Glutamate uptake by the isolated toad brain.

The isolated toad brain accumulates L-glutamate against strong concentration gradients until a tissue-to-medium concentration ratio of about 3000 : 1 is attained. The accumulated glutamate does not equilibrate with most of the endogenous tissue glutamate but is converted rapidly to glutamine and released into the medium. This mechanism may be involved in the preservation of low extracellular levels of cerebral glutamate.

Kinetics of formation and persistence of ethylguanines in DNA of rats and hamsters treated with diethylnitrosamine.

The kinetics of formation and persistence of 7-ethylguanine (e7Gua) and O6-ethylguanine (O6eGua) were determined in rat liver and kidney DNA following i.p. injection with 12.5, 50, 100, or 200 mg DENA per kg body weight. The rate of ethylguanine formation in hepatic DNA was independent of carcinogen dose; however, the maximum level of DNA ethylation reached was linearly related to DENA dose. Persistence of O6eGua but not e7Gua in rat liver DNA appeared to be dose-dependent; the rate of decline in O6eGua concentration slowed as the dose of DENA increased. Ethylation of rat kidney DNA was quantifiable only following treatment with 200 mg DENA per kg body weight, and maximum concentrations of e7Gua and O6eGua were approximately ten times less than those in hepatic DNA of these animals. Nevertheless, elimination of e7Gua and O6eGua from DNA occurred at similar rates in these tissues. Whereas lung DNA from DENA-treated rats contained no detectable ethylguanines, both e7Gua and O6eGua were detected in lung DNA from treated hamsters. The half-life of e7Gua in hamster lung DNA was 28 h, while O6eGua persisted longer, exhibiting a half-life of 91 h. Only trace quantities of e7Gua and O6eGua were detected in hamster kidney DNA, precluding an accurate estimation of the kinetics of DNA alkylation in this tissue. The rate of formation of ethylguanines in hepatic DNA was faster in hamster than in rat, while maximum levels of e7Gua and O6eGua were similar in these two species. Persistence of both e7Gua and O6eGua was markedly different in hepatic DNA of rats and hamsters. e7Gua was eliminated at a faster rate in the hamster (half-life of 20 h), as compared to the rat (half-life of 35 h). Conversely, O6eGua persisted longer in hamster than in rat liver DNA; a half-life of 34 h was found for the hamster, compared to a half-life of 14 h for the rat. The half-lives of e7Gua and O6eGua in hepatic DNA of DENA-treated rats and hamsters were similar to those reported previously for m7Gua and O6mGua in these species, suggesting that the same enzymatic DNA repair systems act upon these structurally related DNA adducts. The formation and prolonged persistence of O6eGua in lung DNA of DENA-treated hamsters may be related to the sensitivity of this species to the induction of respiratory tract neoplasms following exposure to DENA.

In vivo kinetics of O6-methylguanine and 7-methylguanine formation and persistence in DNA of rats treated with symmetrical dimethylhydrazine.

The rates of appearance and removal of 7-methylguanine and O6-methylguanine in DNA from rat liver, kidney, and colon were determined at various intervals up to 120 hr after i.p. administration of 10.2, 40.7, 81.5, or 163 mg 1,2-dimethylhydrazine (SDMH) per kg body weight (one-sixteenth, one-fourth, one-half, or one 50% lethal dose) using high-pressure liquid chromatography and fluorescence spectrophotometry. In most cases, increasing doses of SDMH slowed the rate of methylation of DNA, especially of the liver; colon DNA was methylated at a faster rate than was liver DNA, and kidney DNA was methylated at the slowest rate following SDMH administration. Removal of O6-methylguanine was slow (half-life, 37 to 50 hr) when this base was present in liver DNA at concentrations above 400 mumol/mol guanine; as the concentration fell below 300 mumol O6-methylguanine per mol guanine, the removal rate more than doubled (half-life, 16 to 19 hr). Some evidence was obtained to suggest that in the first 12 hr after maximum DNA methylation following SDMH administration, a rapid time-dependent removal of 7-methylguanine from liver and kidney but not colon DNA occurred. In these instances, then, the rates of formation and removal of aberrant methylated bases did not follow first-order kinetics.

