Ketamine inhibits serotonin uptake in vivo

Anesthetic (120 and 160 mg/kg. i.p.) and subanesthetic (80 mg/kg) doses of ketamine HCl were found to prevent completely the depletion of whole brain serotonin (5-HT) by p-chloramphetamine (PCA). Furthermore, ketamine HCl (160 mg/kg) completely blocked the depletion of 5-HT by PCA in every individual brain region studied (Midbrain-thalamus, hypothalamus, striatum, hippocampus and cortex). Administration of ketamine alone had no effect on brain 5-HT levels. Nialamide (a monoamine oxidase (MAO) inhibitor) and fluoxetine (a selective 5-HT uptake inhibitor) also prevented the depletion of 5-HT by PCA. However, of these three agents, only nialamide prevented the depletion of 5-HT by reserpine. These results suggest that ketamine blocks PCA-induced 5-HT depletion by inhibiting 5-HT uptake and not by inhibiting MAO. Ketamine only weakly affected either [3H]5-HT or [3H]spiroperidol binding to 5-HT1 and 5-HT2 receptors respectively even at concentrations as high as 1 mM. These data support the contention that the primary direct effect of ketamine on serotonergic systems is the blockade of 5-HT uptake and that blockade of 5-HT uptake may mediate some of the behavioral effects of ketamine, such as analgesia.     

Modulation of fluoropyrimidine metabolism in L1210 cells by L-alanosine

L-Alanosine, an analogue of aspartic acid which inhibits the conversion of inosine monophosphate to adenosine monophosphate (AMP), was evaluated in L1210 cells as a modulator of 5-fluorouracil (FUra) and 5-fluorouridine (FUrd) metabolism. L-Alanosine resulted in increased intra-cellular levels of 5-phosphoribosyl-1-pyrophosphate (PRPP), enhanced FUra metabolism to ribonucleotide derivatives, and resulted in more FUra residues incorporated into RNA. Sequential addition of L-alanosine and FUra also resulted in synergistic cytotoxicity as determined by soft agar cloning. Adenine antagonized these biochemical and biological effects of L-alanosine. L-Alanosine also augmented the rate at which FUrd was metabolized and was also associated with a greater incorporation of the FUra residues into RNA. Cytotoxicity after sequential L-alanosine and FUrd was also synergistic. The mechanism by which L-alanosine altered the metabolism of FUrd, however, was different from the way in which it enhanced FUra metabolism in that aspartic acid and not adenine was able to reverse the effects of L-alanosine on FUrd metabolism and cytotoxicity. L-Alanosine appeared to augment the RNA-directed activity of FUra and FUrd in that there was no correlation between the enhanced metabolism and cytotoxicity of these two fluoropyrimidines and either levels of fluorodeoxyuridylate (FdUMP) which inhibits thymidylate (TMP) synthetase or inhibition of the ability of cells to incorporate deoxyuridylate into acid-precipitable material.     

Examination of the metabolism in vitro of parathion (diethyl p-nitrophenyl phosphorothionate) by rat lung and brain.

These studies have indicated there is present in rat lung microsomes a mixed-function oxidase enzyme system capable of metabolizing parathion to paraoxon and to diethyl phosphorothioic acid. In addition, analogous to previous results using rat liver microsomes, the sulfur atom released in the metabolism of parathion to paraoxon was found to covalently bind to lung microsomes. In contrast to liver, the metabolism of parathion by rat lung microsomes is not inducible by pretreatment of the animals with phenobarbital or 3-methylcholanthrene. The metabolism of parathion by lung microsomes is stimulated by NADPH and oxygen and is inhibited by carbon monoxide, anaerobic conditions, SKF-525A and piperonyl butoxide. There is also present in a particulate fraction of rat brain equivalent to microsomes an enzyme or enzyme system capable of metabolizing parathion to paraoxon and to diethyl phosphorothioic acid. The metabolism of parathion to paraoxon by rat brain microsomes is also accompanied by the release and covalent binding of the sulfur atom of parathion. The activity in rat brain microsomes is stimulated by oxygen and NADPH and inhibited by carbon monoxide, anaerobic conditions, SKF-525A and piperonyl butoxide. These data suggest that cytochrome P-450-containing mixed-function oxidase enzyme systems are responsible for the NADPH-stimulaled catalytic activity toward parathion found in rat lung and rat brain microsomes.     

