Pharmacogenetic approaches to drug dependence.

The aim of this volume is to bring together information about both the neurochemical mechanisms associated with the actions of drugs of abuse and the psychopharmacological and behavioural effects of these drugs. One approach that is proving to be quite useful for bringing together these two very diverse fields is the use of classical pharmacogenetic techniques. Pharmacogenetics is defined as the study of genetic and environmental factors underlying individual differences in response to pharmacological or toxicological agents. Much of the early work in this field dealt with genetic differences in drug metabolism and other pharmacokinetic parameters [1]. More recently, however, attention has been directed towards understanding the implications of genetic differences associated with pharmacodynamic parameters mediating the actions of drugs. This paper discusses the use of pharmacogenetic techniques in drug abuse research. It presents an overview of some of the more common genetic techniques available for use in drug research and provides some examples from research projects that have employed these approaches.

Choice of vincristine or 6-mercaptopurine on the basis of polyamine level in tumor-bearing regions of the brain.

The effects of vincristine (VC) and 6-mercaptopurine (6-MP) on body weight, regional weights, and the contents of putrescine, spermidine, and spermine in six regions of the brain were examined in rats that had been given these drugs for 5 consecutive days. VC is recommended for management of tumors in the corpus striatum and/or hippocampus, and cortex although its efficacy is dependent on the doubling time of the tumor cells, whereas 6-MP is recommended for the management of tumors in the cortex, thalamus and/or hypothalamus, and diencephalon. VC and 6-MI are chosen for treatment of the brain tumors because they reduce polyamines which are associated with the reduction of drug-sensitive cells and an inhibition of tumor growth.

One-way cross-sensitization and cross-tolerance to seizure activity from cocaine to lidocaine.

The present study examined the effects of daily treatment with a subconvulsant dose (50 mg/kg) of cocaine or lidocaine on susceptibility to seizures induced by cross-injections of the same dose of the other local anesthetic, and to seizures induced by pentylenetetrazol (PTZ) or N-methyl-DL-aspartate (NMDLA) in ddY mice. Repeated administration of 50 mg/kg cocaine caused the development of sensitization to cocaine-induced seizures during an initial 3 or 4 days, followed by the development of tolerance on days 4-6. The same dose of lidocaine, however, produced little or no seizure activity following repeated administration. In contrast, when injected 24 hr after 2-4 days of cocaine treatment, 50 mg/kg lidocaine produced severe seizures. Interestingly, this cross-sensitization from cocaine to lidocaine diminished upon further cocaine treatment. In contrast, treatment with lidocaine for 2-6 days had no effect on subsequent changes in seizure susceptibility following repeated cocaine injections. Neither treatment with cocaine nor lidocaine for 2 or 5 days influenced susceptibility to seizures induced by a challenge injection of PTZ (50 mg/kg, i.p.) or NMDLA (300 mg/kg, i.p.) 24 hr after treatment. HPLC analyses revealed that the cocaine treatment paradigm used in these studies increased the levels of the polyamines, putrescine and spermidine, in mouse brain, while lidocaine treatment had no effect on cerebral polyamine levels. These results suggest that there are differences in the neural mechanisms underlying the convulsant properties of cocaine and lidocaine in ddY mice.

Role of cerebral spermidine in the development of sensitization to convulsant activity of cocaine and lidocaine.

We have previously shown that daily treatment with subconvulsant dose of cocaine resulted in the elevation of brain levels of polyamines such as putrescine and spermidine and the development of increased susceptibility to cocaine-induced seizures. The present study examined whether exogenously administered polyamines affect seizure activity caused by various doses of cocaine and lidocaine in mice. Thirty minutes after intracerebroventricular treatments with either saline, putrescine or spermidine (1-4 mumol), animals were injected intraperitoneally with cocaine or lidocaine (60-90 mg/kg); then the occurrence of clonic seizures was observed. Spermidine enhanced cocaine-induced seizure activity, while putrescine had no effect on it. Lidocaine-induced convulsions were also dose-dependently potentiated by spermidine. In addition, spermidine significantly enhanced seizure activity following an injection of N-methyl-DL-aspartate. The results suggest that spermidine plays an important role in the development of sensitization to convulsant activity by cocaine and lidocaine via modulation of N-methyl-D-aspartate receptors.

Genetic differences in the time course for the development and persistence of the anticonvulsant effects of carbamazepine against cocaine seizures.

