Quantification of delta-opioid receptors in human brain with N1'-([11C]methyl) naltrindole and positron emission tomography.

The regional binding of N1'-([11C]methyl)naltrindole (MeNTI), a selective delta-opioid antagonist, was studied in healthy human subjects with positron emission tomography (PET). After the bolus intravenous administration of high specific activity [11C]MeNTI, PET was performed over 90 minutes. Arterial plasma samples were obtained during the scanning period and assayed for the presence of radiolabeled metabolites. The data were analyzed with various kinetic (two- and three-compartment models, Patlak graphical analysis) and nonkinetic (apparent volume of distribution and activity at a late scanning time) approaches. This tracer showed irreversible binding characteristics during the scanning period used. The results of the analyses also were compared with the density and distribution of delta-opioid receptors in the human brain in vitro. Additionally, computer simulations were performed to assess the effects of changes in receptor binding and tracer transport changes on the perceived binding parameters obtained with the models. A constrained three-compartment kinetic model was demonstrated to be superior to other quantification models for the description of MeNTI kinetics and quantification of delta receptor binding in the human brain with 11C-labeled MeNTI.

Tracers for imaging melanin with positron emission tomography.

The melanin binding properties of six radioligands were determined in vivo in the eyes of pigmented mice. Binding in the eyes of nonpigmented mice was used to assess nonmelanin binding characteristics. Of these radioligands, 3H-N-methylspiperone showed the best uptake and gave the best signal-to-noise ratio at all time points examined. Its binding appeared essentially irreversible. A PET study with 11C-N-methylspiperone was therefore carried out in a patient with a small ocular melanoma. Increased uptake of 11C-N-methylspiperone was observed in the melanoma. Our studies indicate that PET and radiolabeled NMSP might be used for imaging melanin and for the detection of pigmented melanoma. These results suggest that with a high resolution PET camera it may be feasible to image the melanin-containing cells (dopaminergic neurons) of the substantia nigra in the central nervous system, which could be of interest for the study of Parkinson's disease.

9-Sulfooxymethylanthracene is an ultimate electrophilic and carcinogenic form of 9-hydroxymethylanthracene.

The role of electrophilic hydroxymethyl sulfate esters in the metabolic activation, DNA-damage, mutagenicity, and complete carcinogenicity of polycyclic aromatic hydrocarbons has been elucidated considerably in recent years. The observations are in agreement with a unified hypothesis which predicts that electrophilic hydroxymethyl sulfate esters and closely related aralkylating agents are major ultimate carcinogenic forms of most, if not all, carcinogenic alkyl-substituted and even unsubstituted carcinogenic polycyclic aromatic hydrocarbons. The common final step in a chain of enzymatic substitution reactions is the formation of an aralkylating agent bearing a good leaving group. Activation of hydroxymethyl derivatives, including 9-hydroxymethylanthracene, to electrophilic mutagens has been shown to be catalyzed by 3'-phosphoadenosine-5'-phosphosulfate-dependent sulfotransferase activity. Recent studies, in a complete carcinogenic model, demonstrate that a number of sulfuric acid ester derivatives are more potent than their hydroxymethyl precursors by repeated subcutaneous injection in female Sprague-Dawley rats. In this paper, these observations have been extended to include 9-sulfooxymethylanthracene as an ultimate electrophilic and carcinogenic form of 9-hydroxymethylanthracene.

Autoradiographic and SPECT imaging of cerebral opioid receptors with an iodine-123 labeled analogue of diprenorphine.

