Early induction of reparative hyperplasia in the liver of B6C3F1 mice treated with dichloroacetate and trichloroacetate

Trichloroacetate (TCA) and dichloroacetate (DCA) were administered at concentrations of 0, 300, 1000 or 2000 mg/l in the drinking water of male B6C3F1 and male and female Swiss-Webster mice for up to 14 days. At 2, 5 or 14 days of treatment, mice were injected with [3H]thymidine 2 h prior to sacrifice. The livers were examined histologically and autoradiographically and DNA was isolated and counted. As observed in chronic studies dichloroacetate induced a marked increase in liver weights, but only after 14 days of treatment and local necrosis in both B6C3F1 and Swiss-Webster mice. A significant increase in the labeling index of hepatocytes was observed in animals treated with DCA, but only at 14 days of treatment. No such increases were observed in animals treated with TCA. In contrast, significant increases in [3H]thymidine were observed in the livers of both DCA- and TCA-treated animals after 5 days of treatment. This effect remained apparent with TCA after 14 days of treatment. These data support the hypothesis that the tumorigenic effect of DCA is strongly influenced by necrosis and reparative hyperplasia. On the other hand, the carcinogenic effects of TCA appear to be more closely associated with [3H]thymidine incorporation that can be separated from cell division, suggesting an elevated rate of repair synthesis of DNA. Thus the carcinogenic effects of TCA (and perhaps lower doses of DCA) may involve damage to DNA.     

Hepatic subcellular distribution of [H]T-2 toxin

and . Hepatic subcellular distribution of [³H]T-2 toxin. Toxicon 27, 1307–1311, 1989.—The subcellular distribution of T-2 mycotoxin and its metabolites was studied in isolated rat livers perfused with [³H]T-2 toxin. After a 120-min perfusion, the distribution of radiolabel was to bile 53%, perfusate 38% and liver 7%. Livers were fractionated into mitochondria, endoplasmic reticulum (smooth and rough), plasma membrane and nuclei. Plasma membrane fractions contained 38% of the radiolabel within 5 min, decreasing to < 1% at the end of the 120-min perfusion. Smooth endoplasmic reticulum contained 27% of the radiolabel by 5 min and increased to 43% over the 120-min perfusion. The mitochondrial fraction contained 3% of the radiolabel by 30 min and increased to 10% after 120-min perfusion. Label in the nuclear fraction remained constant at 7% from 30 to 120 min. By 15 min, only the parent toxin was detected in the mitochondrial fraction. In the other fractions, radiolabel was associated with HT-2, 4-deacetylneosolaniol, T-2 tetraol, and glucuronide conjugates. Glucuronide conjugates accounted for radiolabel eliminated via the bile. The time course for distribution of radiolabel in liver suggested an immediate association of [³H]T-2 with plasma membranes and a subsequent association of toxin and metabolites with endoplasmic reticulum, mitochondria and nuclei, the known sites of action of this toxin.     

Evaluation of metabolic endpoints of acute cytotoxicity in V79 fibroblasts

A group of cytotoxicity tests that detect alterations in cell metabolism were applied to obtain a preliminary classification of test chemicals, based on their main mechanism of toxicity. V79 cells were exposed to toxic compounds in ‘acute’ treatments (up to 2 hr) and the specificity of different endpoints of cytotoxicity was compared. We tested five directly acting chemicals: butylated hydroxyanisole, butylated hydroxytoluene, cycloheximide, potassium dichromate [Cr(VI)] and dinitrophenol using four assays measuring; [³H]thymidine uptake and incorporation into DNA, [³H]leucine incorporation into proteins, ATP pool size and cellular energy charge, and oxygen consumption. In the thymidine-uptake test the radioactivity of the nucleotide pool was hardly affected by low concentrations of the toxic chemicals and was reduced by about 20–30% at the 10⁻⁴ concentration, except by butylated hydroxytoluene; the latter which caused a marked inhibition of [³H]thymidine uptake. Thymidine incorporation into DNA was more specifically altered, showing a net inhibition by potassium dichromate, in a concentration-dependent fashion, and by butylated hydroxytoluene. Protein synthesis was more inhibited by potassium dichromate and butylated hydroxytoluene than by the specific inhibitor, cycloheximide. All chemicals reduced ATP concentration, but the energy charge was scarcely affected, reflecting a parallel decrease of the other adenine nucleotides. The assay measuring early changes in oxygen consumption produced the expected results with dinitrophenol, the antioxidants and potassium dichromate, and showed that cycloheximide also inhibits mitochondrial respiration, in agreement with the observed fall of intracellular ATP. All the chemicals tested induced a range of effects on the metabolic parameters analysed but specific pathways of toxicity may be inferred by comparing the results of the individual tests.     

