Sex and strain differences in response to cocaine

After pretreatment with phenobarbital, female B6AF₁ mice showed considerably higher serum glutamic oxaloacetic transaminase (SGOT) elevations and more periportal necrosis from a single injection of cocaine than males. This sex difference was androgen dependent. Castration or treatment with flutamide made males respond like females, while testosterone made females behave like males. There was no significant sex difference in enzymes of cocaine metabolism. When the mice were induced by exposure to pine bedding, males showed higher SGOT elevations and more centrilobular necrosis after cocaine than females. In this case, the sex difference could be attributed to increased levels of cytochrome P-450 and cocaine N-demethylase in liver microsomes. BALB/cBy mice on pine bedding snowed much less liver damage from cocaine than B6AF₁ mice, but they were more sensitive to norcocaine and N-hydroxy norcocaine. This difference was correlated with low levels of cocaine N-demethylase in the BALB/cBy mice. Liver microsomes from phenobarbital-induced BALB/cBy mice had less norcocaine N-hydroxylase activity than those from B6AF₁ mice. These studies demonstrate that the pattern of sex and strain differences in liver damage from cocaine depends on the inducing agent and can be related to a large extent to the microsomal enzymes induced by that agent.     

Metabolism of cocaine and norcocaine to N-hydroxynorcocaine

The mixed function oxidase system of mouse liver microsomes converts norcocaine to N-hydroxynorcocaine (NHNC). This metabolite can be measured by high performance liquid chromatography (HPLC) using an electrochemical detector. Experiments with inducers and inhibitors suggested that the cytochrome P-450 system was responsible for most of the formation of NHNC. NHNC was relatively unstable under physiological conditions, with a T 1/2 of 17 min at pH 7.4 and 37 degrees. HPLC with the electrochemical detector was also used to demonstrate the formation of NHNC in vivo when mice were injected with norcocaine or cocaine.     

Pharmacology of high-affinity binding of [3H](±)2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN) to bovine caudate nucleus tissue

High-affinity binding of [³H](±)2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene ([³H]-ADTN) was improved by use of a subcellular fraction (P₄) of tissue obtained from calf brain. The highest concentration of binding sites was found in caudate nucleus which was evaluated extensively. Binding of 0.5 nM [³H]-ADTN was optimal at 25° and pH 7.5 to 8.0 when a cation-free medium containing antioxidants was used and was 82–87% displaceable (“specific”). The T_{$\text{1}\text{2}$} for association was 20 min, and for dissociation 38 min, under these conditions. Analysis of association-dissociation kinetics and of ligand saturation isotherms revealed an apparent affinity (K_d) of 1–2 nM and a binding maximum (B_max) of 422 fmoles/mg protein. The process proved to be reversible by, and monophasically competitive with, several potent dopamine agonists, with Hill constants dose to unity. Stereoselectivity was found with eight isomer-pairs. Binding of [³H]-ADTN was selective for 3,4-dihydroxyphenethylamines and 10,11-dihydroxyaporphines with potent dopamine-agonist actions, but not for adrenergic catecholamines or other catechols, or blockers of dopamine uptake. Dopamine antagonists competed more weakly and in poor correspondence with their in vivo activities. There was a close correspondence between IC₅₀ values obtained for fifty of the agents tested with both [³H]-ADTN and [³H]-(?)apomorphine (r and slope > 0.9), supporting impressions of the structure-activity characteristics of dopamine agonist binding sites based on prior studies with [³H]-apomorphine.     

