Radioimmunoassay for benzo[a]pyrene.

A benzo[a]pyrene (BP)-bovine serum albumin conjugate was synthesized and used to immunize 2.5- to 3.0-kg New Zealand White rabbits. The resulting antisera to BP bound trace amounts of [3H] (55 pg; 6,000 counts/min). A radioimmunoassay (RIA) to BP was developed by the antiserum first being titered with the [3H]BP and then a standard curve being constructed from the addition of unlabeled BP (0.4-15.8 pmoles; 0.1-4.0 ng/assay tube). The RIA could reliably detect 0.4 pmoles (0.1 ng) BP. The specificity with respect to structurally related polycyclic aromatic hydrocarbons was examined by means of competitive binding. Here concentrations of 4.0-400 pmoles of the compound were used, and the resulting competitive curves were compared for relative cross-reactivity at 50% B/B0 (counts per minute labeled BP bound to antiserum in the presence of corresponding concentrations of unlabeled BP/counts per minute labeled BP bound to antiserum in the absence of unlabeled BP). The antiserum B4-3 was specific to BP with the closest cross-reacting substance being 3-hydroxybenzo[a]pyrene (20% relative cross-reactivity). BP added to pooled human serum was measurable by the RIA at 1 ng/ml. A BP RIA may have potential use for the quantification of the absorbed dose of this carcinogen in man.

Effect of CRF and related peptides on calcium signaling in human and rodent melanoma cells.

Corticotropin releasing factor (CRF) induces a rapid, within seconds, and dose-dependent increase in the intracellular Ca2+ in both human and hamster melanoma cells. This effect is inhibited by depletion of extracellular calcium using 3 mM EGTA and is attenuated by the CRF receptor antagonist, alpha-helical-CRF(9-41). Other peptides of the CRF superfamily, sauvagine and urocortin, also induce increases in cytoplasmic calcium concentration but at higher concentrations than CRF. We conclude that malignant melanocytes express CRF receptors, which are coupled to activation of plasma membrane calcium channels.

A synthetic human Agouti-related protein-(83-132)-NH2 fragment is a potent inhibitor of melanocortin receptor function.

Chemical synthesis of Agouti proteins - Agouti and Agouti-related proteins - is complicated by their large size and by multiple cysteine residues located in the carboxyl terminal regions. Three human Agouti-related protein (AGRP) fragments, two of which correspond to a proposed endoprotease cleavage site between amino acids 82 and 83, were synthesized and tested for anti-melanotropic activity using Xenopus laevis dermal melanophores. Amino-terminal fragments AGRP(25-51) and (54-82) were devoid of significant antagonist activity, whereas the amidated carboxyl-terminal AGRP fragment (83-132)-NH2 was potently active with an inhibitory equilibrium dissociation constant (Ki) of 0.7 nM. The ability to synthesize functionally active AGRP should help unravel its role in the central nervous system and its unusual properties with respect to interaction with the melanocortin family of G-protein coupled receptors.

Stimulation of cell-surface urokinase-type plasminogen activator activity and cell migration in vascular endothelial cells by a novel hexapeptide analogue of neurotensin.

To investigate if neurotensin (NT) could induce activation of urokinase-type plasminogen activator (uPA) in vascular endothelial cells, we utilized the acetyl-NT (8-13) analogue, TJN-950, in which the C-terminal leucine is reduced to leucinol. TJN-950 inhibited the binding of 125I-NT to membranes of newborn rat brains and of COS-7 cells transfected with rat NT receptor cDNA, but at 10(4) higher doses than NT (8-13). However, TJN-950 was as effective as NT in inducing the fibrinolytic activity in bovine vascular aortic and human umbilical vein endothelial cells, and enhanced the migration of vascular endothelial cells. Moreover, administration of TJN-950 induced neovascularization in the rat cornea in vivo. TJN-950 had no effect on expression of uPA, plasminogen activator inhibitor-1 or uPA receptor mRNA. The binding of 125I-TJN-950 to cell membranes was blocked by unlabeled uPA and TJN-950, but not the amino-terminal or 12-32 fragment of uPA. TJN-950 may enhance uPA activity in vascular endothelial cells by interacting with the uPA receptor, resulting in induction of angiogenesis.