Gamma-hydroxybutyric acid (GHB) and gamma-aminobutyric acidB receptor (GABABR) binding sites are distinctive from one another: molecular evidence

γ-Hydroxybutyric Acid (GHB) is thought to be a weak partial agonist at the γ-aminobutyric acid_B Receptor (GABA_BR), but the precise relationship of the GHB receptor (GHBR) to the GABA_BR remains unclear. In order to test the hypothesis that the GHBR is not identical to the GABA_BR, we conducted two groups of experiments. First, GABA_BR subtype 1 (R1) and/or subtype 2 (R2) were over expressed in HEK 293 cells and membrane binding studies on the transfected cells done using [³H]GHB and [³H] (2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene) ethanoic acid ([³H]NCS-382). The latter is a specific antagonist at the GHB binding site. Second, [³H]GHB and [³H]NCS-382 autoradiographic binding studies were done on the brains of mice in which the gene for GABA_BR1a was deleted. Such mice do not have a functioning GABA_BR. There was no detectable specific [³H]GHB or [³H]NCS-382 binding in HEK 293 cells transfected with GABA_BR1, R2, or R1/R2. Binding to [³H]CGP54626A, a high affinity GABA_BR antagonist, was absent in GABA_BR1a^−/− mice. There was no difference in [³H]NCS-382 binding observed in the brains of GABA_BR1a^−/−, GABA_BR1a^+/− or GABA_BR1a^+/+ mice. Specific [³H]GHB binding was observed in the brain of GABA_BR1a^−/− mice but was significantly lower than in wild type mice. These data support the hypothesis that the GHB binding site is separate and distinct from the GABA_BR.     

Effect of lead and chromium on nucleic acid and protein synthesis during sperm-zona binding in mice

Lead and chromium are ubiquitous environmental pollutants and their effects on reproductive physiology as shown by animal experiments and human studies is well documented. The present study was conducted to ascertain the role of these metals on gamete physiology during the sperm-zona binding process, Superovulated ova and capacitated sperm from BALB/c mice were exposed to lead (0.0, 0.2, 0. 5, 1.0 and 2.0 μg/ml) or chromium (0.0, 5.0, 10.0 and 20.0 μg/ml) for 2 h in the culture medium. The sperm that became attached to the ova in the presence of these metals were scored. Synthesis of DNA, RNA and protein was assessed during this process by labelling the cells with [³H]thymidine (20 μCi/ml), [³H]uridine (50 μCi/ml) and [³⁵S]methionine (10 μCi/ml) in the culture medium after exposure to the metals for 2 h. Our studies show a significant dose-dependent decrease in the number of sperm attaching to the ova in both exposed groups along with a decrease in the incorporation of radiolabelled thymidine, uridine and methionine. The results indicate that DNA, RNA and protein synthesis under sperm-zona binding conditions are affected by the presence of these two metals. The physiology of the gametes is altered resulting in a low frequency of sperm attachment to the ova, which in turn leads to a risk of reproductive failure.     

Effects of vinconate on neurotransmitter receptor systems in aged rat brain

We investigated the effects of age and (±)-methyl-3-ethyl-2,3,3a,4-tetrahydro-1 H-indolo[3,2,1-de][1,5]naphthyridine-6-carboxylate hydrochloride (vinconate), an indolonaphthyridine derivative, on neurotransmitter receptor systems in the rat brain using quantitative receptor autoradiography. [³H]Quinuclidinyl benzilate (QNB), [³H]hemicholinium-3 (HC) and [³H]muscimol were used to label acetylcholine receptors, high-affinity choline uptake sites and γ-aminobutyric acid_A (GABA_A) receptors, respectively. [³H]QNB, [³H]HC and [³H]muscimol binding decreased in any brain areas of 24-month-old (aged) rats in comparison with 6-month-old (adult) animals. Chronic treatment with vinconate (10 and 30 mg/kg, i.p., once a day for 4 weeks) partly ameliorated the reduction in [³H]QNB, [³H]HC and [³H]muscimol biding in aged rat brains. This effect was especially noted in [³H]muscimol binding. The results suggest that vinconate may have beneficial effects on age-related changes in neurotransmitter receptor systems.     