2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated depression of rat testicular heme synthesis and microsomal cytochrome P-450

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces hirsutism, alopecia, and chlorance, symptoms that suggest a possible alteration of endocrine function. Therefore, the effects of TCDD on rat testicular cytochrome P-450 content were investigated. Forty-eight hours after a single, oral dose of TCDD (25 μg/kg) testicular microsomal cytochrome P-450 levels were depressed by approximately 24%. Microsomal cytochrome P-450 continued to decrease to 62% of control levels at 4 days and remained at approximately the same levels 7 days following treatment. Testicular microsomal heme content exhibited a similar pattern after administration of TCDD. No alterations in testicular δ-aminolevulinic acid (ALA) synthase were detected. The incorporation of [¹⁴C]ALA into microsomal heme was decreased to approximately 36% of control values at 24 hr after TCDD administration. Testicular weights were not altered during the 7-day experimental period. These data suggest that TCDD depresses cytochrome P-450 levels in the rat testis through an inhibition of the synthesis of testicular heme.     

Nitrilotriacetate (NTA): human metabolism and its importance in the total safety evaluation program

Ten milligrams [¹⁴C]NTA in gelatin capsules was given (po) to 8 male subjects. Blood samples were taken, and samples of urine and feces were collected. In addition, expired CO₂ was collected in 4 of the subjects. NTA is poorly absorbed since approximately 12% of the dose appeared in the urine. The compound is rapidly excreted in the urine since 87% of the absorbed dose was excreted within the first 24 hr post dosing. The blood concentration peak occurred 1–2 hr after dosing. Reverse isotope dilution and thin-layer chromatography showed the urinary radioactivity to be unchanged NTA, and therefore no biotransformation had occurred. These results closely resemble the rat and dog metabolic data except that rats and dogs absorb 4 times more NTA than humans. The metabolic similarities among rats, dogs and humans lends some confidence to the extrapolation of the results derived from rat and dog toxicity tests for the purpose of estimating human safety.     

Comparison of the effects of methyl-N-butyl ketone and phenobarbital on rat liver cytochromes P-450 and the metabolism of chloroform to phosgene

It was previously shown that treatment of rats with methyl-n-butyl ketone (MBK) produced an increase in the total level of liver microsomal cytochromes P-450 and an increase in the rate of metabolism of chloroform (CHCl3) to phosgene (COCl2). In the present study it was found that MBK also produced qualitative changes in the composition of microsomal cytochromes P-450 in rat liver as determined by anion-exchange chromatography. The degree of the chromatographic changes paralleled the effect of MBK on the rate of metabolism of CHCl3 to COCl2 and CHCl3-induced hepatotoxicity, suggesting that MBK potentiated the hepatotoxicity of CHCl3, at least in part, by inducing the formation of cytochromes P-450 that metabolized CHCl3 to the hepatotoxin COCl2. In this regard, reconstitution studies with a form of cytochrome P-450 isolated from rat liver microsomes from rats treated with MBK or phenobarbital (Pb) showed unequivocally that cytochrome P-450 can metabolize CHCl3 to COCl2. Although analysis of rat liver microsomes by SDS-polyacrylamide electrophoresis and anion-exchange chromatography suggested that MBK and Pb had similar effects on the composition of cytochromes P-450, metabolism studies indicated that differences did exist.     

Inhibition of rat testicular heme synthesis and depression of microsomal cytochrome P-450 by estradiol benzoate

Twenty-four hours after a single dose (50 μg, s.c.) of estradiol benzoate (EB), rat testicular microsomal heme and cytochrome P-450 were decreased to 72 and 76% of control levels respectively. Treatment of rats with human chorionic gonadotropin (hCG) resulted in elevated levels of microsomal heme and cytochrome P-450 and increased activity of δ-aminolevulinic acid (ALA) synthase (EC 2.3.1.37). However, the hCG-mediated elevations of testicular microsomal heme and cytochrome P-450 content failed to occur in animals treated with EB. To investigate the possibility that the observed effect of EB was mediated through the pituitary, studies were conducted with hypophysectomized animals. The increased microsomal heme and cytochrome P-450 content mediated by hCG in hypophysectomized animals was again prevented by administration of EB. The elevated activity of testicular mitochondrial ALA synthase produced by hCG in both intact and hypophysectomized animals was not affected by EB. Incorporation of [¹³C]ALA into microsomal heme was depressed 60% 12 hr following a single dose of EB (50 μg, s.c.). These data suggest that EB depresses testicular microsomal heme and cytochrome P-450 content by inhibiting the synthesis of heme at an enzymatic reaction other than ALA synthase.     