The feasibility of imaging cerebral opioid receptors by single photon emission computed tomography (SPECT) has been established in baboon using a novel analog of diprenorphine (DPN) radiolabeled with iodine-123. The radioligand, [123I]-O-IA-DPN (C6-O-[123I]iodoallyl-DPN), was prepared in good yield (80%) with high radiochemical purity (>97%) and high specific radioactivity (>2,400 mCi/micromol). In ex vivo autoradiographic studies, with and without naltrexone blockade, [123I]-O-IA-DPN specifically labeled opioid receptors throughout the mouse brain. Nonmetabolized radioligand accounted for >90% of the signal observed in extracts of whole mouse brain. SPECT imaging trials showed that [123I]-O-IA-DPN selectively localized in regions of baboon brain known to have high densities of opioid receptors, such as striatum, thalamus, and temporal cortex. A much lower level of radioligand uptake and retention was noted for cerebellum, a region with few opioid binding sites. Pretreatment with naltrexone (6.5 pmol/kg) blocked [123I]-O-IA-DPN binding in all brain regions. Using naltrexone blockade to define the nonspecific component for a given region of interest, total to nonspecific binding ratios increased linearly (r > or = 0.98) over the SPECT study with maximal values for striatum (9.8), thalamus (7.1), and temporal cortex (6.9) reached at the last time point investigated (3.5 h). Specific binding for these regions, assessed as the difference between regional SPECT activity for the control and blocked states, proved irreversible over the observation period. By the end of the time course, specific [123I]-O-IA-DPN binding was >85% of total radioactivity in regions rich in opioid receptors and 62% of total radioactivity in cerebellum. The aggregate data are consistent with visualization of multiple opioid receptor types. Thus, [123I]-O-IA-DPN should prove useful for SPECT studies within the constraints imposed by a lack of innate selectivity for a single type of brain opioid receptor.

Carcinogenicity of 1-hydroxy-3-methylcholanthrene and its electrophilic sulfate ester 1-sulfooxy-3-methylcholanthrene in Sprague-Dawley rats.

Previous experiments have demonstrated that the carcinogen 1-hydroxy-3-methylcholanthrene is a metabolite of 3-methylcholanthrene. 1-Sulfooxy-3-methylcholanthrene, prepared by chemical synthesis from 1-hydroxy-3-methylcholanthrene, was shown to be a direct acting electrophilic mutagen and DNA damaging agent. These results imply that 1-hydroxy-3-methylcholanthrene could be metabolically activated to an ultimate electrophilic and carcinogenic form of 1-hydroxy-3-methylcholanthrene and 3-methylcholanthrene in a reaction catalyzed by 3'-phosphoadenosine-5'-phosphosulfate-dependent sulfotransferase activity. 1-Hydroxy-3-methylcholanthrene and its aralkylating reactive ester, 1-sulfooxy-3-methylcholanthrene, were individually administered to groups of 12 female Sprague-Dawley rats at a 0.2 mumol dose, three times weekly, for 20 doses. 1-Sulfooxy-3-methylcholanthrene induced sarcomas at the site of injection in 8 of 12 rats (66%) by 52 weeks, whereas 1-hydroxy-3-methylcholanthrene induced sarcomas at the site of injection in 5 of 12 rats (42%) by 52 weeks. These results, taken together with the results of previous experiments, strongly support the hypothesis that the activated electrophilic mutagen 1-sulfooxy-3-methylcholanthrene plays a major role as an ultimate electrophilic and carcinogenic form of 1-hydroxy-3-methylcholanthrene, a major metabolite of 3-methylcholanthrene.

Tissue distribution and metabolism of the [32P]-labeled oligodeoxynucleoside methylphosphonate-neoglycopeptide conjugate, [YEE(ah-GalNAc)3]-SMCC-AET-pUmpT7, in the mouse.

Development of oligodeoxynucleotides (oligo-dNs) and their analogs as therapeutic agents is complicated by their low rate of transport across cellular membranes, which is required for interaction with the intracellular complementary nucleic acid sequences, and the lack of tissue-specific delivery. To overcome these obstacles, bioconjugates between cell surface receptor ligands and oligodeoxynucleoside methylphosphonates (oligo-MPs) have been constructed containing homogeneous, chemically defined covalent linkages. We have previously established that a model conjugate, [32P]-labeled [YEE(ah-GalNAc)3]-SMCC-AET-pUmpT7 (1), is delivered to Hep G2 cells in a ligand-specific manner, reaching a peak value of 26 pmol per 10(6) cells after 24 hours incubation at 37 degrees C (Hangeland et al., 1995). In this work, the in vivo behavior of this conjugate is explored. Administration of this conjugate to mice via tail vein injection demonstrates rapid uptake in liver to the extent of 69.9 +/- 9.9% of the injected dose after 15 minutes. Thereafter, the conjugate and its metabolites are rapidly cleared via the kidney and urine. Polyacrylamide gel electrophoresis analysis of extracts of Hep G2 cells and mouse liver reveal the conjugate 1 to be extensively metabolized. In contrast, the conjugate found in mouse urine is largely intact. These data show that this novel, biodegradable delivery vehicle represents a viable approach for the delivery of antisense oligo-MPs and other oligo-dN analogs to the liver for therapeutic and diagnostic applications.