Assessment of unscheduled and replicative DNA synthesis in hepatocytes treated in vivo and in vitro with unleaded gasoline or 2,2,4-trimethylpentane

In a recent chronic inhalation exposure study, unleaded gasoline (UG) produced kidney tumors in male rats and liver tumors in female mice, but did not increase the incidence of liver tumors in male mice or rats of either sex. To examine the possible basis for this pattern of hepatocarcinogenesis, unscheduled DNA synthesis (UDS) as an indicator of genotoxic activity and replicative DNA synthesis (RDS) as an indicator of cell proliferation were measured in rat and mouse hepatocytes following in vivo and in vitro exposures to UG and 2,2,4-trimethylpentane (TMP), a nephrotoxic component of UG. Primary hepatocyte cultures, prepared from cells isolated from Fischer-344 rats, B6C3F1 mice, or human surgical material, were incubated with [3H]thymidine and the test agent. UDS was measured by quantitative autoradiography as net nuclear grains (NG). By similar methods, UDS and RDS (S-phase cells) were measured in hepatocytes isolated from rats and mice treated by gavage with TMP (500 mg/kg) or UG (100 to 5,000 mg/kg). A dose-related increase in UDS activity was observed in rat hepatocytes treated in vitro with 0.05 to 0.10% (v/v) UG. These doses were, however, toxic in both mouse and human hepatocyte cultures. Weak UDS activity was observed in hepatocytes isolated from male and female mice treated 12 hr previously with UG. No UDS was induced in rat hepatocytes treated in vivo or in vitro with TMP. Twenty- and fourfold increases in the percentage of cells in S-phase were observed 24 hr after treatment with TMP in male and female mice, respectively, as compared to a fivefold increase in male rats. UG increased the percentage of S-phase cells in male mice by ninefold but failed to induce RDS in females. Thus, there appears to be genotoxic compounds in UG that can be detected in cultured hepatocytes and in the livers of exposed mice. The lack of UDS activity in rat liver was consistent with the reported lack of liver tumors in chronically exposed rats. However, neither the UDS nor the RDS responses in mice exposed by gavage correlated to the sex-specific pattern of liver tumors observed in the 2-year bioassay.     

Liver tumor induction in B6C3F1 mice by dichloroacetate and trichloroacetate

Male and female B6C3F1 mice were administered dichloroacetate (DCA) and trichloroacetate (TCA) in their drinking water at concentrations of 1 or 2 g/l for up to 52 weeks. Both compounds induced hepatoproliferative lesions (HPL) in male mice, including hepatocellular nodules, adenomas and hepatocellular carcinomas within 12 months. The induction of HPL by TCA was linear with dose. In contrast, the response to DCA increased sharply with the increase in concentration from 1 to 2 g/l. Suspension of DCA treatment at 37 weeks resulted in the same number of HPL at 52 weeks that would have been predicted on the basis of the total dose administered. However, none of the lesions in this treatment group progressed to hepatocellular carcinomas. Conversely, the yield of HPL at 52 weeks when TCA treatment was suspended at 37 weeks was significantly below that which would have been predicted by the total dose administered. In this case, 3 of 5 remaining lesions were hepatocellular carcinomas. Throughout active treatment DCA-treated mice displayed greatly enlarged livers characterized by a marked cytomegaly and massive accumulations of glycogen in hepatocytes throughout the liver. Areas of focal necrosis were seen throughout the liver. TCA produced small increases in cell size and much a more modest accumulation of glycogen. Focal necrotic damage did not occur in TCA-treated animals. TCA produced marked accumulations of lipofuscin in the liver. Lipofuscin accumulation was less marked with DCA. These data confirm earlier observations that DCA and TCA are capable of inducing hepatic tumors in B6C3F1 mice and argue that the mechanisms involved in tumor induction differ substantially between these two similar compounds. Tumorigenesis by DCA may depend largely on stimulation of cell division secondary to hepatotoxic damage. On the other hand, TCA appears to increase lipid peroxidation, suggesting that production of radicals may be responsible for its effects.     