Initial studies of the effect of chronic carbamazepine (CBZ) against cocaine-induced seizures indicated that there were genetic differences in both the time course for the development of the anticonvulsant effects of CBZ against cocaine-induced seizures and the persistence of these effects. The present studies were initiated to investigate the time course for the development and persistence of the anticonvulsant effects of chronic CBZ against cocaine seizures in BALB/cByJ, C57Bl/6J and SJL/J mice. The anticonvulsant actions of CBZ were dependent on the duration of CBZ administration, requiring 4-7 days to achieve maximal efficacy. However, once the anticonvulsant effects of CBZ were manifest, the effect persisted for up to 5 days after stopping CBZ treatment depending on the genotype. The levels of CBZ and its active epoxide metabolite were determined in plasma and brain at various time points during and after chronic CBZ treatment. The levels of CBZ and CBZ-10,11-epoxide were substantially reduced over the course of treatment in all three strains, such that the levels of the two compounds in plasma and brain could not account for the decreased susceptibility to cocaine seizures observed following chronic CBZ. These results suggest that the effects of CBZ on cocaine seizures are mediated by relatively long-term changes in one or more biological systems associated with cocaine's convulsant effects.

Increased polyamine levels and changes in the sensitivity to convulsions during chronic treatment with cocaine in mice.

Polyamines have been demonstrated to modulate seizure activity in animals. Repeated administration of a subthreshold dose of cocaine resulted in the development of sensitization to cocaine-induced seizures during an initial 3 or 4 days, followed by the development of tolerance to seizures on days 5 and 6. In the present study, polyamines, such as putrescine, spermidine and spermine, were measured in regions of the brain obtained from mice that showed differential sensitivity in seizure activity during repeated cocaine injections. Animals were sacrificed for polyamine measurements 24 h after the second and the fifth injections of either cocaine or saline (on day 3 and day 6, respectively), and 3 days after the last injection. On day 3, there were significant increases in putrescine in the striatum, hippocampus and cerebellum, and in spermine in the cerebellum of cocaine-treated mice, as compared to saline-treated mice. On day 6, treatment with cocaine significantly increased putrescine in all regions, and spermidine in striatum and hippocampus, as compared to saline treatment. Cocaine treatment had no effect on any polyamine levels measured 3 days after the last injection, except for spermidine in the cortex. Because putrescine has been shown to be an antagonist of the polyamine-binding site on the N-methyl-D-aspartate receptor and to retard the development of amygdala-kindling, the present results suggest that the increases in putrescine content may be associated with the development of tolerance to convulsant effects observed during the later period of repeated administration of cocaine.

Suppressive effect of cycloheximide on behavioral sensitization to methamphetamine in mice.

The effect of a protein synthesis inhibitor, cycloheximide, on behavioral sensitization to methamphetamine was investigated in mice. As indicated by the sensitization tests, repeated injection of methamphetamine (2 mg/kg i.p.) at intervals of 3 and 4 days resulted in a progressive augmentation of the locomotor-stimulating effect of methamphetamine. This phenomenon, called locomotor sensitization, was attenuated by simultaneous treatment with cycloheximide (120 mg/kg i.p.) at the time of stimulant injection. In contrast, when mice were treated with cycloheximide 4 h after stimulant injection, locomotor activity was progressively augmented in the same way as observed in mice receiving repeated injections of methamphetamine alone. On challenge, it was noted that locomotor activity was significantly higher in mice injected repeatedly with the stimulant alone and in those mice treated with the inhibitor 4 h after the stimulant injection compared to the saline-treated control mice. However, mice that had been simultaneously treated with cycloheximide and methamphetamine showed almost the same locomotor activity as the saline-treated control mice. These observations indicated that the locomotor sensitization to methamphetamine was possibly suppressed by simultaneous treatment with cycloheximide. We then examined the dose- and time-dependent nature of the effect of cycloheximide on locomotor sensitization. The stimulation of locomotion observed after repeated injection of the stimulant at a dose of 1.5 mg/kg was significantly attenuated by simultaneous treatment with 120 or 240 mg/kg of cycloheximide, but not by treatment with 60 mg/kg of the inhibitor. However, all the treatments failed to suppress the development of locomotor sensitization elicited by 3 mg/kg of methamphetamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of trihexyphenidyl on place preference conditioning and locomotor stimulant activity of cocaine and methamphetamine.