6-sulfooxymethylbenzo[a]pyrene is an ultimate electrophilic and carcinogenic form of the intermediary metabolite 6-hydroxymethylbenzo[a]pyrene.

Previous experiments have demonstrated that the intermediary metabolite 6-hydroxymethylbenzo[a] pyrene (HMBP) can be activated to the electrophilic mutagen, 6-sulfooxymethylbenzo[a]pyrene (SMBP), by rat and mouse liver PAPS-dependent sulfotransferase activity or by chemical synthesis. This aralkylating metabolite and 6-hydroxymethylbenzo[a]pyrene were individually administered to groups of 12 female Sprague-Dawley rats, at a 0.2 micromol dose three times weekly for 20 doses. SMBP induced sarcomas at the site of injection in 12 of 12 rats by 33 weeks, whereas HMBP induced sarcomas at the site of injection in 12 of 12 rats by 31 weeks. These results, taken together with the results of previous studies, strongly support the hypothesis that the electrophilic mutagen SMBP accounts for most, if not all, of the complete carcinogenicity of the intermediary metabolite HMBP and probably at least some of the complete carcinogenicity of 6-methylbenzo[a]pyrene (MBP), 6-formylbenzo[a]pyrene (formylBP), and even benzo[a]pyrene (BP), all of which are metabolized to HMBP.

7-Sulfooxymethyl-12-methylbenz[a]anthracene is an exceptionally reactive electrophilic mutagen and ultimate carcinogen.

The hypothesis was tested that an ultimate carcinogen of 7-hydroxymethyl-12-methylbenz[a]anthracene (HMBA), a major metabolite of 7,12-dimethylbenz[a]anthracene (DMBA), is a benzylic carbonium ion generated from an exceptionally reactive aralkylating metabolite, such as an electrophilic sulfate ester. In conformity with this hypothesis, sarcomas were rapidly induced in rats following repeated subcutaneous injection of HMBA (67%) or its electrophilic sulfate ester, sodium 7-sulfooxymethyl-12-methylbenz[a]anthracene (SMBA) (100%). It would appear from the results summarized here that the search for a carcinogenic metabolite of DMBA has been successful. In addition, an aralkylating electrophilic mutagen and carcinogen has been prepared from HMBA, which is itself either an ultimate carcinogen or a direct precursor of an ultimate carcinogen, i.e., a benzylic carbonium ion.

7-Sulfooxymethylbenz[a]anthracene is an ultimate electrophilic and carcinogenic form of 7-hydroxymethylbenz[a]anthracene.

The hypothesis was tested that 7-sulfooxymethylbenz[a]anthracene (7-SBA) is an ultimate electrophilic and carcinogenic form of 7-hydroxymethylbenz[a]anthracene. In conformity with this hypothesis, 7-SBA was more carcinogenic than 7-HBA in inducing sarcomas at the site of repeated subcutaneous injection. These metabolites were individually administered to female Sprague-Dawley rats, beginning at 30 days of age, in 0.2 mumol doses given three times each week for 20 doses. One year after the first injection of 7-SBA, seven of thirteen female Sprague-Dawley rats had developed sarcomas. 7-HBA, on the other hand, had induced sarcomas at the site of injection in only two of tweleve rats. No tumors developed either in the control group given sesame oil:DMSO only or in the untreated control group. It would appear from the results summarized here that the search for an ultimate electrophilic and carcinogenic form of 7-HBA has been successful.

1-Sulfooxymethylpyrene is an electrophilic mutagen and ultimate carcinogen of 1-methyl- and 1-hydroxymethylpyrene.

1-Hydroxymethylpyrene and 1-sulfooxymethylpyrene were tested for complete carcinogenic activity by repeated s.c. injection in groups of 12 female Sprague-Dawley rats, respectively. A dose of 0.2 mumol of either 1-sulfooxymethylpyrene or 1-hydroxymethylpyrene was administered every other weekday for 20 doses (i.e., total dose 4 mumol) to each of 12 rats, beginning at 30 days of age. Once a week the rats were weighed then palpated for the appearance of tumors. Tumor-bearing rats were sacrificed 20-50 days after the appearance of first palpable tumor. By 52 weeks, 1-sulfooxymethylpyrene had induced sarcomas at the site of injection in 58% of the rats with an average induction time of 33 weeks. By contrast, 20, 0.2 mumol doses of 1-hydroxymethylpyrene failed to induce tumors at the site of injection in a group of 12 rats. Similarly, neither of the two control groups produced tumors. The present experiment, together with previous data, strongly supports the hypothesis that 1-sulfooxymethylpyrene is either itself an ultimate carcinogen or a direct precursor of an ultimate carcinogen, the highly reactive benzylic carbonium ion, which reacts with DNA to form aralkyl-DNA adducts in a chain of events leading to malignant growth.

