We report the effects of various dopamine receptor-blocking drugs on gene and protein expression, as well as the activity of several hepatic cytochrome P-450 (CYP) enzymes in the male Sprague-Dawley rat. At equipotent doses (with respect to receptor blockade and behavioural tests), the dopamine D2-receptor selective sulpiride and remoxipride gave a conspicuous down-regulation of CYP2C11 and its associated androstenedione 16α-hydroxylation activity as well as of the CYP2C11-specific mRNA. The average immunoidentified CYP2C11 levels correlated with the CYP2C11-specific mRNA levels in all treatment groups (r=0.994), indicating a transcriptional mechanism. The CYP3A protein was also selectively down-regulated. In contrast, androstenedione 5α-reduction was significantly increased. Clozapine, a non-selective neuroleptic, gave the same effects on the steroid metabolism as sulpiride and remoxipride. In contrast, diverging effects were observed for clozapine, compared to sulpiride and remoxipride, on the immunoidentified CYP1A2, CYP2B1, and CYP3A. These proteins were elevated by clozapine, and down-regulated by sulpiride and remoxipride. Our results are of interest for the interpretation of preclinical dose ranging toxicity tests of neuroleptic agents in rats. They may also be relevant in relation to certain interactions and adverse reactions observed in the clinical use of these drugs. The down-regulation of certain CYP enzymes is most likely mediated by an interaction with the growth hormone secretion.

Incubation of the tricyclic antidepressant desmethylimpramine (DMI) with rat liver or brain microsomes in the presence of NADPH or t-butyl-hydroperoxide (TBH) revealed different regiospecificities in the hydroxylation reactions between the tissues. In brain preparations 10-OH-DMI was formed in reactions supported by NADPH or TBH, whereas in the latter case also an unidentified metabolite could be detected. Inclusion of exogenous NADPH-cytochrome P450 reductase in the brain preparations caused a 10-fold higher rate of 10-hydroxylation but no 2-OH-DMI could be detected. By contrast, liver microsomal preparations in the presence of NADPH catalyzed formation of both 2- and 10-OH-DMI, whereas only 10-OH-DMI was formed in TBH-supported reactions. The results indicate that antidepressant drugs can be metabolized in brain with different stereospecificity as compared to liver.

There is a falling longitudinal gradient of P-450IA1 along the length of the small intestine of rats fed a stock diet or a BNF-containing diet. In rats fed a special diet, based on purified ingredients, the gradient was abolished but could be restored by addition of betanaphthoflavone (BNF) to the diet. This indicates that the distribution of P-450IA1 in the rat small intestine is regulated by dietary components. Tape-section autoradiographic analysis showed that exposure to dietary BNF was an important determinant for the disposition of C-14-Trp-P-1. Following dietary BNF treatment, a pronounced retention of radioactivity was observed in the epithelium of the oral cavity, esophagus, forestomach and small intestine, but not in the glandular stomach. This was obvious but less striking in rats given BNF intraperitoneally, whereas no retention could be observed in the gastrointestinal tract of untreated rats.

A specific form of cytochrome P450, P450 IIE1, active in ethanol oxidation, is known to be induced about 10-fold in rat liver following ethanol treatment. This isozyme of P450 participates effectively in the metabolic activation of precarcinogens, such asN-dimethylnitrosamines, and of solvents such as carbon tetrachloride and benzene. In the present investigation, two different polyclonal antisera against P450 IIEl were used in order to map the regional distribution of this P450 form in the rat central nervous system. The presence of P450 IIEl in various brain regions was confirmed by Western blot analysis.

