Rapid liquid chromatographic determination of 5-hydroxyindoles and dihydroxyphenylacetic acid in cerebrospinal fluid of the rat

A stainless steel guide was implanted in the anterior third ventricle of the anesthetized rat and an internal needle shorter than the guide was used to continuously collect cerebrospinal fluid (CSF) at a constant outflow of 1 microliter/min. Five microliter samples were injected directly into a liquid chromatographic column. The mobile phase was adjusted for selective separation of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5-HIAA). Electrochemical detection with a limit of 0.05 pmol was used, 5-HTP and 5-HT concentrations were in the 10(-8) M range in controls while DOPAC and 5-HIAA were in the 10(-7) and 10(-6) M range. Brain aromatic amino acid decarboxylase inhibition with high doses of benserazide corresponded to an increased CSF level of 5-HTP. Monoamine oxidase inhibition with tranylcypromine resulted in a diminution of DOPAC and 5-HIAA. L-Tryptophan loading associated with monoamine oxidase inhibition induced an increase in CSF level of serotonin. These pharmacologically induced changes in serotonin and dopamine metabolite levels exemplify the usefulness of these CSF determinations as indices of brain function.     

The influence of dichlorvos from strips or sprays on cholinesterase activity in chicken

Chickens were exposed to dichlorvos vapour from strips continually or intermittently for three weeks. During exposure dichlorvos in air and cholinesterase activity in plasma were determined. At the end brain-acetyl-cholinesterase activity was assayed.The chicken exhibits a sensitivity towards dichlorvos comparable with man. With plasma cholinesterase activity as a criterion, a single strip was found to produce just no effect if exposure was uninterrupted or lasted 16 h every day. Comparison with dichlorvos aerosol sprays showed that excessive spraying did not produce consistent effects after 5 days. In an experiment of longer duration a decrease of plasma cholinesterase activity was found. With sprays, more dichlorvos was needed to produce the same exposure than with strips.     

d-Cysteine as a selective precursor for inorganic sulfate in the rat in vivo: Effect of d-cysteine on the sulfation of harmol

d-Cysteine, the unphysiological isomer of the sulfur containing amino acid l-cysteine, is not utilized for protein synthesis, glutathione synthesis or taurine production; it was tested as a selective precursor for inorganic sulfate, required for sulfation of xenobiotics. Both cysteine isomers were injected intravenously in the rat, in order to investigate their sulfoxidation to inorganic sulfate. The rates of sulfoxidation were very similar, so that stereospecificity for the amino acid seemed not to play a role. When the rats were fed a low-protein diet (LP-diet; containing only 8% casein as source of amino acids) the serum sulfate concentration decreased to about 20% of control. Under these circumstances the rate of sulfoxidation of both isomers was decreased to the same extent. In order to confirm that both cysteine isomers were equally efficient in providing inorganic sulfate for sulfation of xenobiotics, a constant infusion of harmol (a substrate for sulfation) was given to rats fed the LP-diet. Administration of l- or d-cysteine yielded similar increases in sulfation of harmol under these conditions. These results show that d-cysteine can be used to selectively enhance sulfate availability.     

The relation between the sex-dependency of type I binding of ethylmorphine and the 1-butanol-induced spectral change in mouse liver microsomes

1. A sex difference in the spectral interaction of 1-butanol with liver microsomes from adult mice was observed. In males a profound reverse type I spectrum was elicited, whereas only a small spectral change of irregular shape was apparent in females. This sex difference is the opposite of that observed in the type I binding of ethylmorphine. In immature animals no sex difference was found. Testosterone pretreatment of female mice increased the size of the 1-butanol spectrum concomitantly with a decrease in ethylmorphine binding. 2. Microsomes from males and females did not contain different levels of endogenous substrates. Thus, the presence or displacement of such substrates does not explain the sex differences in type I and reverse type I binding respectively. 3. 1-Butanol was found to interfere with both type II and type I binding. It is concluded that the 1-butanol-induced spectral change consists of at least two components and that the sex difference is due to a sex-dependent type I component.     