A rapid screening system to determine drug affinities for the intestinal dipeptide transporter 1: system characterisation

Purpose: To establish an in vitro system for the rapid assessment of the affinities of potential substrates for the di/tri/oligopeptide transport system (DTS). Methods: Monolayers of Caco-2 cells were cultured in plastic wells for 7–9 days and the uptake of Gly-[³H]L-Pro, a specific and relatively stable substrate for the DTS was used as an affinity probe. Gly-[³H]L-Pro (50 nM), together with excess L-Pro (10 mM), to suppress uptake of any [³H]L-Pro produced by degradation of the probe, was incubated with the test compound (usually 1 mM) at pH 6 for 3 min. The uptake of radiolabel was determined by liquid scintillation counting. Results: High specific-uptake (>85%) of Gly-[³H]L-Pro was obtained with cells grown for 7–9 days. Gly-[³H]L-Pro uptake had a substantial active concentration-dependent component (K_m of 0.39±0.02 mM, V_max of 0.98±0.04 nmol min⁻¹ (mg protein)⁻¹. This process was shown to be specific for the DTS as evidenced by the significant inhibition by compounds reported to be transported by this system and the lack of inhibition by amino acids. The use of low competitor concentrations (1 mM) enabled a range of inhibition values (0–89%) of a series of competitors (amino acids, dipeptides and β-lactam antibiotics) to be estimated, illustrating that structurally similar compounds can be ranked for affinity to the DTS. Conclusion: A screening system, using Caco-2 cells and the dipeptide Gly-[³H]L-Pro as a displaceable probe, was developed to assess a variety of compounds for recognition by the di/tri/oligopeptide transport system. This fully describes the first system that allows structurally related compounds to be ranked on the basis of their affinity for the DTS recognition site.     

Lipid microsphere formulation containing rifampicin targets alveolar macrophages

The study demonstrated that lipid microspheres (LM) containing rifampicin (LM-RFP) could deliver the drug to alveolar macrophages in vitro and in vivo, and that intranasal administration to animals could achieve preferential accumulation in the lungs with less effect on the liver. The LM-RFP particles had a mean diameter of 247.2 ± 75.7 nm, and their size remained stable when stored at 4°C or 25°C for at least 4 weeks. In vitro uptake of [³H]LM-RFP by alveolar macrophages was over 4 times higher than that of unencapsulated [³H]RFP, whereas the in vivo uptake was 30 times higher. Flow cytometric analysis and confocal laser scanning microscopy confirmed that LM could deliver the encapsulated drug effectively to alveolar macrophages in vitro and in vivo. Intranasal administration of [³H]LM-RFP to normal mice resulted in preferential pulmonary uptake of the drug and lower levels in the blood and liver compared with administration of unencapsulated [³H]RFP. In conclusion, LM-RFP could be a promising preparation for delivery via the respiratory tract to tuberculosis (TB) and TB/HIV patients.     

Background DNA repair synthesis in rat hepatocyte cultures used for genotoxicity testing

DNA repair synthesis in primary rat hepatocyte cultures (HPC) was investigated using the bromodeoxyuridine (BrdUrd) density-shift method and autoradiography. Analysis by density-gradient centrifugation of DNA labelled with BrdUrd and [³H]deoxycytidine ([³H]dCyd) for 18–20 hr showed that considerable DNA repair replication occurs in HPC even in the absence of an added genotoxic agent. Repair was demonstrated to be most probably a consequence of DNA damage caused by the collagenase perfusion of the liver during hepatocyte isolation, and by β-decay of the ³H-labelled DNA precursor. Autoradiographic analysis of the distribution of silver grains over nuclei in intact non-S-phase cells, and over isolated nuclei from HPC exposed to [³H]dCyd for 18–20 hr, showed that the vast majority of the radioactivity was incorporated into the nuclei themselves, and not into overlying cytoplasm or mitochondria. In addition, direct localization of mitochondria in hepatocytes using the mitochondrion-specific dye rhodamine 123 showed that very few mitochondria were actually located over the nucleus. The results suggest that cytosolic labelling of control HPC in autoradiographs is mainly caused by mitochondrial DNA synthesis whereas nuclear labelling essentially reflects repair synthesis. They call into question the commonly applied practice of evaluating unscheduled DNA synthesis (UDS) in HPC by subtracting the number of cytosolic silver grains from nuclear grains and expressing repair synthesis as net grains per nucleus.     