Role of nicotinamide adenine dinucleotide in ethanol-induced depressions in testicular steroidogenesis

It is rapidly becoming accepted, without direct evidence, that a change in the NAD+/NADH ratio in the testes produced by the metabolism of ethanol is the principal mechanism involved in its now well-established effects on testicular steroidogenesis. The purposes of the present studies were 2-fold: (1) to examine whether, in fact, in vivo or in vitro ethanol exposure alters the NAD+/NADH ratio in the testes; and (2) to examine the validity of previous reports in which it was found that NAD+ prevented the effects of ethanol on testicular steroidogenesis under in vitro conditions. With regard to the first objective, we found that a large dose of ethanol (2.5 g/kg) markedly reduced gonadotropin-stimulated testicular steroidogenesis in vivo in the male rat, but it did not alter the NAD+ and NADH concentrations in the testes. Similarly, extremely high ethanol concentrations (200 mM) substantially suppressed hMG-stimulated testosterone biosynthesis in in vitro Leydig cell preparations but no change in NAD+ concentration occurred; NADH levels were very low in the Leydig cell preparations (less than 2% of NAD+ levels), but did not appear to change as a function of ethanol exposure. Finally, in contrast to previously published results, we found that NAD+ (1 mM) did not prevent the in vitro effects of ethanol on cAMP-stimulated testicular steroidogenesis. Consequently, our results fail to support the hypothesis that acute in vivo or in vitro ethanol administration inhibits the biosynthesis of testosterone by altering the NAD+/NADH ratio in the testes.     

Pharmacokinetics of inhibition of adenosine deaminase by erythro-9-(2-hydroxy-3-nonyl)adenine in CBA mice

The pharrnacokinetics oferythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) inhibition of adenosine deaminase (ADA) was measured in vivo in CBA mice. The in vivo assay utilized injection of 10–100 nmoles [2-³H]adenosine and measurement of blood ³H₂O 20 min later. A single oral dose of EHNA (50 mg/kg) totally inhibited ADA for 4 hr and caused a large increase in conversion of [2-³H]adenosine to [2-³H]ATP. EHNA (3 mg/kg) decreased deamination by 50% for 2–6 hr, depending on the dose of adenosine used. Mice dosed with EHNA (100 mg/kg) once daily for 7 days showed the same ADA recovery rate as mice dosed only once. High single oral doses of EHNA had no effect on blood ATP and GTP pools.     

Effects of ethanol and barbiturates on Ca2+-ATPase activity of erythrocyte and brain membranes

Exposure to ethanol or pentobarbital in vitro stimulated the ATP-dependent efflux of calcium from human red blood cells (RBC) and the Ca2+-ATPase activity of RBC and rat brain synaptic plasma membranes (SPM). These effects were obtained with concentrations of ethanol (50 mM) and pentobarbital (60 microM) associated with intoxication in vivo. The enhancement of SPM Ca2+-ATPase by ethanol was due to an increase in the apparent affinity of the enzyme for calcium with no change in the maximum velocity. SPM Ca2+-ATPase was also stimulated by an unsaturated fatty acid, cis-vaccenic acid methyl ester (cis-VAME). The membrane-disordering effects of ethanol, four barbiturates and cis-VAME were evaluated in SPM using the fluorescent probe molecule 1,6-diphenyl-1,3,5-hexatriene (DPH). All the compounds decreased the fluorescence polarization of DPH, and these decreases were proportional to the increase in Ca2+-ATPase produced by these drugs. These findings suggest that the increase in Ca2+-ATPase and calcium efflux produced by ethanol and pentobarbital results from the membrane-disordering effects of these drugs.     

Synergism of the toxicity of physostigmine and neostigmine by lithium or by a reserpine-like agent (Ro4-1284)