Antidepressant drugs normalize the increased social behaviour of pairs of male rats induced by short term isolation

In a situation in which a short-term isolated (I) and a group-housed (S) rat were placed together, the influence of various drugs on social behaviour was analysed. It was found that a single intraperitoneal injection of 7.5 mg/kg of the antidepressant drugs clomipramine. nortriptyline and mianserine normalized the increased social interactions of the isolated rat to the level of the group-housed rat, without affecting the social behaviour of that animal. This action of the drugs was not due to changes in locomotor activity. An opiate (morphine), an opiate antagonist (naloxone), a tranquillizer (diazepam), a neuroleptic (haloperidol) and a psychostimulant (amphetamine) did not preferentially influence the social behaviour of the isolated rat. Chronic treatment with the antidepressants did not reduce the increased social interactions of isolated animals. In spite of this it is clear that the increased social behaviour of short-term isolated rats was specifically affected by the antidepressant drugs. This suggests that this behavioural procedure might be useful for predicting antidepressant activity.     

Organ distribution and cellular uptake of methyl mercury in the rat as influenced by the intra- and extracellular glutathione concentration

Intravenous administration of CH₃HgCl (4 μmole/kg) premixed with glutathione or cysteine (8 μmole/kg) to female rats caused a rapid uptake of mercury in the kidney and a depressed content in the liver and blood as compared to CH₃HgCl given alone. GSH depletion in the tissues, produced by injection of diethylmaleate, DEM (3.9 mmole/kg) did not influence the kidney uptake of mercury from administered CH₃Hg⁺-GSH, whereas the uptake of injected CH₃HgCl was depressed. Both GSH and cysteine (8 μmole/kg) promoted the biliary excretion of methyl mercury. In suspensions of rat erythrocytes and isolated hepatocytes, additions of GSH reduced the cellular uptake of CH₃Hg⁺ from the medium, whereas this was increased in the hepatocytes by adding cysteine or methionine. Cysteine addition slightly reduced the uptake of CH₃Hg⁺ in the erythrocytes. GSH-depletion as obtained by DEM pretreatment of the cells, reduced the CH₃Hg⁺ uptake into hepatocytes by 40%, in contrast to only a negligible effect on the erythrocytes. Our results support previous reports that a physiological CH₃Hg⁺-GSH-complexation takes place intracellularly, at least in liver cells. Our results are furthermore consistent with the assumption that biliary excreted CH₃Hg⁺-GSH, which can be reabsorbed, only to a limited extent is taken up by the liver, whereas this GSH-complexation and reabsorption is of importance for the CH₃Hg⁺-uptake in the kidneys.     

Interaction of aromatic aldehydes with isolated rat liver mitochondria

It has been suggested that aromatic aldehydes may reduce cytochrome c [Wolf et al. Fedn Proc.39 (3), 1013 (1980)]. Therefore, interaction of the aromatic aldehydes, p-anisaldehyde, benzaldehyde, p-tolualdehyde, p-carboxybenzaldehyde, p-chlorobenzaldehyde and p-nitrobenzaldehyde, with rat liver mitochondria was examined in vitro. Although both pyruvate/malate- and succinate-mediated respiration, as well as that mediated by other citric acid cycle intermediates, were inhibited by the aromatic aldehydes (0.5 to 1.0 mM), cytochrome c oxidase was not inhibited by aromatic aldehydes (1.0 to 20 mM). There was a marked inhibition of suecinic dehydrogenase and both ADP- and DNP-stimulated respiration by benzaldehyde (2 to 20 mM). Since both pyruvate/malate- and succinate-mediated respiration were inhibited by the aromatic aldehydes without inhibition of cytochrome c oxidase, several sites of inhibition, possibly both at the site of transport of substrates and the active enzymes, may exist. Benzaldehyde, 300 μM, inhibited pyruvate/malate-mediated state 3 respiration by 50% which suggests that no additional functional group or metabolism to another species is required for these inhibitory effects.     