Cocaine produces rewarding and locomotor stimulant effects by increasing extracellular dopamine (DA) levels in the terminal areas of the mesolimbic DA system. Our recent in vitro studies have shown that a muscarinic receptor antagonist, trihexyphenidyl (THP) inhibits the binding of a cocaine analogue to the DA transporter at concentrations that are ineffective in inhibiting 3H-DA uptake, suggesting that THP may attenuate the actions of cocaine selectively. The present study examined whether THP could affect conditioned place preference (CPP) for and locomotor stimulant activity of cocaine and methamphetamine (MAP) in mice. Mice were injected with cocaine (10 mg/kg) or MAP (1 mg/kg) in one compartment of the CPP chamber 4 times every second day. On alternate days the animals received saline in the other compartment of the CPP chamber. Pretreatment with THP was made 10 min before cocaine or MAP injection. The CPP score and locomotor activity were assessed using a novel activity monitor, SCANET. Cocaine and MAP produced CPP for the drug-paired compartment. Pretreatment with THP (0.05-5 mg/kg) had no influence on cocaine-induced CPP at any dose tested. In contrast, MAP-induced CPP was completely antagonized by THP at 5 mg/kg, which produced no CPP by itself. Another muscarinic receptor antagonist, scopolamine (SCP, 3 mg/kg) neither caused CPP by itself nor affected the development of cocaine- or MAP-induced CPP. Both THP and SCP enhanced spontaneous, cocaine- or MAP-induced locomotor activity. Though the present conditioning treatments failed to develop locomotor sensitization to cocaine, THP, but not SCP, acted cooperatively with cocaine to develop locomotor sensitization. The development of locomotor sensitization to MAP was retarded by SCP but was not affected by THP. These results suggest that, contrary to our anticipation, THP has a unique characteristic of specifically counter-acting the rewarding properties of MAP via a non-cholinergic (muscarinic) mechanism.

Genotype-specific blockade of cocaine-induced weight loss by the protein synthesis inhibitor, anisomycin.

Cocaine has been shown to reduce food intake and body weight in rodents and humans. The results of recent research suggest that de novo protein synthesis in the brain is associated with neuroadaptive changes in the central nervous system. The present study reports the effect of anisomycin, a protein synthesis inhibitor with limited toxicity, on the reduction in body weight resulting from repeated daily injections of cocaine (50 mg/kg) to mice from 7 inbred strains (AKR/J, BALB/cByJ, C3H/HeJ, C57BL/6J, CBA/J, DBA/2J and SJL/J). Repeated cocaine administration resulted in substantial weight loss in all but the BALB strain. Anisomycin (5-30 mg/kg), administered 5 min. prior to each daily cocaine injection, significantly attenuated cocaine-induced weight loss in SJL, C3H and CBA mice. The same treatment, however, had no effect on reduction in body weight in C57, AKR and DBA mice. In BALB mice, neither cocaine, anisomycin alone, nor the coadministration of the two drugs, affected weight gain during the experiment. The results suggest that there is a genotype-specific involvement of protein synthesis associated with cocaine-induced weight loss.

Long-term sensitization to the behavioral effects of naltrexone is associated with regionally specific changes in the number of mu and delta opioid receptors in rat brain.

Enhanced sensitivity to some of the behavioral effects of the opioid antagonist naltrexone (NTX) develops following once-weekly injections of cumulative doses of the drug. Rats treated with this regimen of NTX injections show enhanced sensitivity to the operant response rate decreasing effects of NTX and NTX-induced salivation. The enhanced sensitivity is long-lasting and appears to be produced through conditioning processes. We have conducted saturation binding assays to assess possible changes in the number and affinity of mu and delta opioid receptors in cortical, midbrain and hindbrain membrane preparations from Long-Evans rats treated once weekly for 8 weeks with cumulative doses of the drug (1, 3, 10, 30 and 100 mg/kg). 3H-DAMGO (0.5-21 nM) and 3H-pCl-DPDPE (0.04-4 nM) were used to characterize mu and delta receptors, respectively. NTX treatment had no effect on 3H-DAMGO binding in cortex, but decreased binding in midbrain and increased binding in hindbrain relative to saline-treated controls. Saturation analyses revealed that these differences reflected changes in the number, but not the affinity of mu receptors. NTX treatment also increased the amount of 3H-pCl-DPDPE bound to delta receptors in midbrain and hindbrain, but not in cortex. Again, these changes were due to changes in the number of receptors. Thus, chronic NTX differentially affects the number of mu and delta opioid receptors in various brain regions.