Methylated purines in human liver DNA after probable dimethylnitrosamine poisoning.

DNA, isolated from two samples of human liver obtained from a suspected dimethylnitrosamine poisoning, contained 1363 to 1373 micromol of 7-methylguanine per mol of guanine and 273 to 317 micromol of O6-methylguanine per mol of guanine. Liver and kidney DNA obtained from unrelated cases contained no detectable methylated purines. From the DNA methylation levels, it is estimated that the dimethylnitrosamine-poisoning victim had been exposed to a dose of 20 mg or more of dimethylnitrosamine per kg of body weight. The results indicate for the first time that humans, like rodents, appear to activate dimethylnitrosamine metabolically to a strong methylating agent, resulting in methylation of liver DNA at both the 7- and O6 positions of guanine.

Immunoperoxidase demonstration of human serum globulin binding to islet tissue.

Sera from juvenile diabetic and nondiabetic controls were tested, using the immunoperoxidase method, for the presence of islet-binding immunoglobulins. All diabetic sera tested contained an islet-binding protein and on the average the sera were more positive than age-matched controls. Normal adult sera are undifferentiated from juvenile diabetic sera. Most sera from children less than two years of age did not bind to islet tissue and sera from cystic fibrosis patients had a markedly diminished ability to bind to islet tissue. The binding protein appears to be an immunoglobulin which selectively reacts with elements of the islet beta cell.

Anticonvulsant O-alkyl sulfamates. 2,3:4,5-Bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate and related compounds.

Novel sugar sulfamate 1 (McN-4853, topiramate) has been found to exhibit potent anticonvulsant activity analogous to that of phenytoin. In the maximal electroshock seizure test, orally at 2 h in mice, 1 had an ED50 of 39 mg/kg. Orally, 1 had a duration of action in excess of 8 h. Other aspects of the pharmacology of 1, as well as neurochemistry and carbonic anhydrase inhibition, are discussed. The conformational behavior of 1 in solution and in the solid state is discussed. A series of analogues of 1 were synthesized and examined for anticonvulsant properties.

Forskolin carbamates: binding and activation studies with type I adenylyl cyclase.

Three series of analogs were regioselectively prepared from a protected forskolin precursor to afford 7-carbamoyl-7-desacetylforskolins (series 1), 6-carbamoyl-7-desacetylforskolins (series 2), and 6-carbamoylforskolins (series 3). The analogs were pharmacologically evaluated for binding (IC50) to and activation (EC50) of type I adenylyl cyclase in membranes from stably transfected Sf9 cell lines expressing a single adenylate cyclase subtype. The following ranges were determined for the IC50's and EC50's of each individual series: series 1, IC50 = 43-1600 nM, EC50 = 0.5-9.6 microM; series 2, IC50 = 65-680 nM, EC50 = 0.63-6.5 microM; series 3, IC50 = 21-271 nM, EC50 = 0.5-8.1 microM (forskolin IC50 = 41 nM and EC50 = 0.5 microM). Activation paralleled binding; however, some analogs exhibited poor binding and good activation whereas others demonstrated good binding but poor activation. Steric bulk tended to diminish binding and activation when at the 6- or 7-position, although bulk was accommodated at the 6-position if the 7-site was reacetylated. Acylation of the 7-position by the carbamoyl linker or acetyl was important for obtaining good binding and activation; however, the effect was more pronounced with binding. For both binding and activation, small, linear, lipophilic substituents (propyl, allyl, isopropyl) are well tolerated at the 7-position but less so in the 6-position, even when the 7-site is reacetylated. Planar aromatic moieties (phenyl and 2-pyridinyl) demonstrated moderate to good potency for binding and activation when located at either the 6- or 7-positions. There is an overall trend toward increasing potency for both binding and activation with polar substituents.

gamma-Aminobutyrate-A receptor modulation by 3-aryl-1-(arylsulfonyl)- 1,4,5,6-tetrahydropyridazines.