Drug stimulation of putrescine and spermidine syntheses Relationships to enhancement of ribonucleic acid synthesis

The enzymes that synthesize putrescine and spermidine and the rate of uridine-6-[³H] incorporation were measured after a single treatment with various drugs, some of which are known to cause liver enlargement after chronic administration. The drugs examined were phenobarbital, 3-methylcholanthrene, 3, 4-benzpyrene, clofibrate (atromid-S), and heparin. All these drugs produced elevations of both ornithine decarboxylase ( -ornithine carboxy-lyase; EC 4.1.1.17), the enzyme that catalyzes the formation of putrescine from ornithine, and S-adenosyl- -methionine decarboxylase, the enzyme that catalyzes the formation of spermidine from putrescine and S-adenosyl- -methionine. They also stimulated the incorporation of uridine-6-[ H], an RNA precursor. Increased uridine-6-[³H] incorporation followed enhanced ornithine decarboxylase activity and paralleled increased spermidine synthesis in most cases. Cycloheximide administered at the same time as phenobarbital completely abolished the induction of these enzymes.     

Alterations of second messenger systems in the rat brain after 6-hydroxydopamine lesions of the medial forebrain bundle

We studied the sequential changes in second messenger systems in the striatum and substantia nigra (SN) after 6-hydroxydopamine lesions of the medial forebrain bundle in rats. The animals were unilaterally lesioned in the medial forebrain bundle and the brains were analyzed at 1, 2, 4 and 8 weeks postlesion. [³H]Phorbol-12,13-dibutyrate (PDBu), [³H]forskolin and [³H]rolipram were used to label protein kinase C (PKC), adenylyl cyclase and calcium/calmodulin-independent cyclic-AMP phosphodiesterase, respectively. The degeneration of nigrostriatal pathway produced a significant increase in [³H]PDBu binding in the ventromedial part of the ipsilateral striatum from 2 to 8 weeks postlesion. In the contralateral side, [³H]PDBu binding showed a transient increase in the SN only 4 weeks after lesioning. [³H]Forskolin binding showed a significant increase in the ipsilateral and contralateral striatum from 2 to 4 weeks postlesion. In the ipsilateral SN, a significant increase in [³H]forskolin binding was observed at 4 weeks after lesioning. However, no significant change in [³H]forskolin binding was observed in the contralateral SN during postlesion. On the other hand, [³H]rolipram binding showed no conspicuous alteration in the brain during postlesion. These results demonstrate that rats made hemiparkinsonism by unilateral 6-hydroxydopamine injection have a significant increase in [³H]PDBu and [³H]forskolin binding in the striatum and/or SN, whereas no significant change in [³H]rolipram binding is observed in these areas during postlesion. Our findings also suggest that the increase in [³H]forskolin binding is more pronounced than that in [³H]PDBu binding in the brain after unilateral 6-hydroxydopamine injection. Thus, our studies may provide valuable information concerning degeneration of the nigrostriatal pathway such as Parkinson’s disease.     