A single sublethal i.p. dose of lithium chloride (300 mg/kg or 7.1 meq/kg) followed 12 h later by an otherwise sublethal s.c. dose of physostigmine sulfate (1.0 mg/kg) resulted in 90% mortality among male rats following a pronounced cholinergic syndrome, including convulsions. This confirms a previous report of a lethal synergism of physostigmine after subacute dosing with lithium. Mortality could be completely prevented by 1.0 mg/kg of atropine sulfate given 30 min before physostigmine, but was incompletely, if at all, reduced by selective peripheral cholinergic blockers, methylatropine bromide (0.5, 1.5 mg/kg) or glycopyrrolate (1 mg/kg). This suggested a predominantly central site for the toxic interaction. However, a similar synergism of lethality caused by neostigmine methylsulfate (0.3 mg/kg, s.c.) after treatment with lithium, which could be eliminated by methylatropine or glycopyrrolate, indicates that lithium may also produce lethal synergism of a cholinesterase (ChE) inhibitor that does not act centrally. Ro4-1284, an agent that has reserpine-like actions, was tested in combination with physostigmine or neostigmine; it showed synergism of toxicity nearly the same as in the case of lithium plus the cholinergic agents. These findings support the hypothesis that lithium causes the toxic synergism via a reduction of adrenergic activity, leading to an imbalance between adrenergic and cholinergic influences and a consequent failure to tolerate the effects of the ChE inhibitors. A potential hazard for the clinical use of physostigmine and neostigmine, concurrently with lithium or reserpine-like agents, it suggested.     

Effects of 3-methylcholanthrene and phenobarbital on the capacity of embryos to bioactivate teratogens during organogenesis

Pregnant Sprague-Dawley rats were divided into four groups and given ip injections of 3-methylcholanthrene (MC) in corn oil, corn oil only, phenobarbital (PB) in Hank's balanced salt solution (HBSS), or HBSS only. Maternal animals were killed on Day 10 of gestation, and embryos from each group were explanted in medium containing cyclophosphamide (CP), 2-acetylaminofluorene (AAF), or dimethylsulfoxide vehicle. After a 24-hr culture period, embryos from dams treated with HBSS, corn oil, or PB/HBSS exhibited no increase in abnormalities (as compared with controls) when either CP or AAF were added to the media. However, embryos transplacentally preexposed to MC and subsequently treated during culturing with AAF (but not CP) exhibited striking increases in malformation incidence. Commonly observed malformations included abnormally open neural tubes, abnormal flexure rotation, and prosencephalic defects. Homogenates of Day 10 embryos transplacentally preexposed to MC exhibited readily measurable oxidative biotransformation of AAF as assessed with HPLC. Biotransformation of AAF by embryos from the other three groups was virtually undetectable. Incorporation of exogenously supplemented bioactivating systems from livers of mature animals indicated that postmitochondrial supernatant fractions (S-9) from male, MC-pretreated rats effectively catalyzed the conversion of AAF (but not CP) to embryotoxic metabolites. Conversely, hepatic S-9 from adult, male, PB-pretreated rats was highly effective in converting CP (but not AAF) to embryotoxic metabolites. The results indicated the inducerspecific occurrence of embryonic bioconversion of AAF to embryotoxic metabolites via MC-inducible, P-450-dependent, embryonic enzyme systems.     

Further studies on the in vivo effect of diisopropyl 1,3-dithiol-2-ylidenemalonate (NKK-105) on the liver microsomal drug oxidation system in rats

The effect of diisopropyl 1,3-dithiol-2-ylidenemalonate (NKK-105) on microsomal electron transport systems in relation to drug oxidation was studied in rat liver. A single oral dose (250 mg/kg) of NKK-105 increased the ratio of liver to body weight, the microsomal protein content, the cytochrome b₅ content, and the NADPH cytochrome c reductase activity at 24–48 hr after drug administration. The cytochrome P-450 content was decreased at 2–6 hr and slightly increased at 24–48 hr after drug administration. Upon daily administration of NKK-105 at a dose of 250 mg · kg^?1 · day^?1 for 21 days, cytochrome b₅ content and NADPH cytochrome c reductase activity were increased, but cytochrome P-450 content and NADH cytochrome b₅ reductase activity remained unchanged. Despite the increase of NADPH cytochrome c reductase activity, NADPH-dependent lipid peroxidation tended to decrease rather than increase. NADPH stearoyl-CoA desaturase activity increased prior to the increase of cytochrome b₅. Benzphetamine N-demethylase and p-nitroanisole O-demethylase activities were enhanced, accompanied by an increase of cytochrome b₅. Aniline hydroxylase activity was decreased by NKK-105 administration. These results indicate that the induction pattern of liver microsomal electron transport systems by NKK-105 is characteristic.     