The effect of dietary beta-naphthoflavone (BNF) on tissue retention of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was studied in the rat. Female rats, 3 weeks old, were fed a BNF-containing diet for 3 days before being dosed orally or i.v. with 14C-labelled Trp-P-1. The rats were killed at 4, 24 or 48 h after dosage and subjected to tape-section autoradiography. The tissue localization of Trp-P-1-derived radioactivity was compared to that observed in untreated rats and in rats given BNF i.p. Ethoxyresorufin-O-deethylase (EROD) activity and mutagenicity of Trp-P-1 in the Ames test, using S9 prepared from forestomach, glandular stomach, small intestine, liver and lung, were used as in vitro assays to measure the degree of cytochrome P450IA1 and/or P450IA2 induction. Dietary BNF treatment caused a 30- to 40-fold increase in EROD activity in the small intestine, but only a 2-fold increase in the liver and the lung. These inter-organ differences were not observed after i.p. administration of BNF. The increase in mutagenicity of Trp-P-1 in the Ames test could be correlated to the increase in EROD activity. The autoradiographic data showed that the route of administration of BNF as well as of Trp-P-1 were important for the tissue localization of Trp-P-1. Dietary BNF treatment caused a pronounced retention of Trp-P-1-derived radioactivity in the epithelia of the small intestine, forestomach, oesophagus and the oral cavity, regardless of the administration route of Trp-P-1; a similar though less pronounced epithelial retention was observed after i.p. injection of BNF. A clear-cut boundary of accumulated radioactivity between the forestomach and the glandular stomach where the levels were almost non-detectable was observed in rats fed the BNF-containing diet. It is concluded that dietary inducers may be important determinants of metabolism and tissue distribution of toxic compounds.

Polyclonal antibodies against rat cytochrome P-450c (P-450IA1), P-450d (P-450IA2), P-450b (P-450IIB1), P-450h (P-450IIC11) and P-450j (P-450IIE1), were used to probe liver microsomes prepared from species in a number of vertebrate groups ranging from the hagfish, a very primitive vertebrate, to the male rat. Cross-reactivity in Western blots was used to examine the appearance of cytochrome P-450 isozymes structurally related to the rat forms in lower vertebrate groups and to provide information on the evolution of cytochrome P-450 gene families I and II and their regulation in lower vertebrates.

The effects of perinatal or neonatal morphine exposure on the hepatic steroid and xenobiotic metabolism in adult rats were studied. Early morphine exposure did not affect the 16 alpha-hydroxylation or 5 alpha-reduction of androstenedione in either sex, but decreased the 7 alpha- and 6 beta-hydroxylations in both sexes. Morphine exerted a suppressive effect on the ethoxyresorufin-O-deethylase activity and increased the ethoxycoumarin-O-deethylase activity in both sexes. Morphine exposure did not significantly affect its own N-demethylation or the total cytochrome P-450 content in the liver. Neonatal morphine exposure caused a significant decrease in body and testes weight in the adult male rat. We conclude that the effects of morphine are not confined to sex-differentiated pathways and are similar in both sexes.

The effects of neurotensin in vitro (1–100 n m ) on the binding characteristics of [ 3 H]N‐propylnorapomorphine ([ 3 H]NPA) were analysed in striatal membrane preparations of the adult male rat. Subsequently, it was investigated whether the modulatory effects of To nmt neurotensin on [ 3 H]NPA binding were altered by treatment with toluene in vivo (80 p.p.m., 3 days, 6 h day ‐1 ) and in vitro (19 μ mol ml ‐1 ). Displacement of [ 3 H]NPA binding by raclopride (IC 50 about 15 n m ) and SCH 23390 (without effect) indicated that [ 3 H]NPA labelled only D 2 dopamine receptors in the present study. Neurotensin was found to reduce the affinity of D 2 receptors with a maximum response at to ma. At this concentration the KD value was increased by 30–40% without any consistent changes in the number of binding sites. The modulatory effect of neurotensin remained intact also following toluene treatment in vivo and in vitro , although at a higher K D range, since toluene alone increased the KD value of [ 3 H]NPA binding by 40–50%. Thus, the mechanisms mediating the effects of neurotensin and toluene on the D 2 receptor are likely to be different. When neurotensin and toluene treatments were combined, the K D values of [ 3 H]NPA binding were about twice as high as in non‐treated controls. These additive effects may lead to a severely decreased efficiency of dopamine D 2 mediated neurotransmission in vivo .

