Group 1 rats received corn oil as vehicle and served as control. Groups 2, 3, and 4 were administered with Aroclor 1254 [2?mg kg^?1 body weight (b.w.) day^?1] intraperitoneally for 30 days. Groups 3 and 4 received lycopene (2 and 4?mg kg^?1 b.w. day^?1, respectively) orally in addition to Aroclor 1254. After 30 days, animals were euthanized and the skeletal muscles were dissected to determine the following parameters: GLUT4 messenger RNA (mRNA), GLUT4 protein (both plasma membrane and cytosolic fractions), and ¹⁴C-2-deoxyglucose uptake. Though there was no change in GLUT4 mRNA and fasting plasma glucose levels, Aroclor 1254 significantly decreased the GLUT4 protein level in both the subcellular fractions of the gracilis and triceps muscles. Most important, ¹⁴C-2-deoxyglucose uptake showed a significant decrease in Aroclor 1254 alone treated rats, and Aroclor 1254 plus 4?mg lycopene supplementation treatment maintained the same at par with control. Thus, Aroclor 1254 has adverse effects on GLUT4 translocation and ¹⁴C-2-deoxyglucose uptake, and lycopene administered along with Aroclor 1254 has a protective role over it.     

The furofuran-ring selectivity,hydrogen peroxide-production and low Km value are the three elements for highly effective detoxification of aflatoxin oxidase

AFO (aflatoxin oxidase), an enzyme from Armillariella tabescens previously named aflatoxin detoxifizyme, exhibits oxidative detoxification activity toward aflatoxin B₁ and sterigmatocystin. Bioinformatics reveals that AFO is a newly discovered oxidase because AFO does not share any significant similarities with any known oxidase. It is critically important to understand how AFO acts on aflatoxin B₁. In this study, in addition to aflatoxin B₁ (AFB₁) and sterigmatocystin (ST), five other chemicals that have furan or pyran structures were investigated. The results indicated that in addition to AFB₁ and ST, AFO is also able to act on versicolorin A, 3,4-dihydro-2H-pyran and furan. These results suggested that 8,9-unsaturated carboncarbon bond of aflatoxin B₁ is the potential reactive site for AFO. Further findings indicated that the action of AFO is oxygen-dependent and hydrogen peroxide-producing. The simultaneously produced-hydrogen peroxide possibly plays the essential role in detoxification of AFO. In addition, the extremely low K_m value of 0.33 µmol/l for AFO-AFB₁ and 0.11 µmol/l for AFO-ST signifies that AFO is highly selective for AFB₁ as well as ST.     

Screening a Strain of Aspergillus niger and Optimization of Fermentation Conditions for Degradation of Aflatoxin B1

Aflatoxin B₁, a type of highly toxic mycotoxin produced by some species belonging to the Aspergillus genus, such as Aspergillus flavus and Aspergillus parasiticus, is widely distributed in feed matrices. Here, coumarin was used as the sole carbon source to screen microorganism strains that were isolated from types of feed ingredients. Only one isolate (ND-1) was able to degrade aflatoxin B₁ after screening. ND-1 isolate, identified as a strain of Aspergillus niger using phylogenetic analysis on the basis of 18S rDNA, could remove 26.3% of aflatoxin B₁ after 48 h of fermentation in nutrient broth (NB). Optimization of fermentation conditions for aflatoxin B₁ degradation by selected Aspergillus niger was also performed. These results showed that 58.2% of aflatoxin B₁ was degraded after 24 h of culture under the optimal fermentation conditions. The aflatoxin B₁ degradation activity of Aspergillus niger supernatant was significantly stronger than cells and cell extracts. Furthermore, effects of temperature, heat treatment, pH, and metal ions on aflatoxin B₁ degradation by the supernatant were examined. Results indicated that aflatoxin B₁ degradation of Aspergillus niger is enzymatic and this process occurs in the extracellular environment.     