Development of tolerance to amnesic effects of chlordiazepoxide in relation to GABAergic and cholinergic neuronal systems

Chronic administration of benzodiazepines has been reported to produce tolerance in animals and humans. We investigated whether benzodiazepines produce tolerance to the amnesic effects and effects on benzodiazepine receptors, GABAergic and/or cholinergic neuronal systems of repeated administration of chlordiazepoxide, using a passive avoidance task and autoradiographic techniques. Tolerance developed to the amnesic effect of chlordiazepoxide when the drug was administered at a dose of 30 mg/kg (i.p.) once a day for 14 days. Bicuculline (1.0 and 1.5 mg/kg), a GABA_A receptor antagonist, did not induce amnesia in normal mice, but did so in chlordiazepoxide-tolerant mice. Muscimol (0.25 mg/kg), a GABA_A receptor agonist, in combination with a low dose of chlordiazepoxide, induced amnesia in normal mice, but not in chlordiazepoxide-tolerant mice. Scopolamine, an acetylcholine receptor antagonist, induced amnesia in normal mice, but not in chlordiazepoxide-tolerant mice. In the autoradiographical study, although repeated treatment with chlordiazepoxide had no effect on [³H]flunitrazepam and [³H]Ro 15-4513 binding to benzodiazepine receptors, it decreased [³H]muscimol binding to GABA_A receptors, with a decrease in affinity in the cortex and hippocampus. Furthermore, repeated administration of chlordiazepoxide increased [³H]quinuclidinyl benzilate binding to muscarinic acetylcholine receptors in the hippocampus. These results suggest that tolerance develops to the amnesic effects of chlordiazepoxide, and that tolerance may be due to down-regulation of GABA_A receptors and/or up-regulation of acetylcholine receptors.     

Sex differences in DNA damage produced by the carcinogen 2-acetylaminofluorene in cultured human hepatocytes compared to rat liver and cultured rat hepatocytes

Male rats are more susceptible to the induction of liver cancer by the aromatic amine 2-acetylaminofluorene (AAF) than are females. To assess the basis for this difference and to determine whether sex differences in susceptibility to AAF are present in human liver cells, the DNA reactivity of AAF was measured in livers of male and female Sprague–Dawley (SD) rats and in cultured SD rat and human hepatocytes of both sexes. In livers of rats administered oral doses of AAF, the total levels of adducts measured by nucleotide postlabelling at up to 8 weeks were about twofold greater in males than in females. Similarly, the level of AAF-DNA adducts formed in cultured male rat hepatocytes dosed with AAF was about twofold greater than in female rat hepatocytes. Also, the level of DNA repair synthesis was about threefold greater in AAF-dosed cultured male rat hepatocytes compared with female, indicating that the greater adduct levels in males was not due to lesser repair. In contrast, in cultured human hepatocytes of both sexes, AAF produced similar levels of adducts and DNA repair synthesis, which were intermediate between those produced in male and female rat hepatocytes. Thus, the greater susceptibility of male rats to AAF hepatocarcinogenicity is due at least in part to greater bioactivation and formation of AAF-DNA adducts in hepatocytes. Moreover, the data from human hepatocytes suggest that human liver could be less susceptible than male rat liver to the carcinogenic effects of aromatic amine carcinogens of the AAF type.     

Haloperidol treatment differentially regulates [H]DTG and [H](+)-3-PPP labeled σ binding sites

The effect of repeated haloperidol administration on σ binding sites in brain membranes was assessed using [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP) and [³H]1,3-di-o-tolylguanidine (DTG). Administration of haloperidol (1 mg/kg, i.p.) to guinea pigs for 14 consecutive days followed by a 4 day drug-free period prior to sacrifice resulted in 75% and 6% decreases in the specific binding of [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and [³H]1,3-di-o-tolylguanidine, respectively, when measured using a single concentration (2 nM) of radioligand. Scatchard analysis revealed a reduction in both the maximum number of [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding sites and the affinity of these sites for the radioligand; the potency of 1,3-di-o-tolylguanidine to inhibit [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding was also reduced. In parallel studies, the potency of 1,3-di-o-tolylguanidine to inhibit [³H]1,3-di-o-tolylguanidine binding was unaffected by haloperidol treatment, but the potency of (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine against [³H]1,3-di-o-tolylguanidine was reduced 3-fold. Phenytoin, which increased (10-fold) the potency of dextromethorphan to inhibit [³H]1,3-di-o-tolylguanidine binding in control membranes, had no effect in membranes obtained from haloperidol-treated animals. The reduction in [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding was dependent upon the duration of the drug-free period and amounted to 73% and 25% in brain membranes prepared from animals sacrificed 14 days and 28 days, respectively, following cessation of drug treatment. Repeated administration of other antipsychotic and σ agents including DTG, dextromethorphan, spiperone, chlorpromazine and clozapine had no effect on [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine or [³H]1,3-di-o-tolylguanidine binding. These findings suggest that repeated haloperidol administration selectively regulates [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding in guinea pig brain membranes. However, the observations that repeated drug treatment resulted in (1) reduced affinity of (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine for [³H]1,3-di-o-tolylguanidine binding sites, and (2) a loss of the ability of phenytoin to increase dextromethorphan's potency against [³H]1,3-di-o-tolylguanidine, suggest that [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and [³H]1,3-di-o-tolylguanidine binding sites are distinct but functionally coupled.     