A series of 3-aryl-1-(arylsulfonyl)-1,4,5,6-tetrahydropyridazine allosteric modulators of the GABAA receptor was synthesized, and biological activity was examined in vitro and in vivo. Beginning with 1a, stepwise modification of the substituents and conservation of the scaffold yielded a chemical series in which the modulatory activity was enhanced by the presence of GABA. The SAR suggests, but does not establish, that the compounds bind to the steroid binding site on the GABAA receptor. The GABA shift for each compound indicates that all compounds in this series are either agonists or partial agonists.

Pyrroloisoquinoline antidepressants. 3. A focus on serotonin.

A collection of hexahydropyrroloisoquinoline derivatives (1-22), which represent a class of compounds that inhibit the neuronal uptake of dopamine (DA), norepinephrine (NE), and serotonin (5-HT), was investigated in vivo for serotonin-potentiating properties in the mouse head-twitch and rat serotonin syndrome assays. The p-methylthio compound 3b (McN-5652-Z) was found to possess exceptional activity in these assays, and the activity was attributable almost exclusively to the (+)-6S,10bR enantiomer. Ten closely related analogues were synthesized, tested, and compared among themselves and with some previously prepared compounds, both in vivo and in vitro. Several trans diastereomers exhibited strong inhibition of 5-HT uptake and substantial potentiation of 5-HT, while the cis diastereomers (3a, 4a, and 10a) tested were virtually devoid of such activity. Although 3b was only moderately selective in inhibiting the uptake of 5-HT vs NE, its 10-substituted analogues 4b, 7b-9b had improved 5-HT selectivity relative to NE, to the extent of 20-25 times (150-200 times relative to DA). Of these more selective compounds (in vitro), only 4b and 7b had substantial activity in vivo. Sulfoxide 11b appeared to function as a prodrug of 3b in vivo.

Pyrroloisoquinoline antidepressants. 2. In-depth exploration of structure-activity relationships.

A series of pyrrolo[2,1-a]isoquinolines, and related compounds, were examined for antidepressant-like activity, by virtue of their antagonism of tetrabenazine-induced ptosis and sedation, and inhibition of biogenic amine uptake. Thus, we have identified some of the most potent antagonists of TBZ-induced ptosis and some of the most potent inhibitors of the uptake of dopamine, norepinephrine, and serotonin (in rat brain synaptosomes) ever reported. Compounds of particular note, in this regard, are 52b, 29b, 22b, and 48b, respectively. Biological activity was chiefly manifested by the trans isomeric class. Also, through resolution of four compounds, 7b, 24b, 37b, and 48b, biological activity was found to be associated with the (+) enantiomer subgroup (salts measured at 589 nm in MeOH), corresponding to the 6S, 10bR absolute configuration for 7b, 37b, and 48b, and the 6R,10bR configuration for 24b. An X-ray determination on (+)-24b X HBr established its absolute configuration; configurations for the other compounds were verified by enantiospecific synthesis starting with (+)-(R)-2-phenylpyrrolidine. Regarding the pendant phenyl ring, diverse substitution patterns were investigated. Those substitutions that were particularly unfavorable were 3',4',5'-trimethoxy (20b), 2',3',4',5',6'-pentafluoro (34b), 2'-trifluoromethyl (38b), 3',5'-bis(trifluoromethyl) (42b), 4'-n-butyl (44b), 2'-cyano (47b), 4'-methylsulfonyl (50b), and 2'-carboxy (58b). Exceedingly potent compounds, in one way or another, were 10b-12b, 22b, 23b, 25b, 28b, 29b, 33b, 45b, 48b, 51b-53b. The pattern of aromatic substitution had a strong impact on selectivity in the uptake tests (NE vs. DA vs. 5-HT). Activity was significantly diminished by methyl substitution of 7b at the 5 (65, 66), 6 (61b), or 10b (60b) position, by changing the phenyl group of 7b to cyclohexyl (67b), benzyl (68b), or H (72), by moving the phenyl group of 7b to the 5 (69) or 10b (70) position, by expansion of ring B to an azepine (78b), and by modification of ring C to an azetidine (77b), piperidine (75b), or azepine (74b). The interaction of selected analogues with various CNS receptors is reported. Little affinity was shown for the muscarinic cholinergic receptor, suggesting a lack of anticholinergic side effects. Interestingly, 24b and 33b displayed a high affinity for the serotonin-2 receptor, analogous to mianserin and clomipramine. After the body of data was reviewed, derivatives 24b and 48b were chosen for advanced development.

