Studies on the in vitro turnover of histone acetyl groups and the increase of RNA synthesis in rat liver nuclei after in vivo application of alpha-hexachlorocyclohexane (alpha-HCH).

Nuclear uptake of radioactive acetate into rat liver histones in vitro peaked at 2 and 24 hr after α-HCH application, when control values were exceeded by approximately 200%. The magnitudes of acetate uptake into histone fractions of rats treated with α-HCH for 24 hr and of control rats were in the following order: F3 > F2b > F2al > F2a2. Retention of radioactive acetate in F3 and F2al histone fractions of rats pretreated with α-HCH for 24 hr accounted for 61% of the radioactive acetate retained in total histones, which was nearly seven times higher than in histones of control rats. Preincubation of rat liver nuclei isolated 24 hr after α-HCH administration with acetyl-coenzyme A resulted in an enhancement of the already α-HCH stimulated RNA synthesis. This, as well as the coincidence of maximally increased acetate content in histones and increased nuclear RNA synthesis 24 hr after α-HCH, suggests a connection between the two nuclear processes. No effect of preincubation with acetylcoenzyme A was seen on RNA polymerase activities in liver nuclei from rats pretreated for 2 hr with α-HCH which also contain highly acetylated histones or in liver nuclei from control rats. So it is assumed that beside the increase of histone acetate content additional conditions need to be fulfilled to start and to guarantee the α-HCH induced gene activation in rat liver.     

Effects of anionic xenobiotics on rat kidney: I—tissue and mitochondrial respiration

The polar 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) metabolite, 2,2-bis p-chlorophenyl)acetic acid (DDA), and the phenoxyacetic acid herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), were previously shown to be accumulated to high levels in liver and kidney via the organic acid transport system, raising the possibility of organ-specific toxicity secondary to transport. In these studies, accumulation of DDA was shown to depress oxygen consumption by renal cortical slices at high doses (0.1 and 1 mM). Isolated renal and hepatic mitochondria were uncoupled by low doses of DDA (5–10 nmoles/mg mitochondrial protein). Maximal uncoupling was seen at 50–70 nmoles/mg. 2,4-D and 2,4,5-T also produced uncoupling, but at doses of 70 nmoles/mg or higher. All agents were more effective with α-ketoglutarate or pyruvatemalate as substrate than with succinate. With succinate as substrate (but not α-ketoglutarate or pyruvate-malate), all three agents also depressed State 3 (ADP-stimulated) respiration. Again. DDA was more effective than 2,4-D or 2,4,5-T. These results suggest that accumulation of these or other anionic xenobiotics may lead to toxicity in those tissues possessing the organic anion transport system.     

Adrenergic and cholinergic innervation of the spleen of the cod: Gadus morhua

Injections of catecholamines into the splenic artery constricted the cod spleen when perfused in situ at constant pressure. The doses of amines producing 50% of the maximal effect were as follows (mean ± S.E.M.): adrenaline 1.3 ± 0.3 nmoles; noradrenaline 6.3 ± 1.1 nmoles; phenylephrine 10.0 ± 2.3 nmoles; isoprenaline > 10 μmoles. The findings indicate that the response is mediated by α-adrenoceptors. The responses were antagonized competitively by yohimbine (pA₂ = 6.9) and phentolamine (pA₂ = 7.1) and non-competitively by phenoxybenzamine (0.1 μM). No evidence indicating the presence of β-adrenoceptors has been obtained. Acetylcholine similary causes spleen constriction (50% of maximal with 0.8 ± 0.1 nmoles, mean ± S.E.M.); its effect is blocked competitively by atropine (pA₂ = 8.5) but is not affected by hexamethonium or mecamylamine, indicating that muscarinic cholinoceptors independently mediate splenic constriction. Stimulation of the Splanchnic nerve, but not the vagi, constricted the spleen. Attempts to block this effect with selective doses of receptor-blocking agents show that both adrenergic and cholinergic neurones are present, although in different fish the adrenergic/cholinergic ratio varied. Splenic constriction releases previously sequestered red blood cells into the venous circulation. On histochemical examination, nerve fibres in small arteries and trabecular smooth muscle exhibited a weak fluorescence which could be increased by preinjection of noradrenaline: the amine of the fibres is most probably noradrenaline.     

Neuropharmacological effects of isomers of hexachlorocyclohexane. 1. Protection against pentylenetetrazol-induced convulsions