Volatilization of mutagens from beef during cooking.

The process of cooking beef substances which are mutagenic in the Ames Salmonella/microsome bioassay [1,2]. In this study, the formation and disposition of basic mutagens produced by cooking beef at different temperatures were examined. Mutagenic activity increased exponentially with cooking temperature between 137 degrees C and 252 degrees C. However, the amount of mutagenic activity remaining in the meat was only 1--7% of that which was volatilized into the air. The ingested dose of mutagens may therefore be significantly influenced by factors which restrict the dissipation of mutagens from the container, as well as by cooking temperature. Inhalation of airborne mutagens from cooking, as an alternative route of exposure, should be investigated when considered in light of some epidemiological data showing an excess of lung and bladder cancer among cooks and kitchen workers.

Mutagenic activity of marihuana smoke condensates.

Smoke condensates prepared from marihuana cigaretts were mutagenic in strain TA98 of the Ames Salmonella/microsome test, a short-term bioassay which estimates the mutagenic and carcinogenic potential of some chemicals. The mutagens in marihuana smoke condensates required liver enzymes to be activated. The specific mutagenic activity of marihuana smoke condensates were similar to that of tobacco smoke condensates prepared from American cigarettes. Fractionation studies of the marihuana smoke condensates showed that basic components accounted for 76% of the recovered mutagenic activity.

Direct-acting mutagens in automobile exhaust.

Particulate matter in city air contains chemicals which are mutagenic in the Ames Salmonella typhimurium assay. In residential urban areas, the principal mutagens in air do not require liver enzymes to be activated. The source of these liver independent (direct-acting) mutagens may be automobile exhaust because (1) the mutagenic activities were correlated to the lead content or air, (2) the mutagens were found exhaust samples from automobiles and from an experimental CFR single-cylinder gasoline engine, and (3) these mutagens were not found in fuel or unused motor oil, but were found in used motor oil. The strain specificity and the fact that liver enzymes were not required for activation indicated that the exhaust and airborne mutagens were not unsubstituted polycyclic aromatic hydrocarbons (PAH), aromatic amines, alkylnitrosamines or aliphatic epoxides, peroxides and hydroepoxides. A number of nitro-substituted aromatic compounds are direct-acting mutagens in the Ames test, and it is possible that nitration of PAH in exhaust may form the compounds observed here. We synthesized 6-nitrobenzo [a] pyrene and found it to be a potent, direct-acting mutagen with activity comparable to that of benzo-[a] pyrene.

Potent mutagenic impurities in a commercial sample of 3-nitro-9-fluorenone.

A commercial sample of 3-nitro-9-fluorenone was a potent mutagen in the Ames Salmonella assay, producing 1000 TA98 net revertants per plate at 0.76 microgram/plate without the presence of liver homogenates (-S9). After purification by high-pressure liquid chromatography (HPLC), 3-nitro-9-fluorenone was found to be at least 6 times less active than the parent sample. The commercial sample was fractionated by HPLC and the mutagenic impurity peaks collected and subjected to high-resolution mass spectrometry (HRMS). The mass spectra of 2 potent mutagenic fractions showed the principal molecular species to be a dinitrofluorenone and an acetamidomononitrofluorenone. Samples of synthetic 2,7-dinitro-9-fluorenone and 2-acetamido-3-nitro-9-fluorenone had mutagenic activities, HPLC retention times, and mass spectra characteristics similar to the mutagenic impurity fractions collected from the commercial sample.

Antimutagenic properties of liver homogenates, proteins and glutathione on diesel exhaust particulates.