Structure-activity studies on anticonvulsant sugar sulfamates related to topiramate. Enhanced potency with cyclic sulfate derivatives.

We have explored the structure-activity relationship (SAR) surrounding the clinically efficacious antiepileptic drug topiramate (1), a unique sugar sulfamate anticonvulsant that was discovered in our laboratories. Systematic structural modification of the parent compound was directed to identifying potent anticonvulsants with a long duration of action and a favorable neurotoxicity index. In this context, we have probed the pharmacological importance of several molecular features: (1) the sulfamate group (6-8, 22-25, 27, 84), (2) the linker between the sulfamate group and the pyran ring (9, 10, 21a,b), (3) the substituents on the 2,3- (58-60, 85, 86) and 4, 5-fused (30-38, 43, 45-47, 52, 53) 1,3-dioxolane rings, (4) the constitution of the 4,5-fused 1,3-dioxolane ring (2, 54, 55, 63-68, 76, 77, 80, 83a-r, 84-87, 90a, 91a, 93a), (5) the ring oxygen atoms (95, 96, 100-102, 104, 105), and (6) the absolute stereochemistry (106 and 107). We established the C1 configuration as R for the predominant alcohol diastereomer from the highly selective addition of methylmagnesium bromide to aldehyde 15 (16:1 ratio) by single-crystal X-ray analysis of the major diastereomer of sulfamate 21a. Details for the stereoselective syntheses of the hydrindane carbocyclic analogues 95, 96, 100, and 104 are presented. We also report the synthesis of cyclic imidosulfites 90a and 93a, and imidosulfate 91a, which are rare examples in the class of such five-membered-ring sulfur species. Imidosulfite 93a required the preparation and use of the novel sulfur dichloride reagent, BocN=SCl2. Our SAR investigation led to the impressive 4,5-cyclic sulfate analogue 2 (RWJ-37947), which exhibits potent anticonvulsant activity in the maximal electroshock seizure (MES) test (ca. 8 times greater than 1 in mice at 4 h, ED50 = 6.3 mg/kg; ca. 15 times greater than 1 in rats at 8 h, ED50 = 1.0 mg/kg) with a long duration of action (>24 h in mice and rats, po) and very low neurotoxicity (TD50 value of >1000 mg/kg at 2 h, po in mice). Cyclic sulfate 2, like topiramate and phenytoin, did not interfere with seizures induced by pentylenetetrazole, bicucculine, picrotoxin, and strychnine; also, 2 was not active in diverse in vitro receptor binding and uptake assays. However, 2 turned out to be a potent inhibitor of carbonic anhydrase from different rat tissue sources (e. g., IC50 of 84 nM for the blood enzyme and 21 nM for the brain enzyme). An examination of several analogues of 2 (83a-r, 85-87, 90a, 91a, 93a) indicated that potent anticonvulsant activity is associated with relatively small alkyl substituents on nitrogen (Me/H, 83a; Me/Me, 83m; Et/H, 83b; allyl/H, 83e; c-Pr/H, 83j; c-Bu/H, 83k) and with limited changes in the cyclic sulfate group, such as 4,5-cyclic sulfite 87a/b. The potent anticonvulsants 83a and 83j had greatly diminished carbonic anhydrase inhibitory activity; thus, inhibition of this enzyme may not be a significant factor in the anticonvulsant activity. The alpha-L-sorbopyranoses 67, 68, and 80, which mainly possess a skew conformation (ref 29), were nearly twice as potent as topiramate (1). The L-fructose enantiomers of 1 (106) and 2 (107), synthesized from L-sorbose, were found to have moderate anticonvulsant activity, with eudysmic ratios (MES ED50 in mice at 4 h, po) of 1:106 = 1.5 and 2:107 = 3.5. The log P values for 1 and 2 were determined experimentally to be 0.53 and 0.42, respectively, which are less than the optimal 2.0 for CNS active agents. However, analogues with more favorable calculated log P (clogP) values, in conjunction with just minor steric perturbation according to the developed SAR profile, such as 47 (clogP = 2.09), 83m (1.93), and 86 (1.50), did not display improved potency: 47 is less potent than 1, 83m is equipotent with 2, and 86 is less potent than 2. Although the measured log P value for diethyl analogue 31 is 1.52, this did not translate into enhanced potency relative to 1. (ABSTRACT TRUNCATED)