Induction of porphyria in the rat by chronic versus acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin

Chronic oral administration of 1 μg. kg^?1. week^? of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to female rats for 16 weeks resulted in hepatic porphyria. In contrast, administration of single oral doses as high as 30 μg/kg did not produce porphyria, either acutely or 16 weeks later. Activities of hepatic drug-metabolizing enzymes [aryl hydrocarbon hydroxylase (AHH) and glucuronyl transferase] were increased by chronic oral doses of TCDD as low as 0.01 μg. kg^?1. week^?. When animals were dosed with TCDD chronically and then allowed to recover for 6 months, AHH and glucuronyl transferase activities returned toward normal (98 and 86% recovery). However, animals showed only partial recovery from TCDD-induced porphyria. Hepatic porphyrin levels did decrease during this period, but urinary porphyrins and the rate-limiting enzyme in porphyrin synthesis, δ-aminolevulinic acid synthetase, remained maximally elevated during the 6-month recovery period. It is concluded that single doses of TCDD do not produce porphyria in the rat, but that TCDD is porphyrogenic when given chronically. Moreover, when TCDD administration is stopped, recovery from the porphyrogenic effects of TCDD is very slow and does not correlate with the biological half-life of TCDD in the rat.     

The influence of disulfiram and other inhibitors of oxidative metabolism on the formation of 2-hydroxyethyl-mercapturic acid from 1,2-dibromoethane by the rat

The mercapturic acid derivative, $\text{N-}\text{acetyl}\text{-S-2-}\text{hydroxyethyl}\text{-}\text{l}\text{-}\text{cysteine}$, is a major metabolite of 1,2-dibromoethane in vivo. This compound can be formed via two pathways, both involving a potentially dangerous reactive intermediate. One way involves the intermediacy of bromoacetaldehyde, formed by microsomal oxidation, followed by loss of hydrogen bromide. The second pathway, direct conjugation of 1,2-dibromoethane with glutathione, gives rise to S-2-bromoethyl glutathione. Using several inhibitors of microsomal mixed function oxidases, it was found that under these conditions about 10% of the mercapturic acid derivative formed via direct conjugation.Disulfiram, an inhibitor of aldehyde dehydrogenases, but also of microsomal oxidation, also markedly inhibits the excretion of the mercapturic acid, after administration of a single high dose (1 g/kg) or upon chronic treatment with a low dose (50 mg/kg). The inhibitory effect is maximal after 10 days of chronic treatment. Administration of large amounts of 1,2-dibromoethane (>0.20 nmole/rat) following a single lower dose of disulfiram (125 mg/kg) also leads to a lower excretion of mercapturic acid metabolite a phenomenon associated with a decrease in cytochrome P-450 levels. From these results it is concluded that the enhanced carcinogenic effect of the combination disulfiram (chronic)/1,2-dibromoethane is not caused by bromoacetaldehyde, since its formation is completely inhibited under these conditions, but by S-2-bromoethyl-glutathione, although a role for 1,2-dibromoethane itself cannot be excluded.     

The use of competitive inhibition of substrate-binding to cytochrome P450 in the determination of spectral dissociation constants for substrates with multiple types of binding,as illustrated with 1-butanol

A method is described, which allows (a) the detection of competitive inhibition of binding to cytochrome P450 between two substrates which elicit the same type of spectral change, and (b) the estimation of dissociation constants for one type of spectral binding of a substrate which exhibits multiple interaction with cytochrome P450. This method was used to investigate the interaction of 1-butanol with the type I binding site of cytochrome P450 in liver microsomes from female mice. 1-Butanol was found to competitively inhibit the binding of ethylmorphine, and has an apparent dissociation constant for type I binding of 30 mM.     