Pyridine does not induce unscheduled DNA synthesis (UDS) in hepatocytes of male B6C3F1 mice treated in vivo

Pyridine was evaluated in an in vivo/in vitro mouse DNA repair assay. Unscheduled DNA synthesis (UDS) was used as an indicator of DNA damage to hepatocytes from male B6C3F1 mice. Test animals were exposed by oral gavage to pyridine or to the vehicle or positive control articles, and hepatocytes were collected and labeled by incubation in media supplemented with [3H]thymidine. Following labeling, the cultures were processed for autoradiographic analysis. Doses were selected based on a pilot study in which 0, 250, 500, 750, 1000 or 2000 mg kg(-1) pyridine in water was administered by gavage. Mice in the 1000 and 2000 mg kg(-1) dose groups were comatose following dosing and died within 24 h of dose administration. Pyridine dose levels for the UDS determination were set at 175, 350 and 700 mg kg(-1). Pyridine solutions in water were administered to mice 2 or 16 h prior to the scheduled sacrifice. The vehicle control group received water 16 h before sacrifice and the positive control group received 10 mg kg(-1) dimethylnitrosamine (DMN) 2 h before sacrifice. Pyridine did not significantly increase the UDS response in hepatocytes isolated from the treated animals, as measured by the incorporation of [3H]thymidine, using standard criteria for a negative response: less than zero mean net grains in repair (NG) and <20% of cells in repair (% IR; cells in repair have at least 5 NG). The vehicle control group and the low, mid- and high pyridine dose groups yielded less than -8.3 NG and < or =1% IR. The positive control group yielded a positive UDS response, with 10.8 NG and 62% IR. These results indicate that pyridine is non-genotoxic in B6C3F1 mouse liver using the UDS endpoint.     

High-affinity [H]6-nitroquipazine binding sites in rat brain

6-Nitroquipazine is a very potent and selective inhibitor of neuronal 5-hydroxytryptamine (5-HT; serotonin) uptake. We have characterized the specific binding of [³H]6-nitroquipazine to rat brain membranes at 22°C. The present results indicate the presence of a single saturable high-affinity binding component for [³H]6-nitroquipazine. Scatchard analysis revealed an apparent equilibrium dissociation constant (K_d) of 93.0 ± 2.23 pM, and a maximal number of binding sites (B_max) of 831.7 ± 18.7 fmol/mg protein (mean ± S.D., n = 4). The kinetically derived dissociation constant was 74.5 pM. [³H]6-Nitroquipazine binding was inhibited selectively by 5-Ht uptake inhibitors, and a good correlation was demonstrated between the potency of various drugs to inhibit [³H]6-nitroquipazine binding and [³H]5-HT uptake. The highest densities of [³H]6-nitroquipazine binding were obtained in the hypothalamus and midbrain, intermediate binding was observed in the striatum, hippocampus, medulla oblongata and cortex, and the lowest binding was observed in the cerebellum. Lesioning of 5-HT neurons with p-chloroamphetamine resulted in a 72% reduction in [³H]6-nitroquipazine binding compared to controls. These results indicate that the binding site specifically labelled by [³H]6-nitroquipazine is associated with the neuronal 5-HT transporter complex. [³H]6-Nitroquipazine is an excellent radioligand for the study of the 5-HT uptake system.     