Effects of neonatal toluene exposure (80 ppm, day 1–7, 6 h/day) have been studied on regional brain catecholamine levels and utilization, and on serum levels of hypophyseal and adrenocortical hormones in the adult male rat. Catecholamine levels were measured by quantitative histofluorimetry in the forebrain and hypothalamus and by high pressure liquid chromatography with electrochemical detection in the substantia nigra. Catecholamine utilization was evaluated from the decrease in catecholamines seen after tyrosine hydroxylase inhibition using α-methy-p-tyrosine methyl ester hydrochloride (αMT, 250 mg/kg, i.p., 2 h). Serum levels of thyroid stimulating hormone, corticosterone, aldosterone, prolactin and luteinizing hormone were measured by radioimmunoassays. Neonatal toluene exposure produced a reduction of dopamine levels and utilization selectively in the olfactory tubercle and substantia nigra of the adult rat. Furthermore, neonatal toluene exposure produced a significant reduction in the noradrenaline levels and utilization in the substantia nigra and an increase of noradrenaline utilization selectively in the subependymal layer of the median eminence and of the magnocellular part of the paraventricular hypothalamic nucleus. The serum hormone levels were not significantly influenced by neonatal toluene exposure as evaluated in adulthood. However, the αMT induced increase in serum prolactin levels was reduced following neonatal exposure to toluene.

Abstract: The cytochrome P‐450 (P‐450) content of different regions of the rat brain was measured after partial purification of the enzyme from homogenates, and the quantitative contribution of P‐450b,e and P‐450c,d to brain P‐450 was assessed by Western immunoblotting and immunohistochemistry using rabbit antibodies raised against purified hepatic P‐450b and P‐450c (anti‐P‐450b and anti‐P‐450c, respectively). P‐450 could be quantitated by its reduced CO difference spectrum after chromatography of homogenates on p‐chloroamphetamine‐coupled Sepharose. The yield of P‐450 from whole brain was 90 ± 19 pmol/g of tissue, which is ∼ 1% of the level in liver microsomes from control rats. The amount of P‐450 recovered from homogenates of olfactory lobes, hypothalamus, thalamus, striatum, cerebral cortex, and brainstem varied between 40 and 100 pmol/g of tissue. The cerebellum was a region of exceptionally high P‐450 content, with yields of up to 400 pmol/g, whereas the substantia nigra yielded only 16–20 pmol/g. Immunohistochemical studies with anti‐P‐450b and anti‐P‐450c revealed intense staining of a limited number of cells in the cerebellum with both antibodies and in the thalamus only with anti‐P‐450c. In the cerebellum, both anti‐P‐450b and anti‐P‐450c stained the Bergmann glial cells together with their radial processes. Individual glial cells in the granular cell layer were also stained. There was no staining of Purkinje cells. In the thalamus, anti‐P‐450b gave weak staining of certain astroglia, but with anti‐P‐450c, there was intense staining of neuronal somata. Western immunoblots with P‐450 isolated from different brain regions confirmed the distribution of P‐450b,e and P‐450c,d observed with immunohistochemistry. Of all the brain regions examined, P‐450b,e was detected only in P‐450 obtained from the cerebellum and P‐450c only in the cerebellum and thalamus. However, quantitation of the P‐450b,e and P‐450c bands on the immunoblots by 125I‐labeled protein A revealed that these forms of P‐450 account for <1% of the P‐450 in the cerebellum and thalamus. This low content of P‐450b and P‐450c was also reflected in a low level of ethoxycoumarin O‐deethylase activity in the cerebellum and thalamus. From these studies, it is concluded that there are multiple forms of P‐450 in the brain and these different forms of P‐450 are highly selectively localized to certain cells. Furthermore, most of the P‐450 in the brain remains uncharacterized.

Castrated male rats were treated with constant-release implants filled with testosterone, oestradiol-17 beta, 17 beta-hydroxy-5 alpha-androstan-3-one (5 alpha-dihydrotestosterone; DHT), 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-Adiol) or 5 alpha-androstane-3 beta,17 beta-diol (3 beta-Adiol). Only testosterone activated the sexual behaviour of the rats. If combined with oestradiol, DHT or 3 alpha-Adiol induced the behaviour, but 3 beta-Adiol failed to have this effect. Oestradiol inhibited the in-vitro formation of [14C]Adiols from [14C]DHT by combined preoptic and hypothalamic tissue, but only when given in high doses. No effect on the formation of [14C]Adiols from [14C]DHT was found in rats treated in vivo with DHT or with the combination of DHT and oestradiol which effectively stimulated sexual behaviour. These results do not support the suggestion that oestradiol may synergize with androgens to induce sexual behaviour in castrated rats by inhibiting androgen metabolism.

