Molecular dosimetry of 7-alkyl- and O6-alkylguanine in DNA by electrochemical detection.

A methodology is described for the quantitation of 7-alkyl- and O6-alkylguanine in DNA isolated from experimental animals exposed to alkylating agents. Following purification, the DNA is hydrolysed under acid conditions after which 7-alkyl- and O6-alkylguanine are separated from unmodified bases by HPLC using a strong cation exchange column. The fractions containing the methylated purines are subsequently analyzed by HPLC using a reverse phase column coupled to an electrochemical detector (amperometric). This method allows the detection of 10-20 fmoles 7-alkyl- and O6-alkylguanine, when pure markers are analyzed. In practice, the detection limit is 0.5 adducts per 10(6) nucleotides for the methylated and 1 adduct per 10(6) nucleotides for the ethylated form of 7-alkyl- and O6-alkylguanine using 25 micrograms DNA.

4-[123I]iodospiperone as a ligand for dopamine DA receptors: in vitro and in vivo experiments in a rat model.

Radioiodinated spiperone is of interest for dopamine (DA) receptor studies in the living human brain by single photon emission computed tomography (SPECT). Stimulated by data obtained with [11C]-N-methyl-spiperone we synthesized 4-[123I]iodospiperone and investigated the in vitro binding characteristics of this ligand to the striatal membrane of the rat and the in vivo distribution over various rat brain regions. The in vitro binding experiments showed that this radioligand displays about 10 times less affinity for the DA receptor than spiperone and specific binding, as shown with [3H]spiperone, was not observed. Displacement by butaclamol was not observed. The in vivo studies demonstrated that both 4-[123I]iodospiperone and [3H]spiperone concentrate in striatal tissue, respectively, 1.9 and 3.5 times as high as in cerebellar tissue. Haloperidol pretreatment largely prevented this accumulation. In view of the obtained target-to-non-target ratios we believe, however, that this accumulation in brain areas rich in DA-receptors does not offer prospects for clinical receptor imaging with SPECT.

The use of microwave irradiation with low formalin concentrations to enhance the conversion of dopamine into norsalsolinol in rat brain: a pilot study.

The fixation of the neurotransmitter dopamine in the central nervous system by perfusion with formalin solutions seems to take place mainly via the formalin-induced condensation product norsalsolinol. In the present investigation the influence of microwave irradiation of the formalin-induced condensation of dopamine was studied in vitro and in vivo by making use of different, relatively low, formalin concentrations. It appeared that in vitro and in vivo the dopamine conversion was complete with 4% formalin and no influence of microwaves was noted. However, by making use of much lower formalin concentrations (0.2% and 0.4%) the condensation of dopamine was strongly augmented, in vitro (200%) and in vivo (at least 500%) using microwave techniques. There was a considerable loss in non-microwaved tissue (30%) after perfusion in vivo. This was lower (10%) in microwaved tissue. In experiments with perfused brain tissue which allowed a more complete calculation, a loss was found. This might be caused by a strong binding of dopamine and/or norsalsolinol to tissue components or to side reactions that could not be traced by the present experimental techniques.

Repeated administration of HA-966 and haloperidol to rats: similar tolerance to striatal dopamine accumulation after HA-966 challenge, but dissimilar effects on striatal [3H]spiperone binding.

Repeated administration of 1-hydroxy-3-amino-pyrrolidone-2 (HA-966) to rats induces tolerance, as shown by a decreased, drug-stimulated accumulation of dopamine (DA) in the striatum. In the present study we compared the adaptive response of the striatal dopaminergic system to repeated administration of HA-966 with the adaptive response observed after repeated haloperidol. These treatments deprive dopamine (DA) receptors from their agonist and cause a blockade of DA receptors, respectively. Tolerance to HA-966 was not accompanied by a change in the specific binding of [3H]spiperone to striatal membranes. This is in contrast to the well-documented up-regulation of DA receptors that occurs with tolerance to haloperidol. Repeated haloperidol pretreatment also diminished DA accumulation following a challenge dose of HA-966, to a similar extent as that caused by repeated pretreatment with HA-966. These similar effects of pretreatment with HA-966 or haloperidol on the response to the HA-966 challenge are in line with, and strengthen, the idea that an increased sensitivity of presynaptic DA receptors is responsible for the decreasing effect of HA-966 after its repeated administration. Haloperidol and HA-966 clearly have different effects on postsynaptic DA receptors, as is shown by their differential effects on striatal [3H]spiperone binding.