2-Acetylaminofluorene metabolism in rat liver microsomes: formation of 9-hydroxy-2-acetylaminofluorene and effect of hepatic enzyme modifiers.

9-Hydroxy-2-acetylaminofluorene [N-(9-hydroxy-9H-fluoren-2-yl)acetamide; 9-OH-2-AAF] was isolated and identified as a metabolite of 2-acetylaminofluorene (2-AAF) in rat liver microsomes. Evidence for this metabolite was provided by thin-layer chromatography (tlc), inverse isotope dilution analysis, pH partition, and nmr spectroscopic studies. 9-Oxo-2-acetylaminofluorene [N-(9-oxo-9H-fluoren-2-yl)acetamide; 9-OXO-2-AAF] was also detected and identified (by tlc) as a microsomal metabolite of 2-AAF. Microsomal conversion of 2-AAF to 9-OH-2-AAF and 9-OXO-2-AAF was dependent upon the presence of NADPH and was inhibited by a carbon monoxide atmosphere. Increased formation of 9-OH-2-AAF was noted in microsomes obtained from rats treated with the hepatic enzyme modifiers, phenobarbital and pregnenolone-16α-carbonitrile and inhibited in the presence of metyrapone. In contrast, treatment with 3-methylcholanthrene or chlordane did not modify 9-OH-2-AAF or 9-OXO-2-AAF formation. Also, rates of 9-OXO-2-AAF formation were increased only by prior administration of phenobarbital and were unaffected by metyrapone. These data provide evidence for the involvement of specific types of cytochrome P-450 in the 9-carbon hydroxylation reaction of 2-AAF.     

The relationship between binding to cytochrome P-450 and metabolism of n-alkyl carbamates in isolated rat hepatocytes

The binding constants of an homologous series of n-alkyl (C₂-C₁₀) carbamates

to the cytochrome P-450 of suspensions of isolated, viable rat hepatocytes have been measured. All the carbamates except ethyl and propyl carbamate produced type I difference spectra and their binding affinities (1/K_s) were found to be directly dependent upon their lipophilicity. These binding affinities were similar to those determined in rat liver microsomes. Maximum development of the binding spectrum in hepatocytes was always within one second of the addition of each carbamate, indicating that for these carbamates membrane permeability was not rate limiting for access to, and metabolism by, cytochrome P-450 and that much of the cells' cytochrome P-450 was unoccupied by endogenous substrates. The major metabolites of C₄-C₈ carbamates were unconjugated ω-1 oxidation products. Below hexyl carbamate only the ω-1 hydroxylated metabolite was observed but for the more lipophilic carbamates the keto metabolite was also a major product. The same products were found in blood after i.p. dosing of rats with hexyl carbamate. A direct relationship was observed between the affinity constant of the carbamate for cytochrome P-450 and the total rate of oxidative metabolism in the ω-1 position. Hydrolysis of the carbamate group was a minor metabolic pathway in contrast to the in vivo situation.     

Analysis and differentiation of mechanism of tolerance to methadone in methadone-fed rats.