Effects of taprostene,a stable prostacyclin analogue,on haemodynamics,platelet function and arachidonate metabolism in healthy volunteers

Summary In order to assess the effect of taprostene on haemodynamics, platelet function and arachidonate metabolism in 4 healthy volunteers an intravenous infusion of 25 ng · kg^–1 · min^–1 was given for 6 h.During the infusion period systolic blood pressure dropped from 130 to 111 mm Hg and diastolic blood pressure from 77 to 69 mm Hg. The heart rate rose from 77 to 84 beats/min.During the taprostene infusion the slope and height of the ADP and collagen induced platelet aggregation curves were significantly inhibited and the sensitivity of platelets to PGI₂ and PGE₁ was increased.Plasma and serum thromboxane B₂, conversion of exogenous radiolabelled arachidonic acid, WU-test, circulating endothelial cell count, concentration of platelet factor 4, -thromboglobulin, malondialdehyde and the PGI₂-synthesis stimulating plasma factor did not show any clear drug-related alteration.It is concluded that infusion of taprostene 25 ng · kg^–1 · min^–1 caused measurable inhibition of platelet function ex vivo.     

In vitro cytotoxicity of polychlorinated biphenyls (aroclors 1016, 1242, 1254 and 1260) and their effect on phospholipid and neutral lipid composition of Chinese hamster ovary (CHO-K1) cells

Cytotoxicity of 4 Aroclors (1016, 1242, 1254 and 1260) was compared in Chinese hamster ovary (CHO-K1) cells in Ham's F-12 medium. When parameters of toxicity were cell numbers or tissue protein, 50% lethality occurred at Aroclor concentrations between 30 and 45 ppm. An in vitro clonal assay with CHO-K1 cells was a sensitive indicator of cytotoxicity of the polychlorinated biphenyls (PCBs). From EC₅₀ values (concentration that allowed 50% survival of formed colonies), cytotoxicity was lower with Aroclor 1016 (32 ppm) and higher with Aroclors 1254 (27 ppm) and 1260 (28 ppm). In cells exposed 24 h to a marginally cytotoxic dose (20 ppm) of each Aroclor, phospholipid (PL) thin-layer chromatography (TLC) showed an increase in phosphatidylcholine (PC) and a decrease in phosphatidylethanolamine (PE) and diphosphatidylglycerol (DPG). Neutral lipid (NL) TLC of cells given Aroclors 1242, 1254 or 1260 showed a 3–4-fold increase in triglyceride (TG) and a similar reduction in cholesteryl esters (CE); in contrast to Aroclor 1016 which produced no change in TG and a smaller (2-fold) reduction in CE. Cholesterol and free fatty acid fractions were unaffected by any of the Aroclors. The TG:PL ratio remained unchanged in cells given Aroclor 1016, but increased 3–4-fold with Aroclors 1242, 1254, or 1260. Compared to total values in the untreated controls, CHO-K1 cells contained less neutral lipid and more phospholipid only with Aroclor 1016.These results support the concept that differences in the behavior of Aroclor 1016 are related to its PCB composition. Changes in membrane PL and NL components, observed at marginally cytotoxic levels of each Aroclor, provided further evidence that the PCBs may affect membrane integrity and associated metabolic functions.     

Protein kinase C-mediated inhibition of transmembrane signalling through CCKA and CCKB receptors