Effects of methotrexate and cyclophosphamide on polyamine levels in various tissues of rats.

The effects of methotrexate (MTX) and cyclophosphamide (CYP) on body weight, organ weight, and the concentration of putrescine, spermidine, and spermine in 14 different tissues were measured in rats that had been given these compounds for 5 consecutive days. These three polyamines in both the thymus and spleen of rats treated with MTX and CYP showed a statistically significant decrease. Further, putrescine in the seminal vesicles, kidney, liver, and small intestine of MTX-treated rats, and in the prostate, seminal vesicles, kidney, heart, liver, small intestine, and lung of CYP-treated rats, spermidine in the prostate, seminal vesicles, testis, thymus, spleen, kidney, heart, small intestine, and skeletal muscle of CYP-treated rats, and spermine in the prostate, seminal vesicles, kidney, heart, small intestine, and stomach of CYP-treated rats showed statistically significant decreases. Recognition of the significance of polyamine levels and attention to their response in anti-cancer drug therapy may have clinical implications.

Effect of ethanol on fatal carbon monoxide poisoning in awake mice [correction of rats].

The effect of ethanol on fatal carbon monoxide (CO) poisoning was investigated in mice injected intraperitoneally with ethanol. Ethanol (1.5 and 3.0 g/kg) was injected 15 min prior to exposure to gas containing 6.6% CO. The survival period was significantly lengthened with ethanol in proportion to the doses injected, although the carboxyhemoglobin (CO-Hb) saturation level in postmortem blood was almost the same in all groups. On the other hand, the CO-Hb level in the blood of mice injected with ethanol was significantly lower than that of control mice during the early exposure period when all mice were still alive. Our results showed that the acute ethanol injection did not influence the CO-Hb saturation level in blood at death, but did affect the duration of survival, probably because of ethanol's ability to decrease blood flow and CO intake.

Urinary excretion of p-hydroxylated methamphetamine metabolites in man. II. Effect of alcohol intake on methamphetamine metabolism.

The effect of drinking alcoholic beverages on methamphetamine metabolism was investigated in man. The subjects, 97 males and 9 females, were divided into three groups by evaluation of their urinary pH; i.e., acidic, subacidic and neutral groups. The subjects in each group were further divided into ethanol-positive subjects and ethanol-negative subjects, depending on the presence or absence of ethanol in their urine. Gas chromatographic analysis showed the urinary concentrations of methamphetamine in the ethanol-positive subjects to be higher than those in the ethanol-negative subjects in both the acidic and subacidic urinary pH groups. Liquid chromatography, on the other hand, showed the urinary concentrations of p-hydroxymethamphetamine and p-hydroxyamphetamine for the ethanol-positive subjects to be lower than those for the ethanol-negative subjects in all three groups. The relative proportions of p-hydroxylated metabolites to unchanged methamphetamine in urine, therefore, were severely reduced in the ethanol-positive subjects. These results suggest that drinking alcoholic beverages probably results in a suppression of methamphetamine metabolism in man.

Simultaneous monitoring of conditioned place preference and locomotor sensitization following repeated administration of cocaine and methamphetamine.

The paradigm of conditioned place preference has been widely used to demonstrate the rewarding properties of psychomotor stimulants. Such drugs also stimulate locomotor activity. Repeated administration of low doses of psychomotor stimulants causes progressive increases in the locomotor stimulating effect, a phenomenon termed behavioral sensitization. Using a new activity monitor (SCANET MV-10LD) that simultaneously measures the amount of time spent and the distance traveled in each side of a two-compartment chamber, the present study assessed place preference conditioning and locomotor sensitization following repeated administration of cocaine or methamphetamine (MAP) in mice. We examined the effect of environmental factors on these activities using two different types of chamber: one having a single cue, and the other having dual cues for the discrimination of compartments. In both types of chamber, cocaine (5-20 mg/kg) and MAP (1-2 mg/kg) similarly produced conditioned place preference. However, repeated cocaine administration caused the development of locomotor sensitization only in the single-cue chamber. On the other hand, repeated administration of MAP resulted in the development of sensitization in both types of chamber. The findings indicate that environmental factors differentially affect the development of locomotor sensitization, but not place preference conditioning, following repeated administration of cocaine or methamphetamine. The advantages of this new system will be discussed.

Modification of behavioral responses to methamphetamine evoked by the stimulant's metabolite p-hydroxynorephedrine in rats.