1. Ethoxyresorufin O-deethylase (EROD) activity, aldrin epoxidase (AE) activity, cytochrome P-450 content, and levels of cytochrome P-450E (the major BNF-inducible P-450 form and primary EROD catalyst in scup) or its homologues were measured in hepatic microsomes isolated from Fundulus heteroclitus, scup (Stenotomus chrysops) and brook trout (Salvelinus fontinalis) treated with β-naphthoflavone (BNF) or phenobarbital (PB).2. In all three teleost species, BNF treatment caused expected increases in P-450 content, EROD activity and P-450E level; but either no change or a slight decrease in AE turnover rate (nmol/min/nmol P-450).3. Polyclonal antibodies to P-450E did not inhibit AE activity in microsomes from BNF-treated scup, confirming that this major BNF-inducible P-450 form does not catalyze AE activity in fish.4. In contrast, PB treatment did not affect hepatic AE activity, P-450 content or levels of “P-450E” in F. heteroclitus, but did variably affect EROD activity which was suppressed in one experiment and elevated in another.5. The results indicate that (i) contrary to previous reports, neither PB nor MC-type inducers increase AE activity in F. heteroclitus, (ii) MC-type inducers do not affect AE activity in the other teleost species examined, and (iii) AE activity is not a reliable indicator of P-450 induction by environmental chemicals.6. We emphasize the need to establish the mechanism of PB action, and the nature of any fish P-450 forms analogous to PB-inducible forms in mammals, in order to conclusively evaluate PB-responses in fish.

The effects of chronic toluene exposure (CTE) (80 ppm, 6 h/day, 5 days/week, 3 months) were studied on neuropeptide and 5-hydroxytryptamine receptors, on protein phosphorylation levels and on catecholamine levels in various brain regions in the 15-month-old male rat. Behavioral parameters and serum levels of hypophyseal hormones and corticosterone were also analyzed. CTE selectively reduced [3H]neurotensin ([3H]NT) binding in the basal layers of the orbital cortex. Instead, CTE increased the binding of [3H]etorphine in the nucleus accumbens and of [125I]vasoactive intestinal polypeptide ([125I]VIP) in the area postrema and hypoglossal nucleus. Acute treatment with the irreversible monoamine receptor antagonist N-ethoxycarboxyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) increased the binding of [3H]NT in the orbital cortex in toluene exposed rats as compared with the reduced [3H]NT binding obtained in air exposed rats treated with EEDQ. Furthermore, the EEDQ induced increase in [125I]VIP binding in the area postrema and the hypoglossal nucleus was replaced by a reduced binding of [125I]VIP in EEDQ-treated CTE rats. CTE produced an overall increase in calcium-induced back phosphorylation and an overall decrease in cyclic adenosine monophosphate-induced back phosphorylation in the frontoparietal cortex. Noradrenaline stores tended to be reduced within various hypothalamic subnuclei and the serum prolactin levels were increased following CTE. However, no marked effects of CTE were seen on the behavioral parameters. In conclusion, the regional selectivity of CTE in disturbing [3H]NT and [125I]VIP binding may be due to the demonstrated vulnerability of monoamine-neuropeptide interactions to toluene.