Inhalation toxicity of acetaldehyde in rats. I. Acute and subacute studies

The 4-h LC₅ of acetaldehyde in rats was determined and found to be 13 300 ppm (24.0 g/m³ air). In a 4-week study groups of 10 male and 10 female rats were exposed to 0, 400, 1000, 2200 or 5000 ppm acetaldehyde for 6 h/day, 5 days/week. Treatment-related changes observed at the 5000 ppm level included dyspnoea and excitation during the first 30 min of each exposure, yellow-brown fur, severe growth retardation, more neutrophils and less lymphocytes in the blood, a reduced productioon of urine with a high density, increased lung weights, and severe degenerative, hyperplastic and metaplastic changes of the nasal, laryngeal and tracheal epithelium. Major lesions seen at 1000 and 2200 ppm comprised growth retardation and an increased production of urine in males, slight to moderate degeneration with or without hyper- and metaplasia of the nasal epithelium, and only at 2200 ppm, minimal epithelial changes in the larynx and trachea. The only change observed at the 400 ppm level that could be attributed to acetaldehyde was slight degeneration of the nasal olfactory epithelium seen as loss of microvilli and thinning and disarrangement of the layer of epithelial cells.     

Inhibition of centrally-evoked pressor responses by neurohypophyseal peptides and their fragments

Intracerebroventricular administration of fragments of [arginine⁸]-vasopressin (AVP) such as AVP_1–6 and AVP_7–9 attenuated the pressor response evoked by electrical stimulation of the mesencephalic reticular formation in urethane-anaesthetized rats. Oxytocin (OXT) and the fragment OXT_7–9 were also active, although OXT_1–6 did not affect the pressor response. These peptides did not influence the bradycardia accompanying the rise in blood pressure, nor the basal blood pressure. The inhibition of the pressor response was shown for OXT_7–9 to be dose-dependent up to 25 ng. These data suggest that oxytocin, vasopressin and some neuropeptide fragments have an inhibitory role in the regulation of blood pressure. Both the covalent pressinoic ring structure and the C-terminal linear portion of vasopressin contain active sites, while the activity of oxytocin appears to be present in the C-terminal tripeptide Pro-Leu-Gly.     

An evaluation of methods to decrease the availability of inorganic sulphate for sulphate conjugation in the rat in vivo

The concentration of inorganic sulphate in serum of the rat (about 0.9 mM) could be lowered in the following three different ways. (1) Oral administration of sodium chloride (8 mmol/kg) decreased serum sulphate within 2 hr to 0.5 mM. Eight hours after administration serum sulphate had returned to the control level. (2) Feeding of a low-protein diet (8 per cent casein, without supplements of sulphur-containing amino acids or inorganic sulphate salts) reduced urinary sulphate excretion in 2 days to 10 per cent of control. Concomitantly, serum sulphate was decreased to half the control level. (3) Paracetamol (1.0 or 1.5 mmol/kg, orally), a substrate of sulphation, reduced serum sulphate within 3 hr to 30 per cent of control. Eight hours after administration the sulphate concentration tended to rise again. Fasting initially increased serum sulphate; after 3 days of fasting still considerable amounts of inorganic sulphate were excreted in urine (50–70 per cent of control). Even after 3 days serum sulphate was not yet significantly decreased below control. Lowering of the serum sulphate concentration results in a decreased availability of inorganic sulphate.Sulphation of a high dose of phenol (266 μmol/kg) was decreased at a serum concentration of sulphate of 0.3 mM, presumably because sulphate was depleted by the high dose of phenol. Feeding the low-protein diet, however, caused no decrease in sulphation at a tracer dose of [¹⁴C]-phenol (1.25 μmol/kg), while paracetamol pretreatment did cause a decrease in the fraction of the dose that became sulphated, probably because remaining unconjugated paracetamol competed with phenol for sulphation; the tracer dose of [¹⁴C]-phenol did not further deplete sulphate. The findings are discussed in relation to implications for the toxicity of many xenobiotics that are eliminated as sulphate conjugates.     