Rats fed methadone in drinking water (0.5 mg/ml) for 1 or 2 weeks developed tolerance to the analgesic effect of methadone as measured by the hot-plate method. This decreased analgesia was accompanied by an increased methadone N-demethylase activity measured in vitro and an increased methadone metabolism in vivo as judged by increases in the percentages of total ¹⁴C as ¹⁴C-water-soluble metabolites in the liver and urine 3 hr after administration of [¹⁴C]methadone. Two-day oral methadone administration and 4-day phenobarbital pretreatment also increased methadone metabolism both in vitro and in vivo and decreased the analgesic effect of methadone but not of morphine, indicating that increased methadone metabolism contributed to the development of tolerance to methadone analgesia (dispositional tolerance). Methadone-fed rats also developed cross-tolerance to morphine-induced analgesia. Two-week feeding caused tolerance to the analgesic effects of methadone and morphine to a greater degree. However, there was no significant difference between the increases in methadone metabolism produced by 1- and 2-week feedings. These results indicate that dispositional tolerance did not account for all of the tolerance seen after chronic methadone feeding. This suggests that chronic methadone feeding also caused cellular adaptive tolerance. This suggestion is further supported by the evidence that 4-day phenobarbital pretreatment, enhanced methadone metabolism more than 2-week methadone feeding, but the decrease in methadone analgesia caused by phenobarbital pretreatment was not as great as that caused by 2-week methadone feeding. It is concluded that tolerance to methadone analgesia as a result of chronic methadone feeding arises due to both increased metabolic inactivation of methadone and cellular adaptation to the drug in the brain.     

Inductive effect on hepatic enzymes and acute toxicity of individual polychlorinated dibenzofuran congeners in rats

Toxicological assessment of 13 individual polychlorinated dibenzofurans (PCDFs) with varying chlorine substitutions was made in young male Wistar rats. All the 9 congeners having at least three chlorine atoms in the ring positions at 2, 3, 7 and 8, such as 1,2,7,8-, 2,3,6,7-, and 2,3,7,8-tetrachlorodibenzofurans (TCDFs), 1,2,3,7,8-, 1,2,6,7,8-, 2,3,4,7,8-pentachlorodibenzofurans (PenCDFs), 1,2,3,4,6,7- and 1,2,3,4,7,8-hexachlorodibenzofurans (HCDFs), exhibited a typical 3-methylcholanthrene (MC)-type induction, i.e., a marked increase in activity of aryl hydrocarbon hydroxylase (AHH) and DT-diaphorase in the liver. The most potent congeners were 2,3,7,8-TCDF and 2,3,4,7,8-PenCDF, both of which significantly induced the AHH and DT-diaphorase even at a single dose of 1 μg/kg. On the contrary, the congeners having two or less chlorine atoms in the lateral ring positions, such as 2,8-dichlorodibenzofuran, 1,3,6,7- and 1,3,6,8-TCDFs, and 1,2,4,6,8-PenCDF, did not show any inductive effect on these hepatic enzymes at the single dose of 5 or 10 mg/kg. All the MC-type PCDFs except for 1,2,7,8-TCDF caused a marked atrophy of the thymus and a hypertrophy of the liver in rats, while no toxic signs were observed in animals treated with the congeners lacking the MC-type inducing ability. The ranking of toxic potencies of the MC-type PCDFs coincided well with their inducing abilities. The hepatic disposition of the congeners varied markedly. For example, 2,3,7,8-TCDF and 2,3,4,7,8-PenCDF showed an equipotent toxicity, however, the hepatic concentration of PenCDF was about 20-fold higher than that of TCDF. These results clearly indicate that the acute toxicity of PCDF congeners is well correlated with their MC-type inducibility, but not with their hepatic distribution.     

Interference of thiosulfate with potentiometric analysis of cyanide in blood and its elimination

Studies with cyanide in combination with various antidotal regimens indicated that sodium thiosulfate interfered with the potentiometric determination of cyanide. The basis for this interference is ascribed to an enhanced biotransformation of thiosulfate in the presence of blood. A microdiffusion technique, coupled with a silver/sulfide ion-specific electrode, caused falsely elevated cyanide levels when samples contained thiosulfate. The contaminant causing the falsely elevated cyanide level is believed to be sulfide anion. This sulfide contaminant can be removed by oxidation with hydrogen peroxide, and the excess hydrogen peroxide subsequently can be eliminated with sodium sulfite. The toxicologic implication of the potentiometric determination of cyanide in the presence of sodium thiosulfate is important because sodium thiosulfate is employed widely as an antidote in cyanide poisoning.     