Activity of aromatic substituted phenylpiperazines lacking affinity for dopamine binding sites in a preclinical test of antipsychotic efficacy.

Generally, antipsychotic agents are dopamine receptor blocking agents that also block conditioned avoidance responding (CAR) in the rat. Recently, however, both (Q-methoxyphenyl)piperazine (OMPP, 1h) and (m-chlorophenyl)piperazine (MCPP, 1o) have been reported to block conditioned avoidance responding in the rat although neither has dopamine receptor blocking properties. The present paper examines the behavioral and biochemical profile of a number of additional substituted phenylpiperazines. None of the phenylpiperazines tested demonstrated high affinity for either dopamine D-1 or D-2 receptor sites, yet many were effective in blocking CAR. The results suggest that the phenylpiperazines may be effective antipsychotic agents without blocking dopamine receptors. Moreover, the active compounds did demonstrate activity in displacing ligand binding to serotonin receptors. Receptor binding profiles were determined for 5-HT-1A and 5-HT-1B binding sites as well as for 5-HT-2 sites. The data from this preclinical test suggest these phenylpiperazines might be effective antipsychotic agents acting via a nondopaminergic mechanism of action.

Orally active benzamide antipsychotic agents with affinity for dopamine D2, serotonin 5-HT1A, and adrenergic alpha1 receptors.

New antipsychotic drugs are needed because current therapy is ineffective for many schizophrenics and because treatment is often accompanied by extrapyramidal symptoms and dyskinesias. This paper describes the design, synthesis, and evaluation of a series of related (aminomethyl)benzamides in assays predictive of antipsychotic activity in humans. These compounds had notable affinity for dopamine D2, serotonin 5-HT1A, and alpha1-adrenergic receptors. The arylpiperazine 1-[3-[[4-[2-(1-methylethoxy)phenyl]-1-piperazinyl]methyl]benzoyl]p ipe ridine (mazapertine, 6) was chosen because of its overall profile for evaluation in human clinical trials. The corresponding 4-arylpiperidine derivative 67 was also highly active indicating that the aniline nitrogen of 6 is not required for activity. Other particularly active structures include homopiperidine amide 14 and N-methylcyclohexylamide 31.

DNA ethylation in target and non-target organs of hamsters and rats treated with diethylnitrosamine.

Kinetics of ethylation of target and non-target organ DNA in vivo by diethylnitrosamine (DEN) was compared in rats and Syrian golden hamsters, since published reports indicate a single dose of DEN induces both kidney and liver tumors in rats and almost exclusively respiratory tract tumors in hamsters. Following treatment with 200 mg DEN/kg, 7-ethylguanine (7-etG) was lost more rapidly from hamster than from rat liver DNA, while O6-ethylguanine (O6-etG) persisted longer in hamster than in rat liver DNA. DNA ethylation was not detected in rat lung (non-target organ), while both 7-etG and O6-etG were quantitated in hamster lung (target organ) following DEN treatment. DNA ethylation in rat kidney DNA was approximately 1/10 of that in liver by 200 mg DEN/kg, and the persistence of 7-etG and O6-etG differed only slightly in these tissues. Ethylation of hamster liver DNA by DEN at doses between 20 and 200 mg/kg, as measured by 7-etG and O6-etG was proportional to the dose of carcinogen up to 160 mg/kg; at larger doses DNA ethylation sharply increased. Differences in the persistence of O6-etG between DEN-treated rats and hamsters cannot solely account for species differences in the organotropism of DEN carcinogenesis.

Etoperidone, trazodone and MCPP: in vitro and in vivo identification of serotonin 5-HT1A (antagonistic) activity.