Effects of triethylenetetramine upon the metabolism and toxicity of 63NiCl2 in rats

Triethylenetetramine (TETA, 0.75 mmol/kg, im) was administered to Fischer rats immediately prior to ⁶³NiCl₂ (0.068 or 0.10 mmol/kg, ip or im) to determine (a) effects of TETA upon ⁶³Ni-kinetics, and (b) antidotal effects of TETA upon Ni-induced nephrotoxicity and hyperglycemia. TETA markedly reduced plasma ⁶³Ni concentrations and greatly increased urine ⁶³Ni excretion during 6 hr after injection of ⁶³NiCl₂, compared to values in control rats that received only ⁶³NiCl₂. In rats killed 6 hr after injections of TETA and ⁶³NiCl₂, ⁶³Ni concentrations in liver, kidney, spleen, lung, and heart averaged 3.4, 0.72, 0.27, 0.22, and 0.12 times corresponding ⁶³Ni concentrations in organs from control rats that received only ⁶³NiCl₂. The results supported the hypothesis that combined administration of TETA and ⁶³NiCl₂ resulted in partition of the body pool of ⁶³Ni between two major ⁶³Ni-components: (a) ⁶³Ni-TETA complex, and (b) nonchelated-⁶³Ni, and that each ⁶³Ni-component was eliminated from plasma according to its respective clearance. The renal clearance of ⁶³Ni-TETA complex was estimated to be >20 times that of nonchelated-⁶³Ni. Administration of TETA substantially reduced Ni-induced proteinuria and aminoaciduria. TETA did not prevent Ni-induced hyperglycemia and hyperglucagonemia, but TETA did partially inhibit Ni-induced hyperinsulinemia.     

Degradation of isolated deoxyribonucleic acid mediated by nitroso-chloramphenicol: Possible role in chloramphenicol-induced aplastic anemia

Reduction of the nitro group of chloramphenicol (CAP) gives rise to more highly reactive intermediates which may be involved in the aplastic anemia associated with CAP use. One such intermediate, nitroso-chloramphenicol (NO-CAP), has been found to be a potent agent for mediating degradation of isolated DNA. In a reaction mixture containing 100 μM NO-CAP, 100 μM CuCl₂, and 5 mM NADH, 7 μg of Escherichia coli [³H]DNA was completely degraded to acid-soluble fragments in 30 min. Damage to DNA was in the form of single-stranded scissions. The requirement for copper was specific, and copper chelating reagents blocked the degradation. The need for a reducing agent could be met equally well by NADH or NADPH, but not by sulfhydryl reagents such as glutathione, dithiothreitol and 2-mercaptoethanol. Oxygen was also necessary for the NO-CAP-mediated DNA damage, with reduced forms of oxygen participating in the reaction. A role for H₂O₂ was indicated by the inhibition of the degradation seen when catalase was included in the mixture. Hydroxyl radicals are known to be produced in the reaction of H₂O₂ with certain transition metals. Scavangers of hydroxyl radicals also inhibited strand-scission, suggesting that the radicals may be the primary agents in DNA degradation. The importance of the nitroso moiety of NO-CAP was evidenced by the lack of DNA damage seen when NO-CAP was replaced by CAP under the conditions tested.     