Effects of DDMP and DDEP on the deoxyribonucleoside triphosphate concentrations in human cells

Both DDMP and DDEP were found to lower the free deoxythymidine triphosphate concentration and raise the free deoxyadenosine triphosphate concentrations and uptake of tritiated thymidine into DNA in normal human phytohaemagglutinin-stimulated lymphocytes. With both drugs, these effects could be detected after as little as 15 min incubation and with DDMP at concentrations as low as 10^?8M and the effects could be reversed by the reduced folates 5-methyltetrahydrofolate or 5-formyltetrahydrofolate. The effects of these drugs closely paralleled those of methotrexate and show that both drugs are powerful inhibitors of dihydrofolate reductase in normal human cells.     

Malathion and phenthoate carboxylesterase activities in pulmonary alveolar macrophages as indicators of lung injury

Malathion and phenthoate carboxylesterase activities were investigated in pulmonary alveolar macrophages (PAM) in Sprague-Dawley rats. PAM was found to be capable of hydrolyzing phenthoate at a faster rate than malathion. Oral administration to rats with O,O,S-trimethyl phosphorothioate (OOS-Me), a pneumotoxic impurity present in technical grades of malathion and phenthoate, increased the activities of these esterases in PAM without affecting an activity in lung microsomal carboxylesterase. The time course study indicated that this increase was maximal on Day 1 following treatment with OOS-Me at 20 and 40 mg/kg of doses. To assess the usefulness of measuring these esterases in PAM as an indicator of lung damage, paraquat and bromobenzene were administered to rats with treatment regimens which have been shown previously to result in histopathologically demonstrable pneumotoxicity. Malathion and phenthoate carboxylesterase activities in PAM were increased by two- to threefold following treatment with paraquat or bromobenzene. These treatments also increased lung microsomal malathion carboxylesterase activity by threefold. Furthermore, infection of rats with Pseudomonas aeruginosa by intratracheal inoculation increased malathion and phenthoate carboxylesterase activities in PAM by two- to threefold without increasing these activities in lung microsomes. These results indicate that PAM may play a significant role in detoxifying airborne malathion and phenthoate when inhaled. Furthermore, the activities of malathion and phenthoate carboxylesterases may be useful for detecting lung injury produced by pneumotoxic chemicals as well as bacterial infection.     

Neurochemical and behavioural correlates of the interaction between amphetamine and Δ9-tetrahydrocannabinol in the rat

A single dose of Δ⁹-tetrahydrocannabinol (THC) (10 or 100 mg/kg) was administered via stomach gavage following the single injection intraperitoneally of an acute dose of amphetamine (1 or 3 mg/kg) in the rat. The THC effects, which included a reduction in spontaneous locomotor activity, the development of prostration, and circling behaviour, were antagonized in a dose-dependent manner by pretreatment with amphetamine. Although no change in the endogenous levels of noradrenaline, dopamine and serotonin in hypothalamus, hippocampus, caudate-putamen or the rest of brain occurred with THC, it appears that THC may have increased dopamine turnover in the brain. The dose-dependent amphetamine antagonism to the behavioural effects of THC was paralleled by a dose-related increase in brain dopamine concentration. This elevation in dopamine levels may be instrumental in triggering the neuronal events responsible for the amphetamine antagonism of the actions of THC. The subtle interaction of THC with brain dopamine neural systems may be involved in the reinforcing properties of the self-administration of THC.     

Induction of aryl hydrocarbon hydroxylase by 3-methylcholanthrene in liver,lung and kidney of gonadectomized and sham-operated wistar rats

Detailed dose-response curves have been obtained for the induction of aryl hydrocarbon hydroxylase (AHH) m liver, lung and kidney by intraperitoneally administered 3-methylcholanthrene (3-MC) in Wistar rats. The effects of gonadectomy also were studied. Lung was the tissue most sensitive to induction, followed by liver, and then kidney in all cases. Although liver exhibited the greatest overall activity, the per cent increase over control AHH levels was much higher in the two extrahepatic tissues. Gonadectomy did not affect either control or induced AHH activity in any of the three organs in the female, or in the male lung. However, castration of the males decreased control liver enzyme levels and increased these levels in kidney. The AHH levels reached after maximal 3-MC induction were the same in castrated and sham-operated male rat livers. A different pattern was seen in male kidney enzyme levels where significantly increased maximal induction was observed in castrated animals.