Mechanism of aralkyl-DNA adduct formation from benzo[a]pyrene in vivo.

Three different pathways have been proposed for the metabolic activation of the ubiquitous polycyclic aromatic hydrocarbon, benzo[a]pyrene (BP). The most widely accepted activation mechanism is based on ring oxidation to diol epoxides; the other two relatively less studied pathways involve radical cation formation and benzylic electrophilic ester formation arising from a chain of substitution reactions. The present study was undertaken to test for the existence of the latter mechanism in vivo. Female Sprague-Dawley weanling rats were injected subcutaneously with 320 mumol of BP/kg body weight, and the formation of DNA adducts was examined. 32P-Postlabeling analysis of the subcutaneous tissue DNA under newly developed chromatography conditions exhibited two sets of adduct profiles: one resulting from alkyl substitution and the other from ring oxidation. One major and two minor aralkyl-DNA adducts were detected. The relative adduct labeling (adducts/10(10) nucleotides) remained constant at around 2 during the first 5 days of treatment and then increased to 6.4 +/- 2.6 at day 7. The corresponding total values of the known ring oxidation (e.g., diol epoxide) adducts were 15-50 times higher. When animals were injected with 6-methyl-BP, 6-(hydroxymethyl)-BP, and 6-(acetoxymethyl)-BP, the known or proposed intermediates in the benzylic ester pathway, each of these and the parent compound showed chromatographically identical profiles of aralkyl-DNA adducts. Cochromatography in multiple solvents of these in vivo adducts with standards prepared by reaction of 6-(bromomethyl)-BP with individual nucleotides showed that the predominant in vivo aralkyl-DNA adduct was derived from guanine while the second major adduct was from adenine.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo labeling of nicotinic cholinergic receptors in brain with [3H]cytisine.

[3H]Cytisine was evaluated as an in vivo ligand for the nicotinic cholinergic receptor (nAchR) in mouse brain. The tracer was injected intravenously, and radioactivity in brain regions was analyzed. Radioactivity peaked in the brain at 30 minutes. It was highest in the thalamus, intermediate in the superior colliculi, prefrontal cortex and hippocampus, and low in the cerebellum. Pretreatment with unlabeled cytisine inhibited binding in the thalamus, but not in the cerebellum. Binding was displaced by l-nicotine, but not by d-nicotine or dexetimide. The results suggest that cytisine, appropriately labeled with a positron emitting radionuclide, may be useful for study of nicotinic cholinergic receptors in humans by emission computed tomography.

Bioalkylation of benz[a]anthracene, 7-methylbenz[a]anthracene, and 12-methylbenz[a]anthracene in rat lung cytosol preparations.

Benz[a]anthracene (BA) and the monomethyl meso-anthracenic or L-region derivatives 7-methylbenz[a]anthracene (7-methylBA) and 12-methylbenz[a]anthracene (12-methylBA) underwent a bioalkylation substitution reaction in rat lung ctyosol preparations, fortified with S-adenosyl-L-methionine to form the more potent carcinogen 7,12-dimethylbenz[a]anthracene. The methyl groups of the highly reactive L-region methylated metabolites also underwent enzymatic hydroxylation in rat lung cytosol preparations to yield the corresponding hydroxymethyl derivatives, 7-hydroxymethylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene, and 7,12-dihydroxymethylbenz[a]anthracene. The biooxidation reaction took place enzymatically, and exclusively, or nearly so, at the reactive methyl groups attached to the meso positions or L-region of the hydrocarbon. Bioalkylation and biooxidation reactions did not occur when the hydrocarbons were incubated with a boiled cytosol preparation, indicating the need for enzymatic activation of the L-region methyl groups. Also, the bioalkylation reaction did not occur in the absence of S-adenosyl-L-methionine. Furthermore, the S-adenosyl-L-methionine-dependent reaction was inhibited by S-adenosyl-L-homocysteine, suggesting that the reaction is catalyzed by a cytosolic S-adenosyl-L-methionine-dependent methyltransferase.