The aim of the present investigation was to evaluate whether the responses of central catecholamine (CA) neurons to CA receptor blockade by haloperidol are altered upon toluene exposure. Male rats were exposed to air or toluene (80 ppm) for 5 and 4 days, 6 h day-1. CA levels and utilization were determined in discrete regions of the forebrain and hypothalamus as well as in the substantia nigra (SN) and anteromedial frontal cortex (AMFC). Serum levels of corticosterone, thyroid stimulating hormone, luteinizing hormone and prolactin were determined by radioimmunoassay procedures. Toluene exposure led to increased dopamine (DA) utilization in the AMFC and increased CA utilization in the paraventricular hypothalamic nuclei. In air-exposed rats haloperidol (1 mg kg-1, i.p., 2 h before killing) increased DA utilization in the marginal part of the nucleus caudatus putamen (CAUD). In toluene-exposed rats, haloperidol induced significant depletions of DA stores in the SN and in the medial and central parts of the CAUD. In the posterior nucleus accumbens (ACC) DA utilization was significantly increased. Combined haloperidol and toluene treatment selectively decreased DA levels in the ACC and SN, and significantly increased DA utilization in the CAUD, as compared with the air-exposed control group. Furthermore, after combined treatment, there was a specific increase in noradrenaline (NA) utilization in the SN and in CA utilization in the medial palisade zone of the median eminence. Serum prolactin levels were substantially raised in both the air and toluene groups after the haloperidol treatment. In conclusion, acute haloperidol treatment preferentially reduces DA levels and increases DA and NA utilization in the SN and in discrete tel- and diencephalic areas in rats exposed to toluene.

A quantitative assessment of the levels of cytochromes P-450 b and P-450 c in the brains and pituitary glands of untreated and beta-naphthoflavone (BNF)-pretreated rats was made with polyclonal antibodies raised against hepatic P-450 b and c and the sensitive fluorometric assay of P-450 catalytic activity, namely, the O-deethylation of ethoxycoumarin (ETC). In the microsomal fraction of brains of untreated rats, the rate of formation of 7-hydroxycoumarin from ETC ranged between 0.1 and 20 pmol/min/mg of microsomal protein, which is approximately 0.01-2% of the level of hepatic microsomes of phenobarbital-induced rats. This brain activity was completely inhibited by anti P-450 b antibodies but was unaffected by anti P-450 c antibodies. As with hepatic P-450 b, metyrapone and chloramphenicol (100 microM) were good inhibitors of catalytic activity, whereas alpha-naphthoflavone (1 microM) was a poor inhibitor. No ETC O-deethylase activity was detectable in microsomes prepared from the pituitary glands of untreated rats. Upon pretreatment of rats with BNF, there was induction of ETC O-deethylase activity in the pituitary gland to a level of 3.3 +/- 1.5 pmol/min/mg of microsomal protein, but there was no significant increase in the level of activity in brain microsomes. Despite this, there was evidence of induction of P-450 c in both the brain and pituitary of BNF-pretreated rats since anti P-450 c antibodies inhibited brain activity by 55% and pituitary activity by 84%. The regional distribution of P-450 b and c in the hypothalamic-preoptic area and olfactory bulbs was examined. The level of ETC O-deethylase activity in the hypothalamic-preoptic area was not different from that in the whole brain, but in the olfactory bulbs activity was higher than that in whole brain, with a range of 0.1-52 pmol/min/mg of microsomal protein. The catalytic activity in the whole brain and in the olfactory bulbs was inhibited by anti P-450b but not by anti P-450c antibodies. Neither estradiol, testosterone, dehydrotestosterone, nor 5 alpha-androstane,3 beta,17 beta-diol (100 microM) competitively inhibited ETC O-deethylase activity, indicating that P-450 b is not responsible for the steroid hydroxylations previously reported in the brain. BNS pretreatment of rats did not cause a consistent increase in ETC O-deethylase upon BNF induction. However, there was an induction of P-450 c in the olfactory bulbs since catalytic activity was inhibited with anti P-450c antibodies.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of treatment with toluene in vivo (80 ppm, 3 days, 6 h/day) and in vitro (19 μmol/ml) were analyzed on the binding characteristics of [3H]neurotensin in rat striatal membranes. Exposure to toluene in vivo did not produce any significant effects on the binding characteristics of [3H]neurotensin. However, the addition of toluene in vitro caused a trend for a decreased Bmax value and produced a significantly reduced KD value of [3H]neurotensin binding. The absence of effects at 80 ppm indicates that the neurotensin receptor is relatively insensitive to toluene exposure, in contrast to, e.g. the dopamine agonist binding sites. Furthermore, the toluene response of the neurotensin receptor, as seen after treatment in vitro, is different from the responses seen in many monoamine receptors, which show decreased affinities following toluene exposure. It is possible that toluene is mediating its effects on the neurotensin receptor by changing the lipid micro-environment in which the receptor is situated. Another explanation would be that toluene selectively acts on the monoamine receptors, e.g. the more sensitive dopamine receptors, which through receptor-receptor interactions would cause the response seen in the neurotensin receptor. However, it cannot be excluded that the suggested receptor-receptor interaction itself is affected by toluene.