Diesel exhaust particulates contain mutagens which are active in the Ames Salmonella typhimurium assay. The mutagens do not require liver enzymes for activation and, in fact, the addition of liver homogenates (S-9) to the Ames system decreases the mutagenicity of diesel exhaust samples. We have examined here the properties and components of S-9 that account for its antimutagenic effect. The antimutagenic effect of S-9 is not changed by heating S-9 in a boiling water bath for 5 min or by omission of the NADPH-generating system in the cofactor mixture. These experiments showed that the antimutagenic effect on S-9 is not enzymatic. The antimutagenicity of S-9 disappeared after the protein component of S-9 was removed by filtration. Exogenous albumin added to the Ames system mimicked the antimutagenic effect. Analysis of the albumin content of liver cytosol showed that 90% of the antimutagenic effect could be accounted for by the amount of albumin present. Glutathione added to diesel exhausts reduced mutagenicity, but the qualities of glutathione present in S-9 are too small to account for the antimutagenic effect of S-9. We conclude that the antimutagenic effect of S-9 is non-enzymatic and is most likely the result of non-specific protein binding of mutagens to liver albumin. The antimutagenic effect of glutathione on diesel exhausts suggests that the mutagens present are electrophiles.

Inhibition of shaking movements in rats by central administration of cholinergic and adrenergic agents.

Shaking movements of the body, similar to that made by a dog when wet ('wet-dog shakes'), occur in rats in response to pharmacological stimuli and in response to stimuli associated with cold and skin irritation. In this study, shaking movements, elicited by a variety of stimuli, were inhibited by central administration of nanomolar doses of drugs that act as agonists on muscarinic, adrenergic, and opiate receptors. The brain regions that mediate the drug inhibition of shaking appear to be located in the medial preoptic area and in structures lining the aqueduct and fourth ventricle.

Inhibition of the shake response in rats by adenosine and 2-chloroadenosine.

Shaking movements, similar to those made by a dog when wet, were elicited in rats by immersion in ice-water, injections of icilin, a chemical that produces sensations of cold, and naloxone-precipitated morphine withdrawal. Adenosine and 2-chloroadenosine produced dose-dependent inhibition of shaking to ice-water and icilin. The 2-chloroadenosine effect was mediated centrally because the ICV dose required to produce inhibition was not effective when given IP. Caffeine antagonized the inhibitory effects of adenosine and 2-chloroadenosine. 2-Chloroadenosine suppressed morphine-abstinence shaking as well as the body weight loss that normally accompanies withdrawal.

CRF and related peptides as anti-inflammatory agonists.

The permeability of endothelial surfaces increases in response to injury. We have shown that vascular leakage in experimental models of tissue injury can be inhibited by CRF and by a novel class of peptides that we call mystixins. Binding sites for iodinated-Tyro-CRF have been revealed in mucous membranes, and immunoreactive CRF-like materials have been found in inflamed tissues. Perhaps the breakdown of cytoskeletal intermediate filaments after insult generates or exposes peptide domains similar to mystixins. Endogenous CRF-like or mystixin-like peptides, if activated or released locally in injured tissues, may function as agonists to counteract the immediate inflammatory response. If this is so, the peripheral actions of these peptides add a new dimension to the idea that CRF and related substances organize and regulate an organism's response to stress.

Potent inhibition of thermal edema in rat by des-Tyr-Dynorphin A.

In an earlier study, dynorphin A(1-13) [Dyn A(1-13)] was shown to inhibit heat-induced edema in the anesthetized rat's paw but the potency of this action was low, with effective doses in the range of 3-4 mg/kg i.v. In this study, Dyn A and related fragments were tested. Thermal edema was elicited in anesthetized male albino rats by immersion of the hindpaw in 58 degrees C water for 1 min. The median effective dose (ED50 and 95% confidence limits) in mg/kg i.v. for inhibition of edema were: Dyn A, Dyn A(2-17), and Dyn A(1-13), 1.7 (1.2-2.4), 0.15 (0.09-0.24), and 3.2 (1.9-5.5), respectively. The ED50 values of [D-Ala2]Dyn A, [D-Ala2]Dyn A(2-17), and [D-Ala2]Dyn A(2-17)-amide were found to be 0.92 (0.40-2.10), 1.25 (0.60-2.63), and 0.65 (0.36-1.16) mg/kg i.v., respectively. Dyn A(2-17), 0.5 mg/kg i.v., also inhibited pulmonary edema produced by i.v. injection of epinephrine. The anti-edema action of Dyn A(2-17) was not blocked by naloxone, an opioid receptor antagonist, or dependent on the hypotensive action of this peptide. It is postulated that the antiedema activity of Dyn A resides in the core fragment Dyn A(6-12). Two peptides, N-acetyl-Dyn A(6-12)-amide and N-acetyl-[D-Leu12]Dyn A(6-12)-amide, were synthesized and, when tested, were effective in reducing thermal edema with ED50 values of 1.4 (0.6-3.7) and 2.2 (1.2-4.1) mg/kg i.v., respectively.