The Ki values for etoperidone, trazodone and MCPP (m-chlorophenylpiperazine dihydrochloride) at 5-HT1A sites (using rat cerebral cortical synaptosomes and [3H]8-OH-DPAT) were determined to be 20.2, 23.6 and 18.9 nM, respectively. In an effort to elucidate the functional nature of the interaction at 5-HT1A sites in vivo, the ability of each compound to elicit reciprocal forepaw treading (RFT) or to block the RFT induced by 8-OH-DPAT in reserpinized rats was tested. Specifically, 8-OH-DPAT (1.0 mg/kg SC)-challenged or non-challenged (control) reserpinized (1.0 mg/kg SC) rats were administered etoperidone, trazodone or MCPP (IP) and scored for the elicitation of RFT (indicative of 5-HT1A agonistic activity) or for block of RFT induced by 8-OH-DPAT (indicative of 5-HT1A antagonistic activity). Reference compounds confirmed the specificity of the test. We report that etoperidone, trazodone and MCPP inhibited 8-OH-DPAT-induced RFT (ID50 = 17.4, 23.8 and 13.4 mg/kg, respectively). Only marginal RFT was produced in non-challenged animals by etoperidone and trazodone at a high dose (40 mg/kg). Taken together, the results suggest a predominant antagonistic activity of etoperidone, trazodone and MCPP at 5-HT1A receptor sites in rat central nervous system. However, one cannot rule out the possibility that these compounds are weak partial agonists. This activity may be relevant to the antidepressant action of these compounds.

Topiramate promotes neurite outgrowth and recovery of function after nerve injury.

Topiramate is a structurally novel neurotherapeutic agent with a unique combination of pharmacological properties and currently is available in most world markets for treating several seizure disorders. Because its pharmacological profile was suggestive of possible activity as a neuroprotectant, topiramate was evaluated and found to be active in several animal models of stroke or neuropathic pain. This prompted an evaluation of topiramate as a possible neurotrophic agent. In this study, topiramate enhanced the recovery of facial nerve function after injury when administered orally at therapeutically relevant doses, and significantly increased neurite outgrowth in cell cultures derived from fetal rat cortical and hippocampal tissues.

[3H][D-Ala2,NMePhe4,Gly-ol5]-enkephalin (mu-opioid) binding in beige-J mice.

Tritiated [D-Ala2,NMePhe4,Gly-ol5]-enkephalin ([3H]DAGO) was used to examine mu-opioid receptor number and mu-ligand binding in brain synaptic membranes (P2 fraction) from C57BL/6J-bgJ/bgJ (beige-J) mice, a strain with combined deficiencies in immunological function (resembling Chediak-Higashi syndrome) and analgesic response to mu-opioid agonists such as morphine and DAGO. As controls, white mice, beige-J littermates (normally responsive to mu-opioid agonists), and a known mu-deficient strain (CXBK) were also examined. Neither the KD (0.47 to 0.49 nM) nor the Bmax (153 to 168 fmol/mg protein) determined for beige-J mice was significantly different from values determined for littermates or white mice. In contrast, the Bmax of CXBK mice (66 fmol/mg protein) was clearly less than that of the other strains. The analgesic defect of beige-J mice, therefore, is not likely due to an insufficient number of mu-opioid receptors, as it presumably is in CXBK mice. Carbachol (200 micrograms/ml), which partly corrects the analgesic defect of beige-J mice, had no effect on [3H]DAGO binding either acutely in vitro or chronically ex vivo after administration to beige-J mice for three weeks. Hence, the analgesic defect of beige-J mice appears to be due to some defect in the mu-opioid receptor-effector coupling mechanism or to some endogenous substance that inhibits binding of mu-opioid ligands to otherwise functional receptors.

In vivo microdialysis and liquid chromatography/thermospray mass spectrometry of the novel anticonvulsant 2,3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate (topiramate) in rat brain fluid.