Effect of hypoxia on carbon tetrachloride hepatotoxicity

The effect of hypoxia on carbon tetrachloride-induced hepatotoxicity was studied. Male rats were exposed to carbon tetrachloride for 2 hr in the presence of differing oxygen concentrations. Serum glutamate-pyruvate transaminase (SGPT) activities were measured 24 hr after the end of the exposure. Exposure of rats to 5000 ppm carbon tetrachloride in the presence of 100, 21,12, or 6% oxygen resulted in SGPT activities of 489, 420, 3768, and 1788 I.U./l respectively. Exposure of rats to air and 0, 1250, 2500, 5000, or 7500 ppm carbon tetrachloride gave SGPT activities of 35, 32, 69, 420, and 2188 I.U./l respectively; when 12% oxygen was used, the corresponding SGPT activities were 32, 665, 691, 3768, and 4200 I.U./l respectively. Exposure of rats to hypoxia produced histopathologically detectable condensation of hepatic cytoplasmic material, and exposure to 5000 ppm carbon tetrachloride in the presence of air produced mild centrilobular necrosis, which was much more severe when rats were exposed to 5000 pm carbon tetrachloride in the presence of 12% oxygen. Hepatic microsomal conjugated diene concentrations were increased by hypoxia and by exposure to carbon tetrachloride, but no synergistic interaction was observed. Hepatic microsomal cytochrome P-450 concentrations were decreased after exposure to carbon tetrachloride, but were the same after exposure to carbon tetrachloride and 12 or 21% oxygen. Hepatic carbon tetrachloride concentrations were the same in rats exposed to carbon tetrachloride in the presence of 12 or 21% oxygen; hepatic chloroform concentrations were higher in rats exposed to carbon tetrachloride in the presence of air than in the presence of 12% oxygen. The covalent binding of [¹⁴C]carbon tetrachloride metabolites to hepatic microsomal lipids and proteins was increased markedly by hypoxia as compared with normoxia. The covalent binding of metabolites of carbon tetrachloride to cellular macromolecules may play a role in the potentiation of carbon tetrachloride toxicity by hypoxia.     

Effects of the chiral isomers of erythro- and threo-9-(2-hydroxy-3-nonyl)adenine on purine metabolism in sarcoma 180 cells

The effects of the chiral isomers of erythro- and threo-9-(2-hydroxy-3-nonyl)adenines (EHNA and THNA) on purine metabolism in Sarcoma 180 cells have been determined. At concentrations of 10–80 μM [10- to 1000-fold greater than their K_i values with adenosine deaminase (ADA)], all isomers inhibited purine salvage and biosynthesis de novo. Although (+)-EHNA, the most potent ADA inhibitor, exerted the greatest effects, there was no direct correlation between the potency of ADA inhibition and the secondary effects on purine metabolism, e.g. (+)-EHNA is about 2-fold more inhibitory than (?)-EHNA in blocking purine base incorporation but about 250-fold more potent as an inhibitor of ADA (K_i of (+)-EHNA = 500nM[Bessodes et al., Biochem. Pharmac.31, 879 (1982)]). All the isomers inhibited the incorporation of radiolabeled purine bases (adenine, guanine and hypoxanthine) and nucleosides (guanosine and inosine) into acid-soluble nucleotides and of glycine into 5′-phosphoribosyl-formylglycineamide. Unlike the results of Henderson et al. [Biochem. Pharmac.26, 1967 (1977)] with Ehrlich ascites cells, the incorporation of adenosine into nucleotides was only slightly inhibited in Sarcoma 180 cells. (+)-EHNA did not inhibit the activities of 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase, purine phosphoribosyltransferases or nucleotide kinases in cell extracts. Accumulation of PRPP was inhibited only under conditions that fostered rapid synthesis.     