1. The rat CCK_A and CCK_B receptors were stably expressed in Chinese hamster ovary (CHO-09) cells in order to compare modes of signal transduction and effects of protein kinase C (PKC) thereupon.
2. Spectrofluorophotometry of Fura-2-loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cells. Sulphated cholecystokinin-(26-33)-peptide amide (CCK-8-S) increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in CCK_A cells, measured as an increase in Fura-2 fluorescence emission ratio, 1000 fold more potently than its non-sulphated form (CCK-8-NS) (EC₅₀ values of 0.19 nM and 0.18 μM, respectively). By contrast, CCK-8-S and CCK-8-NS were equally potent in CCK_B cells (EC₅₀ values of 0.86 nM and 1.18 nM, respectively). The CCK_A receptor agonist JMV-180 increased [Ca²⁺]_i only in CCK_A cells. Likewise, pentagastrin increased [Ca²⁺]_i only in CCK_B cells. Finally, CCK-8-S-induced Ca²⁺ signalling through the CCK_A receptor was most potently inhibited by the CCK_A receptor antagonist L364,718, whereas the CCK_B receptor antagonist L365,260 was more potent in CCK_B cells.
3. Receptor-mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3-isobutyl-1-methylxanthine. CCK-8-S and, to a lesser extent, CCK-8-NS, but not JMV-180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCK_A cells. By contrast, none of these agonists increased cyclicAMP in CCK_B cells.
4. Short-term (3 min) pretreatment with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) evoked a rightward shift of the dose-response curve for the Ca²⁺ mobilizing effect of CCK-8-S in both cell lines. In addition, short-term TPA pretreatment markedly reduced CCK-8-S-induced cyclicAMP accumulation in CCK_A cells. In both cases, the inhibitory effect of TPA was abolished by the PKC inhibitors, GF-109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4-α-phorbol 12-myristate 13-acetate.
5. During prolonged TPA treatment, the cells gradually recovered from phorbol ester inhibition and in the case of CCK-8-S-induced Ca²⁺ mobilization complete recovery was achieved after 24 h of TPA treatment. Western blot analysis revealed that this recovery was paralleled by down-regulation of PKC-α, suggesting the involvement of this PKC isotype in the inhibitory action of TPA.
6. This study demonstrates that following expression in CHO cells (i) both CCK_A and CCK_B receptors are coupled to Ca²⁺ mobilization, (ii) only CCK_A receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.

     

Induction of immunotoxicity in mice by polyhalogenated biphenyls

Acute administration of Aroclor-1254 (500 mg/ kg) or 3,4,5,3,4,5-hexabromobiphenyl (HBB) (2–6 mg/ kg) IP, profoundly inhibited the plaque forming response to subsequent challenge with sheep erythrocytes in Ah locus positive (C57B1/6N or B6C3F1N) mice. These studies showed: 1) the immunotoxicity results paralleled enzyme induction results insofar as HBB was approximately 100 times more potent than Aroclor 1254; (2) neither Aroclor nor HBB treatment caused significant induction in the Ah locus negative DBA/2N mice; 3) when B6C3F1 mice were challenged with sheep red blood cells (SRBC) 6 or 16 weeks post Aroclor 1254 treatment, substantial recovery of a PFC response was observed; 4) when these compounds were administered to older (76-week-old) (B6C3F1 mice, severe depression of a PFC response was observed. In contrast to its profound depression of a PFC response, Aroclor-1254 (up to 1250 mg/kg) caused slight increases in lymphocyte proliferation induced by either T or B cell mitogens. A single 500 mg/kg dose of Aroclor-1254 also suppressed the ability of recipient B6C3F1 animals to reject a challenge with either the syngenic fibrosarcoma (PYB6) or the gram negative pathogen (Listeria monocytogenes).     

Endothelial cells contain beta adrenoceptors

Summary The direct identification of beta adrenoceptors in endothelial cell cultures has not been possible until the advent of a new beta-adrenergic radioligand, [¹²⁵I]iodocyanopindolol ([¹²⁵I]ICYP). Using [¹²⁵I]ICYP, we report thes successful identification of a beta adrenoceptor in cultured bovine aortic endothelial cells. At 37°C, specific binding is saturable, stable and reversible. There is a single class of binding sites (21,500±2,900 sites/cell) with an equilibrium dissociation constant (K _d) of 109±26 pM. The rate constant of association, k ₁, is 1.22×10⁹ M^–1 min^–1 and of dissociation, k _–1, is 0.01 min^–1. Binding studies on monolayers of endothelial cells grown in microtiter plates yield similar data (K _d=53±9 pM, B _max=20,000±1,900 sites/cell). Stereoselectivity of binding for the (–)-isomer is demonstrable for both agonists and antagonists. A series of adrenergic agonists competes with [¹²⁵I]ICYP for binding with an order of potency suggesting beta₂ subselectivity; isoproterenol (0.73 M) > epinephrine (15 M) > norepinephrine (71 M). Furthermore, the beta₂ inhibitor butoxamine is more potent than the beta₁ inhibitor practolol (7.7 M vs 22 M respectively). The GTP analogue, Gpp(NH)p, reduces isoproterenol affinity to 1.9 M and increases the Hill coefficient from 0.62–0.90.