Tritium isotope effect in reversed-phase high-performance liquid chromatographic analysis of dopamine and dihydroxyphenylacetic acid.

A tritium isotope effect has been demonstrated in the high-performance liquid chromatographic analysis of dopamine and its acidic metabolite dihydroxyphenylacetic acid. The chromatographic system consisted of tributyl-n-phosphate, bound to a ChromSpher C8 column, as stationary phase, and a citrate buffer, containing the ion-pairing agent perchlorate, as the mobile phase. For detection we used continuous electrochemical monitoring (for the total amount of solutes) and discontinuous liquid scintillation counting (for radiolabelled molecules) of the column effluent. [3H]Dopamine and [3H]dihydroxyphenylacetic acid were biosynthesized by incubation of homogenates of striatal tissue from rat brains with 3H-labelled L-tyrosine. The tritium-labelled compounds were eluted before the corresponding unlabelled analogues. The capacity factor reduction increased with the number of tritium atoms incorporated in the molecules: for single, double and triple tritium-labelled dopamine the separation factors amounted to 1.015, 1.028 and 1.033, respectively. No isotope separation was observed for 7-14C-labelled dopamine and dihydroxyphenylacetic acid. The isotope effect observed is ascribed to a decrease in lipophilicity following tritium substitution for hydrogen.

Monitoring the specific activities of dopamine and its metabolites in striatum and olfactory tubercle after intravenous administration of l-[(3)H]tyrosine: Complex relations, indicating more than two compartments.

It is still a matter of debate whether in dopaminergic nerve endings dopamine (DA) is present in different functional and/or metabolic compartments. To investigate this, DA metabolism was studied in vivo by measuring the specific activity of DA and its metabolites after intravenous administration of l-[3,5-(3)H]tyrosine (200 ?Ci/rat) to freely moving animals. The incorporation of (3)H into DA and metabolites was determined in striatum and olfactory tubercle at 5, 10, 20, 40, 60 and 80 min after [(3)H]tyrosine administration. In both structures the level of [(3)H]tyrosine initially declined monoexponentially, but deviated from that pattern later on. The curves representing the formation in time of [(3)H]DA and [(3)H]metabolites were very similar in both structures, although as a whole, the levels in the olfactory tubercle were higher. The relative patterns of the specific activities of DA and those of its metabolites, a possible clue to DA compartmentation, neither indicated a clearcut metabolic one-compartment, nor a two-compartment system. The flow of radioactivity through DA metabolism could in fact only be explained by assuming more complex metabolic relations.

Effects of morphine on dopamine metabolism in rat striatum and limbic structures in relation to the activity of dopaminergic neurones.

Administration of morphine results in efflux of dopamine provided that the nerve impulse flow of the dopaminergic neurones is impaired. In the present study we investigated whether the morphine-induced increase in dopamine metabolite levels is related to impulse flow in a similar way. Pretreatment with gamma-butyrolactone to impair nerve impulse flow, abolished the effect of morphine on the concentrations of dihydroxyphenylacetic acid and homovanillic acid. Pretreatment with apomorphine had a similar effect, as well as combined pretreatment with gamma-butyrolactone and apomorphine. Since gamma-butyrolactone and apomorphine both reduce nerve impulse flow, but gamma-butyrolactone increases while apomorphine decreases dopamine biosynthesis, it would appear that the antagonism of morphine-induced increases in dopamine metabolites is due to the common property of impulse flow reduction. It was also shown, however, that pretreatment with alpha-methyl-paratyrosine, which inhibits dopamine biosynthesis, resulted in antagonism of morphine's effect on dopamine metabolite levels. It is concluded, therefore, that morphine-induced dopamine efflux is observed under conditions when no effect on dopamine metabolism is observed, and vice versa. Three effects of morphine on dopaminergic neurones can be distinguished: an increase in impulse flow in nigrostriatal dopaminergic neurones, increased dopamine biosynthesis and catabolism, and efflux of dopamine. The first effect probably is effected in the cell body areas, while the latter two effects may be produced at the level of the nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of serotonin and derivatives by reversed-phase ion-pair partition chromatography with fluorometric and electrochemical detection.