Effect of inhibitors of arachidonic acid metabolism on α-aminoisobutyric acid transport in human lymphocytes

The role of arachidonic acid metabolism (or metabolites) in the modulation of α-aminoisobutyric acid transport in resting and concanavalin A-stimulated human peripheral blood lymphocytes was evaluated using previously characterized inhibitors of arachidonic acid metabolism. Nordihydroguairetic acid (a nonselective antioxidant), 5,8,11,14-eicosatetraynoic acid (an inhibitor of lipoxygenase and cyclooxygenase activities), indomethacin and acetylsalicylic acid (selective cyclooxygenase inhibitors), and 1-benzylimidazole, Ro-22-3581 and Ro-22-3582 (thromboxane synthetase inhibitors) proved to be potent inhibitors of amino acid transport activity in normal resting and lectin-activated lymphocytes at concentrations known to decrease thromboxane A₂ production. The rank order of effectiveness of these various inhibitors compared favorably with their relative potencies as inhibitors of thromboxane B₂ synthesis under the same conditions, as determined by radioimmunoassay. Inhibitory effects noted were not due to overt cytotoxicity and seemed to involve changes primarily in the V_max and not the K_m of the transport process. Drug-induced alterations in the magnitude of concanavalin A binding were not observed. These results suggest that the activity of amino acid transport systems can be influenced by certain arachidonic acid metabolites, probably thromboxanes, in both stimulated and unstimulated lymphocytes. In addition, these findings may provide a partial explanation for the observation that inhibitors of thromboxane formation prevent lymphocyte mitogenesis [J. P. Kelly, M. C. Johnson and C. W. Parker, J. Immun.122, 1563 (1979)].     

Circulating concentrations of di(2-ethylhexyl) phthalate and its de-esterified phthalic acid products following plasticizer exposure in patients receiving hemodialysis

The degree of exposure to the plasticizer di(2-ethylhexyl) phthalate (DEHP) was assessed in 11 patients undergoing maintenance hemodialysis for the treatment of renal failure. The amount of DEHP leached from the dialyzer during a 4-hr dialysis session was estimated by monitoring the DEHP blood concentration gradient across the dialyzer. Circulating concentrations of the biologically active products of DEHP de-esterification, viz., mono(2-ethylhexyl) phthalate (MEHP) and phthalic acid, were also determined during the dialysis session. On the average, an estimated 105 mg of DEHP was extracted from the dialyzer during a single dialysis session, with a range of 23.8 to 360 mg. The rate of extraction of DEHP from the dialyzer was correlated with serum lipid content as expressed by the sum of serum cholesterol and triglyceride concentrations (r = +0.65, p less than 0.05). Time-averaged circulating concentrations of MEHP during dialysis (1.33 +/- 0.58 micrograms/ml) were similar to those of DEHP (1.91 +/- 2.11 micrograms/ml). Blood concentrations of phthalic acid (5.22 +/- 3.94 micrograms/ml) were higher than those of the esters. The length of time patients had been receiving regular dialysis treatment was not a determinant of circulating concentrations of DEHP or MEHP. In contrast, time-averaged circulating concentrations of phthalic acid correlated strongly with the duration (in years) of dialysis treatment (r = +0.92, p less than 0.001). The results indicated substantial exposure to DEHP during hemodialysis and that the de-esterified products of DEHP are present in significant concentrations in the systemic circulation. Further study is needed to assess the contribution of these metabolites to the biological actions of DEHP in man.     

Antigenicity and cutaneous toxicity of N-(4′-(9-acridinylamino)-3-methoxyphenyl)-methanesulfonamide (AMSA; NSC-249992) in guinea pigs and rabbits

Dermal rashes appearing in personnel involved in the bulk formulation of the antineoplastic agent AMSA prompted this investigation of the allergenicity and cutaneous irritation potential of the drug in rabbits and guinea pigs. Maximization testing of AMSA in guinea pigs resulted in a 100% skin sensitization rate. A 14-day cutaneous irritation study using a modified Draize test did not show any potential of AMSA to produce skin irritation on intact or abraded skin. AMSA did not induce antibodies in rabbits which could be detected by passive hemagglutination, cutaneous anaphylaxis, Arthus reactivity, or delayed hypersensitivity in rabbits. AMSA also failed to sensitize guinea pigs for systemic anaphylaxis.