Mechanism of butylated hydroxytoluene-associated modification of diethylnitrosamine-induced squamous stomach carcinoma

The metabolism of butylated hydroxytoluene (BHT) and the effect of BHT on the metabolism of diethylnitrosamine (DEN) was studied in male and female BALB/c mice to further understanding of the selective protection of BHT on the incidence of DEN-induced squamous-stomach carcinomas in female (but not in male) mice. Following intragastric administration of [¹⁴C]BHT, the antioxidant was covalently bound to tissue macromolecules. The relative distribution of this bound BHT varied with time; 8 hr after [¹⁴C]BHT administration, most of the covalently bound BHT was associated with the protein components; at 96 hr the nucleic acid components bound more BHT than did the protein components. Animals pretreated with BHT and given [¹⁴C]DEN intragastrically had lower blood levels of radioactivity and eliminated a larger percentage of DEN and/or its metabolites in the urine and as carbon dioxide than animals given [¹⁴C]DEN alone. The binding of DEN and/or its metabolites to cellular macromolecules of the squamous stomach of female animals was decreased following pretreatment with BHT. However, the BHT-associated decrease in DEN binding was also observed in the squamous stomach of male animals and in the liver of both sexes, although the tumour incidence in these target organs for DEN carcinogenesis is not modified by BHT. These results suggest that the BHT-associated decrease in the binding of DEN to DNA is of a generalized rather than a selective nature, and may be insufficient to account for the protective effect of BHT. Two parameters that were found to parallel the susceptibility of DEN target tissues to the anticarcinogenic effects of BHT were the relative degree of inhibition of DEN bound to RNA species and the relative amount of BHT bound to DNA. Thus the anticarcinogenic properties of BHT may be more complex than an induction of enzymes that detoxify the carcinogen and/or an inhibition of enzymes that activate the carcinogen with a resulting decrease in the quantity of carcinogen available for electrophilic reactions.     

Involvement of reversible binding to alpha 2u-globulin in 1,4-dichlorobenzene-induced nephrotoxicity

Similarly to unleaded gasoline, 1,4-dichlorobenzene (1,4-DCB) administered for 2 years caused a dose-related increase in the incidence of renal tumors in male but not in female rats or in either sex of mice. Unleaded gasoline and 2,2,4-trimethylpentane (TMP), a component of unleaded gasoline, increased protein droplet formation and cell proliferation in male but not in female rat kidneys. These protein droplets contained, alpha 2u-globulin, a male rat-specific low-molecular-weight protein and 2,4,4-trimethyl-2-pentanol, a metabolite of TMP that was reversibly bound to this protein. Studies were undertaken to determine if 1,4-DCB produced similar effects; 1,2-DCB was used for comparison since it did not produce renal carcinogenesis in male rats. Gel filtration chromatography of a 116,000g supernatant prepared from kidneys of 1,4-[14C]DCB-treated rats showed that radiolabel coeluted with alpha 2u-globulin as one sharp peak as opposed to a multipeak pattern observed for 1,2-[14C]DCB; the maximal quantity of radiolabel for 1,4-DCB was twice that for 1,2-DCB. Equilibrium dialysis of kidney cytosol in the presence or absence of sodium dodecyl sulfate demonstrated that the radiolabel was reversibly bound to alpha 2u-globulin; the amount for 1,4-[14C]DCB-treated rats was almost twice as much as that for 1,2-[14C]DCB-treated rats. 1,2-DCB was also shown to be covalently bound to renal alpha 2u-globulin, and covalently bound to liver and plasma high-molecular-weight proteins. 1,4-DCB and, to a minor extent, 2,5-dichlorophenol, the major metabolite of 1,4-DCB, were reversibly bound to renal alpha 2u-globulin from 1,4-DCB-treated rats. 1,4-DCB increased protein droplet formation in male but not in female rat kidneys, whereas equimolar doses of 1,2-DCB showed no effect in either sex. Renal cell proliferation, measured by [3H]thymidine incorporation into renal DNA, was increased after 1,4-DCB but not after 1,2-DCB treatment. Nephrotoxicity and biochemical alterations induced by 1,4-DCB resemble those of unleaded gasoline and suggest that a similar mechanism is involved in the induction of alpha 2u-globulin nephropathy in male rats.