A flexible and efficient system is described for reversed-phase ion-pair partition-chromatographic analysis of serotonin, its precursors tryptophan and 5-hydroxytryptophan, its main metabolite 5-hydroxyindoleacetic acid, and tryptamine. The chromatographic system consists of tri-n-butylphosphate as stationary phase and buffered water-methanol mixtures, containing perchlorate, as mobile phases. Retention can be selectively influenced by means of the pH, the perchlorate concentration, and the methanol content of the mobile phase, as well as the temperature of the phase system. The compounds of interest can be separated within 10 min and no interference from catecholamines and derivatives was observed. Compared with electrochemical detection, fluorometric detection yielded more favourable detection limits and was more selective when supernatants of brain tissue homogenates were directly injected. Both detection systems showed inadequate selectivity if urine samples were directly injected, but 5-hydroxyindoleacetic acid could readily be assayed.

Dopaminergic and adrenergic effects on plasma LH levels in rats with hypothalamic islands.

In female rats hypothalamic islands were prepared by making a cut around the mediobasal hypothalamus. Three weeks later the rats were ovariectomized. Two weeks after ovariectomy, part of the animals showed elevated blood LH levels with oscillations, although the mean LH levels were significantly lower than in sham-operated ovariectomized animals. There was a significant correlation between the occurrence of elevated blood LH levels after ovariectomy and the inclusion of at least part of the suprachiasmatic nucleus within the islands. Nor-adrenaline content of the hypothalamus was decreased significantly, but there was no significant change in hypothalamic dopamine content. Injection of doses of apomorphine, ranging form 2.5 microgram/kg to 25 mg/kg sc into island-bearing and sham-operated animals caused a dose-dependent decrease of plasma LH. However, the magnitude of the decrease was smaller in rats with hypothalamic islands than in controls. Even the highest doses of apomorphine did not decrease plasma LH further than to about 100 ng of LH-RP-1/ml. Phenoxybenzamine (10 and 40 mg/kg ip) and phentolamine (20 mg/kg ip) caused a decrease of plasma LH in ovariectomized control rats, but in ovariectomized rats with hypothalamic islands these drugs caused an increase. Clonidine (100 microgram/kg sc followed by 100 microgram/kg ip) had no effect in ovariectomized control rats, but in ovariectomized rats with a hypothalamic island an increase was induced. These results may have been caused by an increased sensitivity of hypothalamic alpha-receptors resulting from severing afferent noradrenergic fibres.

Differential effects of acute and subacute HA-966 treatment on storage and release of striatal dopamine.

Acute injections of HA-966 (100 mg/kg) into rats caused a rapid elevation of dopamine (DA) content in the striatum. 3,4-Dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels increased after a latency period of 0.5 h and 1 h respectively. Repeated ("subacute") HA-966 treatment produced a smaller DA increase than did single administration, while DOPAC and HVA rose at the same rate after both treatments. In HA-966-pretreated rats no tolerance for DA increase followed a lesion of the dopaminergic fibres. Acute as well as subacute HA-966 prevented the disappearance of DA after alpha-methyl-p-tyrosine for about 3 h. In both cases DOPAC and HVA levels dropped sharply after HA-966. HA-966 had no influence on the decline of DOPAC and HVA levels after monoamine oxidase inhibition. It is concluded that the rises of DOPAC and HVA after HA-966 did not occur because the capacity of the vesicular DA stores was exceeded. Instead, HA-966 affects the storage mechanism for newly formed DA. Possible explanations for the observed tolerance to DA accumulation after HA-966 are discussed.

Electrical stimulation of the nigrostriatal pathway after HA-966 block of triatal dopamine release.

Striatal dopamine (DA) of rats increased twofold 1 h after blockade of DA release by either an electrolytic lesion of the substantia nigra (SN) or systemic administration of 1-hydroxy-3-amino-pyrrolidone-2 (HA-966). If the treatments were combined, no further increase was observed. The decrease in DA induced by alpha-methyl-p-tyrosine (alphaMT) in otherwise untreated animals was not enhanced by a 20 min electrical stimulation of nigrostriatal fibres. If, however, in addition to synthesis blockade by alphaMT, DA release was interrupted by a lesion of the SN and/or by HA-966, electrical stimulation caused a significant decrease in striatal DA. It is assumed that HA-966 exerts its action in the dopaminergic nigrostriatal system by way of blockade of the nerve impulse flow at the level of the SN.