The concentration of a novel anticonvulsant, 2,3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate (topiramate), was determined in the extracellular fluid of rat brain by in vivo microdialysis combined off-line with liquid chromatography/thermospray mass spectrometry. A microdialysis probe was stereotaxically implanted in the nucleus accumbens region of the rat brain. The maximum concentration of topiramate in the brain dialysate for a dose of 50 mg kg-1 i.v. was approximately 10 microM and occurred 45 min post-injection. The detection limit of topiramate in the extracellular fluid of rat brain was in the 0.1 microM range using selected ion monitoring techniques. The base peak, which was the ammonium adduct ion [M + NH4]+, was used for detection. An internal standard of d12-labeled topiramate was utilized for quantitation by isotope dilution analysis.

DNA-damaging effects of genotoxins in mixture: modulation of covalent binding to DNA.

Modulation of DNA adduct formation by pre-existing adducts was examined in synthetic oligonucleotides and genomic DNA (calf thymus); genotoxins studied were N-acetoxy-acetylaminofluorene (N-AcO-AAF), aminofluorene (AF), aflatoxin B1-8,9-epoxide (AFB1-8,9-epoxide), and dimethylsulfate (DMS). Oligodeoxynucleotides containing either guanine-C8-AAF (Gua-C8-AAF) or Gua-C8-AF adducts and a neighboring unadducted guanine (G) (target G), located 1, 2, or 4 nucleotides from the adduct, were reacted, as single- (ss) or double-stranded (ds) substrates, with dimethylsulfate (DMS) or AFB1-8,9-epoxide. A modified Maxam-Gilbert technique showed that the presence of the AAF adduct lowered the extent to which AFB1-8,9-epoxide, but not DMS, reacted with target G. Binding of AFB1-8,9-epoxide to the target G was attenuated (> or =5-fold) when the target was located immediately adjacent to an AAF, but not AF, adduct in ds-DNA. Reaction with AFB1-8,9-epoxide increased when the target G was located 2 or 4 nucleotides from the AAF adduct. Pretreatment of calf thymus DNA with AAF (0-1.8% nucleotides modified) reduced levels of Gua-N7-AFB1 adducts formed after subsequent treatment with AFB1-8,9-epoxide. Pretreatment of calf thymus DNA with AFB1 did not alter levels of adducts formed after subsequent treatment with N-AcO-AAF. The supposition that aflatoxin B1-binding to DNA may be altered by conformational changes in the helix, due to the presence of a pre-existing AAF adduct, is supported by the absence of an effect by AF and confirmation of local denaturation of the oligomer helix by use of chemical probes hydroxylamine and diethylpyrocarbonate. Nonetheless, the importance of changes in the nucleophilicity of neighboring nucleotides and local steric effects cannot be ruled out.

DNA-damaging effects of genotoxins in mixture: nonadditive effects of aflatoxin B1 and N-acetylaminofluorene on their mutagenicity in Salmonella typhimurium.

Most animal genotoxicity studies have used exposures to single chemicals; humans, however, are potentially exposed to mixtures of genotoxins. Cancer and developmental toxicity risks associated with genotoxins in mixture are generally estimated by assuming additivity of the components. Two or more genotoxins acting sequentially or simultaneously may present a greater or lesser hazard than that predicted by simple addition of their potencies. Previously, we studied the effect of one genotoxin on the binding of a second genotoxin to DNA in an in vitro system and demonstrated that consecutive binding of the two toxins was not additive. In the present study, the effect of one genotoxin on the mutagenicity of another was evaluated for two well-known genotoxins using the Salmonella assay. Pretreatment of frameshift strains TA98 and TA1538 with AFB1-8,9-epoxide (17.3 ng/plate) enhanced the mutagenicity induced by subsequent exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) approximately 2-3 times above theoretical values for additivity. Pretreatment of base-substitution strain TA100 with N-AcO-AAF (0.1 microg/plate) inhibited the mutagenicity following subsequent exposure to AFB1-8,9-epoxide by 3 times below the theoretical additive value. Concentration-response relationships for these enhancing or inhibitory effects were demonstrated using increasing concentrations of the first genotoxin during pretreatment. These results demonstrate effects, other than additive, of sequential exposures to two genotoxins on the induction of mutations in a bacterial system.