Pro-Leu-Gly-NH2 and pareptide inhibit development of tolerance to haloperidol catalepsy in the mouse.

Single doses of MIF-1 (0.03-2.0 mg/kg, SC) and chronic pretreatments with MIF-1 (0.03-2.0 mg/kg, SC, BID, 3 1/2 days) or pareptide (0.25 mg/kg, SC, BID, 3 1/2 days) did not affect the acute cataleptic response to haloperidol in the mouse. Chronic pretreatment with haloperidol (8.0 mg/kg, IP, BID, 3 days) decreased the duration of catalepsy in mice given smaller challenge dose of haloperidol (2.0 or 3.0 mg/kg, IP) 15 hours after the last pretreatment injections. Administration of either MIF-1 or pareptide to mice also chronically pretreated with haloperidol antagonized the development of tolerance.

CRF-evoked bradycardia in urethane-anesthetized rats is blocked by naloxone.

CRF, injected IV at a dose of 6 nmol/kg, produced a fall in blood pressure and in heart rate in urethane-anesthetized rats. The CRF-bradycardia was not obtained in hypophysectomized animals, in animals pretreated with dexamethasone, or in animals pretreated with the narcotic antagonist, naloxone (1 mg/kg, IV). By contrast, the hypotensive effects of CRF were not affected by these procedures. Vagotomy or pretreatment with a low dose of N-methylnaloxone did not affect the CRF-bradycardia, indicating that the slowing of the heart was not due to parasympathetic stimulation or due to a peripherally mediated opioid chemoreflex. The results suggested that the CRF-bradycardia was mediated by the release of opioid peptides from the pituitary.

Corticotropin-releasing factor inhibition of substance P-induced vascular leakage in rats: possible sites of action.

Substance P (SP), 40 micrograms/kg SC, induced protein leakage in the skin, muscle, trachea and esophagus of the anesthetized rat as measured by Monastral blue B labeling of small blood vessels. CRF, 30 micrograms/kg SC, injected 30 min before SP, decreased the SP-induced dye leakage. To locate where CRF might act, autoradiographic studies of [125I]-CRF binding to esophageal segments were conducted and displaceable binding of [125I]-CRF to submucosal elements in the esophageal epithelium were revealed, suggesting that CRF acts on selective sites to reduce vascular leakage.

Pharmacological activities of the MIF-1 analogues Pro-Leu-Gly, Tyr-Pro-Leu-Gly and pareptide.

The pharmacological activities of the related free acid analogues of MIF-1, Pro-Leu-Gly (PLG) and Tyr-Pro-Leu-Gly (YPLG), were investigated because of the possibility that they may be formed during the digestion of milk and wheat proteins in vivo. The amino acid sequences -Tyr-Pro-Leu-Gly- and -Pro-Leu-Gly- are present in proteins from these foods. Chronic administration of either PLG (0.25 mg/kg, SC, BID) or the control substance, pareptide (0.25 mg/kg, SC, BID), antagonized the development of tolerance to the cataleptic effects of haloperidol in mice. The effect of YPLG (0.25 mg/kg, SC, BID) on the development of this tolerance was borderline and not statistically significant. Nanomolar concentrations of PLG, YPLG, and pareptide each increased the in vitro binding of 3H-apomorphine to rat striatal receptors. In this in vitro system, bell-shaped dose response curves were observed for each peptide. The effects of these peptides on tolerance development and apomorphine binding are similar to those previously reported for MIF-1 and demonstrate that amidation at the carboxyl terminus is not required for biological activity.