Toluene (4.7–150 μmol per ml) was added for 30 or 60 min to astroglial and neuronal primary cell cultures from rat striatum and changes in cell morphology were analyzed by light microscopy. After 60 min incubation in 40 μmol toluene/ml, the cell bodies of the astrocytes appeared contracted, and their processes and nuclei were clearly visible. At higher doses of toluene the astrocytes seemed to be flattened and major cell damage was visualized by the uptake of vital dyes. The neurons, however, became affected and judged by morphological criteria only at the higher toluene doses.

Male and female Wistar rats were given an initiating i.p. injection of diethylnitrosamine (DEN; 200 mg/kg body wt). Two weeks later the rats were given a diet containing 0.02% (w/w) 2-acetylaminofluorene (2-AAF) for 2 weeks. In the middle of the 2-AAF treatment a 70% partial hepatectomy (PH) was performed. In order to identify the pituitary hormone responsible for the previously observed sex difference (male greater than female) in and influence of ectopic pituitary grafts on focal growth during 2-AAF/PH selection of enzyme-altered foci, male rats were treated with a continuous infusion of bovine growth hormone (bGH; 6 micrograms/h) or ovine prolactin (oPrl; 6 micrograms/h) by way of osmotic minipumps. Hormonal treatment was started 1 week after initiation and was finished 1 week after the 2-AAF selection period. All rats were killed 6 weeks after initiation and liver sections were stained for gamma-glutamyltransferase. The number of foci/cm2 as well as the area per focus and area ratio (mm2 foci/cm2 liver section) were calculated. Whereas no significant differences in the number of foci/cm2 were observed between the different groups of rats, bGH treatment of male rats decreased both the area/focus and the area ratio down to the female level. No significant effects were seen following oPrl administration when compared with control males. In vitro studies of subcellular preparations from the liver lobes obtained at PH showed that the sexually differentiated N-hydroxy-2-AAF sulfotransferase activity (male greater than female) in male rats was 'feminized', i.e. decreased, by bGH administration, but not by infusion of oPrl. The present investigation strengthens the view of growth hormone as an important determinant of sex differences in chemical carcinogenesis in rat liver, possibly via an influence on carcinogen metabolism.

Effects of subacute toluene exposure (80 p.p.m. toluene in air, 5 + 4 days, 6 h day-1) were analysed on calcium (Ca2+)- and cyclic adenosine monophosphate (cAMP)-induced protein phosphorylation levels in membrane preparations from the frontoparietal cortex and the striatum of the adult male rat. After protein separation by gel electrophoresis, the amount of radioactive phosphate incorporated from adenosine 5'-[gamma-32P] triphosphate, tetra-(ethylammonium) salt ([32P]ATP) was measured indirectly by autoradiography. The 21 most phosphorylated protein bands were then analysed by computerized image analysis. In the frontoparietal cortex no protein bands were significantly affected after cAMP-induced back phosphorylation, while after Ca2+ stimulation there was a decreased incorporation of [32P]ATP in a 22,000 protein band. In the striatum there was a reduced incorporation of [32P]ATP in a 26,000 protein band after cAMP-induced back phosphorylation, and in four bands of 20,000, 21,000, 52,000 and 134,000, respectively, after Ca2+ stimulation. The reduced incorporation of [32P]ATP in these proteins indicated increased original phosphorylation levels after toluene exposure. A comparison between the frontoparietal cortex and the striatum showed a selective vulnerability of phosphorylation processes in striatal membrane protein bands. In conclusion, toluene exposure at low doses augments membrane protein phosphorylation levels in the rat forebrain and especially in the striatum, probably leading to changes in information handling and/or metabolic changes.