Methyl-substitution of benzene and toluene in preparations of human bone marrow.

The metabolism of benzene and toluene was investigated in preparations of human bone marrow incubated with S-adenosyl-L-methionine. Benzene undergoes a methyl-substitution reaction to yield toluene as a metabolite. Furthermore, toluene undergoes methyl-substitution in preparations of human bone marrow incubated with S-adenosyl-L-methionine to yield o-xylene, m-xylene, and p-xylene. Metabolites were detected by gas chromatography and mass spectroscopy. No metabolism of either benzene or toluene was detected when a boiled bone marrow preparation was used in the incubation, demonstrating the enzymatic nature of the S-adenosyl-L-methionine dependent methylation of both benzene and toluene.

Metabolism of chrysene, 5-methylchrysene, 6-methylchrysene and 5,6-dimethylchrysene in rat liver cytosol, in vitro, and in rat subcutaneous tissue, in vivo.

The polynuclear aromatic hydrocarbon chrysene undergoes a bioalkylation substitution reaction in vitro, in rat liver cytosol preparations, and in vivo, in rat dorsal subcutaneous tissue to yield 6-methylchrysene as a metabolite. In addition, both 5-methyl- and 6-methylchrysene were found to undergo a dealkylation reaction in these tissues to yield chrysene as well as both a biooxidation reaction to yield the corresponding hydroxyalkyl substituted chrysene and a bioalkylation reaction to give a dimethyl substituted chrysene. 5-Methylchrysene enzymatically cyclized to the 4,5-methylenechrysene derivative, an analog of benzo[a]pyrene in these tissues. 5,6-Dimethylchrysene was metabolized to monomethyl chrysenes, chrysene, and the hydroxyalkyl substituted chrysenes. The results suggest that chemical or biochemical substitution of a methyl group at the center of highest biochemical reactivity may be a necessary step in the metabolic activation and carcinogenicity of these compounds and their methylene bridged metabolites.

Rules of molecular geometry for predicting carcinogenic activity of unsubstituted polynuclear aromatic hydrocarbons.

The rules of molecular geometry for predicting carcinogenic activity of polynuclear aromatic hydrocarbons (PAH) have been applied to a series of 50 unsubstituted PAH, and predicted carcinogenic activity is in good agreement with the results of testing for complete carcinogenic activity in mice and/or rats. The rules were developed from a knowledge of the center or centers of highest chemical or biochemical reactivity and are consistent with a unified hypothesis which states that the first step in the metabolic activation of unsubstituted PAH is the biochemical introduction of a methyl group. This bioalkylation reaction 1) takes place between certain PAH and S-adenosyl-L-methionine and is catalyzed by cytosolic methyltransferase, 2) offers a means of probing for centers of reactivity in PAH, 3) provides a biochemical link between unsubstituted preprocarcinogens of aromatic type ArX and alkyl-substituted procarcinogens of aromatic type ArCH2X (where X = H), and 4) makes it possible to include compounds of both aromatic types, in a consistent theory of aromatic hydrocarbon activation which incorporates alkyl substitution. The present study reveals that there are structural determinants of carcinogenicity.

Metabolism of the carcinogen 3-methylcholanthrene in human bone marrow preparations.

The metabolism of 3-methylcholanthrene was investigated by incubating the hydrocarbon with human bone marrow preparations in air for 60 min at 37 degrees C. The major metabolites were identified by HPLC and GC/MS as 1-hydroxy-3-methylcholanthrene, 1-keto-3-methylcholanthrene, and cholanthrene. The results demonstrate that the potent carcinogen, 3-methylcholanthrene, can undergo biochemical reactions in preparations of human bone marrow, giving rise to the formation of metabolites which are known to be carcinogenic in rats and mice.

The site of substitution of the methyl group in the bioalkylation of benzo[a]pyrene.

The major product of the S-adenosyl-L-methionine methyltransferase dependent substitution reaction (bioalkylation) of benzo[a]pyrene in preparations of rat liver cytosol was isolated by solvent extraction and high performance liquid chromatography and its structure determined by GC/MS and NMR. The results indicate that the methyl group is introduced at the center of highest chemical reactivity (or carbon atom at which it is easiest to localize pi electrons) in the anthracene nucleus, giving rise to 6-methylbenzo[a]pyrene. These data, unequivocally demonstrate the site of substitution of the methyl group in the bioalkylation of benzo[a]pyrene.