D-amino acid-substituted analogs of corticotropin-releasing hormone (CRH) and urocortin with selective agonist activity at CRH1 and CRH2beta receptors.

The activities of corticotropin-releasing hormone (CRH)-related peptides and several analogs were examined in cells transfected with either CRH1 or CRH2beta receptors, in suppression of heat-induced rat paw edema in pentobarbital-anesthetised animals and in stimulation of release of immunoreactive corticotropin (ir-ACTH) from rat anterior pituitary tissue in vitro. The peptides tested were human/rat (h/r)-CRH, r-urocortin, h-urocortin, white sucker fish or maggy sole urotensin I and some analogs of these peptides substituted with D-amino acids at residues 4 (urocortin), 5 (CRH and urotensin I) and 20 (CRH). In cells transfected with CRH1 receptors, these peptides were similar in potency in stimulation of cAMP accumulation. By contrast, at CRH2beta receptors peptides of the urocortin and urotensin series were more potent than h/r-CRH while [D-Glu20]-h/r-CRH was 6.5-fold less active than h/r-CRH. I.v. administration of h/r-CRH or related peptides 10 min prior to a thermal stimulus produced a significant dose-dependent inhibition of rat paw edema formation. Comparison of the ED50's showed that urocortins ([D-Ser4]-h-urocortin, h-urocortin, [D-Pro4]-r-urocortin, r-urocortin) were approximately 2 to 3 times more active than h/r-CRH, but [D-Glu20]-h/r-CRH was 18.5-fold less active. In the assay for ir-ACTH release, the activity of h/r-CRH and [D-Glu20]-h/r-CRH was similar but [D-Pro5]-h/r-CRH and [D-Pro4]-r-urocortin was less potent than the native peptide. These results provide further evidence that D-amino acid substitution at residue 20 reduces the potency of h/r-CRH at endogenous (anti-edema effect) and transfected (cAMP accumulation) CRH2beta receptors whilst activity at the CRH1 receptor is retained (ACTH-release and cAMP accumulation). On the other hand substitutions at residues 4 or 5 in r-urocortin or h/r-CRH respectively appear to decrease activity at CRH1 but not CRH2beta receptors The modified CRH and urocortin analogs described here may provide clues for the further design of receptor selective ligands.

Dynorphin A(6-12) analogs suppress thermal edema.

Dynorphin A (Dyn A) is a 17-residue opioid peptide derived from prodynorphin precursors found in mammalian tissues. Removal of Tyr1 from Dyn A produces a peptide that is more potent than Dyn A in attenuating the acute phase of the inflammatory response, as measured by inhibition of heat-induced edema in the anesthetized rat's paw (exposure to 58 degrees C water for 1 min). Dyn A(2-17), however, no longer interacts with opioid receptors. It was postulated that the non-opioid anti-inflammatory actions of Dyn A(2-17) may reside in Dyn A(6-12); that is, Arg-Arg-Ile-Arg-Pro-Lys-Leu. here we report on the activities of Dyn A(6-12) analogs modified by substitutions on the N terminus, by single N-methyl substitution and by single replacement of residues by alanine. The results indicated that the minimal sequence required for an anti-edema ED50 of <1.0 micromol/kg i.v. was anisoyl-Arg6-Arg7-Xaa8-Arg9-Pro10)-Xaa11-+ ++Xaa12-NH2. A prototype, p-anisoyl-[D-Leu12] Dyn A(6-12)-NH2, with an ED50 of 0.20 micromol/kg i.v. compared to an ED50 of 0.08 micromol/kg i.v. for Dyn A(2-17), was selected for further tests of biological activity. This analog, like Dyn A(2-17), lowered blood pressure in anesthetized rats. In a model of neurogenic inflammation, produced by antidromic stimulation of the vagus in the anesthetized rat, p-anisoyl-[D-Leu12] Dyn A(6-12)-NH2, 0.23 micromol/kg i.v., attenuated the negativity of tracheal tissue interstitial pressure (Pif), which normally develops after nerve stimulation. Modulation of interstitial pressure may be the mechanistic basis for the anti-edema properties of these Dyn A(6-12) analogs.