The development of sex differences in the demethylation of ethylmorphine and in its interaction with components of the hepatic microsomal cytochrome P450 system in mice.

The development of sex differences in ethylmorphine N-demethylation and several components of the reaction chain were studied in hepatic microsomes from mice of the CPB-SE strain between 3–11 weeks of age. Sex-specific changes were observed in demethylation rate, type 1 spectral interaction, cytochrome P450 content, and ethylmorphine-induced stimulation of NADPH-cytochrome P450 reductase activity. These changes occurred mainly between weeks 3–7 and were confined to females. It is concluded that the development of the cytochrome P450 system is repressed by androgen during sexual maturation. The kinetic constants of demethylation developed differently from ethylmorphine binding constants. Changes in demethylase were mainly restricted to K_m, whereas the changes in type 1 binding only involved the maximum spectral change. In combination with differences observed between the developmental patterns of demethylation rate and cytochrome P450 reductase activities, this demonstrated that the reduction of cytochrome P450-substrate complex is not rate-limiting in ethylmorphine demethylation. The type 1 spectral change was correlated with the amount of cytochrome P450 only when a large portion of the cytochrome was considered inactive in ethylmorphine binding. It is suggested that immature animals possess a low basal level of ethylmorphine binding type 1 sites, which is elevated selectively in females during sexual maturation.     

Sites of action of Ca2+ channel inhibitors

Ca2+ channel inhibitors are viewed as a subgroup of Ca2+ antagonists. Most of the currently used Ca2+ channel inhibitors are thought to act by reducing Ca2+ entry into the cell through Ca2+ channels. There is substantial electrophysiological evidence that the major site of action of verapamil, nifedipine and diltiazem in cardiac cells is a sarcolemmal Ca2+ channel. Cytosolic sites of action may contribute to their effects but probably only at higher than therapeutic concentrations. The recent ligand binding studies also tend to support the view that the sarcolemma is the site of action of Ca2+ channel inhibitors in smooth muscle. High affinity binding sites for 1,4-dihydropyridines without any established function are found in fast skeletal muscle and some neuronal membranes. The binding of [3H]nitrendipine to membranes from cardiac, skeletal and smooth muscle, and from brain is saturable, reversible and of high affinity; it is sensitive to cations and other drugs that interact with Ca2+ channels. Inhibition of [3H]nitrendipine binding and blockade of K+ responses in guinea pig ileum by 1,4-dihydropyridines are well correlated, supporting the view that the observed binding is to Ca2+ channel. In contrast, blockade of Ca2+ channels in cardiac and skeletal muscle and in brain synaptosomes occurs only at higher concentrations than needed to saturate the high affinity binding sites. The therapeutic success of Ca2+ channel inhibitors in the treatment of angina pectoris, hypertension, peripheral vascular diseases, and many other disease entities is based on selective inhibition of Ca2+ entry into smooth muscle cells. The specificity of some of these drugs for Ca2+ channels in different cell types, organs, or vascular beds is probably determined by receptor modulation and the effect of reflex mechanisms, which in turn determine the indications for their therapeutic use.     

Effects of phenobarbital and diphenylhydantoin on acute vitamin D3 toxicity in the rat.

Methylene chloride is biotransformed in vivo to carbon monoxide (CO), and increased carboxyhemoglobin (COHb) concentrations occur in animals and man following exposure. Binding of CO to hemoglobin causes a shift to the left in the position of the oxyhemoglobin dissociation curve (ODC), theoretically resulting in a diminished ability to release oxygen in tissue. This study was designed to determine the effect of methylene chloride alone and in combination with CO on altering the ODC. Methylene chloride and CO, alone and in combination, were incubated with rat and human blood in vitro and the position of the ODC was measured by the P50 value. The results indicate clearly that methylene chloride alone had no effect on changing the P50 value of the ODC. It was concluded that any shift to the left of the ODC following exposure to methylene chloride is due solely to CO formed from the biotransformation of methylene chloride. Thus, speculation that methylene chloride increases the affinity of Hb for CO is not supported.     