The central effect of p-hydroxynorephedrine (OH-NE), one of the p-hydroxylated metabolites of methamphetamine (MAP) and amphetamine (AMP), was investigated in rats. Locomotion and stereotypy were examined after SC injections of 0.5-5 mg/kg of MAP or 0.02-0.5 mg/kg of apomorphine (APO) in animals treated with either saline or 5-50 mg/kg of OH-NE IP 20 hr before behavioral assessment. The locomotor stimulating effect of both 0.5-2 mg/kg of MAP and 0.2 mg/kg of APO was enhanced by 5 mg/kg of OH-NE. On the other hand, 30 mg/kg of OH-NE severely suppressed the stimulating effect of MAP but had no influence on that induced by 0.2 mg/kg of APO. The stereotypy induced by 5 mg/kg of MAP or 0.5 mg/kg of APO was enhanced and prolonged in the OH-NE-treated rats. Subsequently, examinations were performed to determine whether OH-NE had any effect on the dopaminergic mechanism. Hypomotility induced by 0.02 mg/kg of APO was alleviated by 5 mg/kg of OH-NE, but was aggravated by 30 mg/kg. These results suggest that OH-NE administered prior to SC injections of MAP or APO influences their behavioral effects via the dopaminergic mechanism. The possibility that other neural mechanisms may be involved in this OH-NE-induced behavioral modification is also discussed.

Differential effects of NMDA receptor and dopamine receptor antagonists on cocaine toxicities.

Cocaine produces not only euphoric effects but also a wide range of detrimental effects, including seizures and lethality. The present study examined the involvement of the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptors and the dopamine D1 and D2 receptors in seizure activity and lethality observed following single and repeated injections of cocaine in ddY mice. Repeated injections of 60 mg/kg cocaine resulted in the development of sensitization to the convulsant effects of cocaine during an initial 3 or 4 days, followed by the development of tolerance at day 5 and day 6. Repeated injections of 90 mg/kg cocaine augmented the lethal effect of cocaine progressively over the course of treatment. Treatment with 0.1-0.4 mg/kg of the noncompetitive NMDA receptor antagonist, MK-801, prevented the development of sensitization to cocaine-induced seizures in a dose-dependent manner, and attenuated partially the cocaine-induced lethality. In contrast, treatment with 10-50 mg/kg of the dopmaine D2 receptor antagonist, sulpiride, had no effects on the development of sensitization and tolerance to cocaine-induced seizures. On the other hand, treatment with 0.1-0.5 mg/kg of the dopamine D1 receptor antagonist, SCH 23390, not only prolonged the latency to 90 mg/kg cocaine-induced seizures but also delayed the development of sensitization to the convulsant effects of cocaine. The increased lethality observed following repeated injection of cocaine was unaffected by treatment with SCH 23390, but was severely aggravated by treatment with sulpiride.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of heating on Y-chromosome detection.

The effect of heating on Y-chromosome detection was investigated. Heat-treated blood was divided into four groups according to the hemolysis pattern obtained by the coil planet centrifuge system. A significant difference between the Y-positive nuclei of males and females was observed up to group III of the hemolysis pattern, and it was possible to determine the sex of the blood donor in spite of karyolysis and degeneration of blood components. However, sex determination of blood in group IV, indicating complete hemolysis, was impossible because of overlapping between male and female Y-chromosome counts. Practical application of this sex determination method was successful even with severely burned cadavers. However, it was suggested that putrefaction together with heat damage made the identification nearly impossible.

An enzyme-linked immunosorbent assay for plasma-paraquat levels of poisoned patients.

To investigate the efficacy of medical treatments, plasma-paraquat levels were measured in patients who had ingested the liquid weedkiller, Gramoxon, containing paraquat. We determined the levels by an enzyme-linked immunosorbent assay (ELISA) using a murine paraquat-specific monoclonal antibody developed by Niewola et al. (Clin. Chim. Acta, 148 (1985) 149-156). Using this ELISA, concentrations of paraquat in the range 1.56-100 ng/ml could be measured. This assay method had good precision and was suitable for measurement of low levels of paraquat. The therapeutic treatments were most effective on the day of admission. The plasma-paraquat levels of poisoned patients decreased significantly. However, the levels tended to increase again during the pause period of the haemoperfusion and this increase was serious for fatal cases. It is clear that elimination of poison not only from blood but also from tissues is necessary to lower the mortality rate.