Stimulation of Na+,K+-ATPase of isolated smooth muscle membranes by the Ca2+ channel inhibitors,nimodipine and nitrendipine

Nimodipine (0.015 to 1.5 μM) increased Na⁺, K⁺-ATPase activity by 70–120% in isolated smooth muscle membranes. At 0.015 μM, nitrendipine, but not nifedipine, verapamil or diltiazem, also activated this enzyme. Nimodipine stimulated this Na⁺, K⁺ATPase three times more than nitrendipine at 15 nM. Marked stimulation of Na⁺,K⁺-ATPase by nimopidine was seen in membranes from rat and guinea pig aorta and rat vas deferens, but not in membranes from guinea pig heart or brain. Although it is not known whether these results are applicable to intact cells, the results are consistent with the hypothesis that vasodilation produced by nimodipine and nitrendipine may be due not only to inhibition of Ca²⁺ entry but also to the stimulation of the Na^? pump.     

A biochemical study of the cotton pellet granuloma in the rat: Effects of dexamethasone and indomethacin

The subcutaneous implantation of a cotton pellet into a rat results in the formation of a granuloma at the site of the implant. The early events comprise an accumulation of fluid and proteinaceous material together with an infiltration of neutrophils. The granuloma formed by day 7 is characterized by the formation of a vascularized fibrous capsule containing fibroblasts and infiltrating mononuclear cells which are rich in $\text{N-}\text{acetyl}\text{-β-}\text{d}\text{-glucosaminidase}$ (NAG). Granuloma development was quantitated by dry weight measurements, and its cellular content was measured by assaying activity of NAG and total nucleic acid content. Nucleic acid determinations showed that cell infiltration into the granuloma took place at a virtually constant rate over a 7-day period. In contrast, the NAG activity did not change significantly until after day 5 when a large increase in the amount of enzyme extractable from the granuloma was seen. Systemic treatment of the animal with dexamethasone or indomethacin resulted in an inhibition of granuloma weight gain, NAG activity and nucleic acid levels. The data suggest that the two drugs acted during the early phase of granuloma development at the level of cell infiltration. Both drugs given on days 0–3 alone suppressed granuloma formation, whereas treatment on days 4–7 was without effect.     

Selective induction of renal microsomal cytochrome P-450-linked monooxygenases by 1,1-dichloroethylene in mice

Intraperitoneal administration of a single dose of 1,1-dichloroethylene (DCE) to C57 B1/6N mice (125 mg/kg) caused a selective 6- to 10-fold increase in renal microsomal 7-ethoxyresorufin O-deethylase ( EROD ) and 7-ethoxycoumarin O-deethylase ( ECOD ), without affecting benzo[a]pyrene hydroxylase activity (AHH) or total microsomal cytochrome P-450 content. The observed increases did not result from in vitro activation of the enzymes or from any analytical artifact. Moreover, studies with actinomycin D and cycloheximide demonstrated that the increases resulted from de novo enzyme synthesis. Maximal enzyme induction was observed after a DCE dose of approximately 125 mg/kg, and the induced enzyme decayed rapidly, returning to control levels in about 3 days. Compared to female mice, male mice had higher basal levels of renal EROD and ECOD and were more responsive to the inductive effects of DCE; this correlated with corresponding differences in microsomal cytochrome P-450 levels. Starvation of mice for 24 or 48 hr increased renal EROD and ECOD activities in both male and female mice, but not the extent observed after DCE. The present results support the view of multiple renal cytochrome P-450 isozymes.