Isomers of 1,2,3,4,5,6-hexachlorocyclohexane (HCH) have been reported to reduce the sensitivity of rats to the convulsant action of pentylenetetrazol (PTZ). The effect has been reexamined with a view to assessing whether γ-HCH, the insecticide lindane, and its nonconvulsant isomers act alike as PTZ antagonists. In a preliminary survey, the kinetics of HCH concentrations in brain were established after oral doses of the isomers α through δ. Brain:blood concentration ratios, estimated at the time of peak brain content, were smaller for β-HCH than the other isomers. Approximate half-lifes, recorded in females, for the elimination from brain were, in days: δ-HCH, 0.5; γ-HCH, 1.5; α-HCH, 6; β-HCH, 20. PTZ-antagonistic action was tested for and was confirmed to be a property of all four isomers, using as a measure of effect the PTZ concentration in brain after the onset of the first seizure that developed while the drug was infused iv at a constant rate. Minimally effective concentrations (MEC) of HCH in brain indicated that the order of potency was β-HCH ～ γ-HCH > δ-HCH > α-HCH. The MEC were three to five times lower than brain concentrations associated with signs of neurotoxicity, except for γ-HCH for which the difference was negligible. For single oral doses of α-, β-, and δ-HCH onset, intensity and duration of the PTZ-antagonistic effects were related to brain content of HCH. After γ-HCH, the state of lowered sensitivity to PTZ was preceded by a transient state of heightened sensitivity while brain content of HCH was rising. Once developed, the PTZ-antagonistic effect of γ-HCH was unrelated, in its intensity and duration, to the HCH concentration in brain. It is concluded that the effects of γ-HCH and of its nonconvulsant isomers have different mechanisms, α-, β-, and δ-HCH antagonizing PTZ in a manner analogous to that of certain kinds of anticonvulsant. In distinction to the state after treatment with α-, β-, or δ-HCH, the brains of rats given γ-HCH contained substantial quantities of metabolites. The major one of these, (3,6/4,5)-1,2,3,4,5,6-hexachlorocyclohexene-1, was found to have PTZ-antagonistic action. However, evidence is presented to show that metabolites are unlikely to contribute to the development or maintenance of the effect produced by the parent compound.     

Effect of a selective beta 2-adrenoceptor agonist, procaterol, on tissue cyclic AMP level. Its determination after tissue fixation by microwave irradiation.

Intravenous injection of procaterol (1μmole/kg of body wt). a selective β₂-agonist, into conscious rats caused increases in tissue cyclic AMP, with a peak level at 2 min after injection. Two- to 3-fbld increases were induced by procaterol in heart and liver, while 6- to 10-fold increases were induced in trachea, lung and skeletal muscle. Procaterol-induced alterations in cyclic AMP levels in trachea and skeletal muscle were characterized by their long duration (over 2 hr). The duration of the procaterol action reflected sustained stimulation of β-adrenoceptor-linked adenylate cyclase, because the tissue level of cyclic AMP subsided rapidly to the base-line level when propranolol (10 μmoles/kg) was injected intravenously immediately after the peak level was obtained with procaterol. 3-Isobutyl- 1-methylxanthine (50 μmoles/kg, s.c.), an inhibitor of phosphodiesterase, was very effective in enhancing the procaterol-induced increases in cyclic AMP in trachea, lung and skeletal muscle. Procaterol caused increases in tissue cyclic AMP levels in skeletal muscle. trachea, lung and heart at doses of 1, 10, 10 and 100 nmoles/kg, respectively, in a dose-dependent manner. It was concluded that procaterol was a long-acting and selective β₂-agonist which was effective in increasing tissue cyclic AMP, as well as in increasing bronchodilator and metabolic alterations. Determination of tissue cyclic AMP using rapid tissue fixation by microwave irradiation could serve as a useful means in vivo for the interaction of β-agonists or antagonists with β₁- or β₂-adrenoceptors.     

Comparative study on the distribution of α- and γ-hexachlorocyclohexane in the rat with particular reference to the problem of isomerization

1. Blood levels and tissue distributions of α-, β-, γ- and δ-hexachlorocyclohexane (HCH) were studied following oral administration of α-HCH and γ-HCH to rats.

2. Following administration of α-HCH, there was no evidence of β-HCH, γ-HCH or δ-HCH, nor could transformation into α-HCH, β-HCH or δ-HCH be detected after exposure to γ-HCH.

3. After eight weeks of administration, tissue retention of α-HCH was 10–20 times greater than that of γ-HCH.

4. γ-HCH was eliminated to a much greater extent than α-HCH from the tissues, and in particular from fatty tissue.

5. α-HCH accumulated in fat and brain, while γ-HCH showed very low affinity for lipid.     

Effects of the interferon inducing agents tilorone and polyriboinosinic acid · polyribocytidylic acid (poly IC) on the hepatic monooxygenase systems of pregnant and fetal rats

Interferon inducing agents, including tilorone and polyriboinosinic acid polyribocytidylic acid (poiy IC), are known to depress hepatic cytochrome P-450-dependent monooxygenase systems and the induction of these systems by phenobarbital (PB) and 3-methylcholanthrene (MC) in mature male rats. The current study investigated the effects of tilorone and poly IC on the cytochrome P-450 systems of non-induced, PB-induced, MC-induced and pregnenolonecarbonitrile (PCN)-induced pregnant rats and their fetuses. Pregnant rats received either tilorone or poly IC and saline, PB, MC or PCN, and microsomes from their livers and those of their fetuses were examiued for cytochrome P-450 content, aminopyrine (AP) N-demethylase activity and benxo[a]pyrene (BP) hydroxylase activity. The generalixation can be made from these studies that, when the interferon inducing agents caused changes in cytochrome P-450 content or monooxygenase activities of either induced (PB, MC or PCN) or non-induced (saline) animals, decreases were seen in maternal livers and increases in fetal livers. Thus, in maternal livers tilorone depressed cytochrome P-450 and AP N-demethylase activity in non-induced and PB-, MC- and PCN-induced rats and BP hydroxylase activity in the induced animals; BP hydroxylase activity was not depressed in non-induced maternal livers. Poly IC depressed cytochrome P-450 and AP N-demethylase activity in non-induced and PB-induced rats but not in PCN-induced animals. BP hydroxylase was depressed by poly IC in both PB- and PCN-induced animals. Fetal hepatic cytochrome P-450 and monooxygenase activities were increased by tilorone in PB- and PCN-induced rats but not in non-induced or MC-induced animals. Poly IC increased cytochrome P-450 and both monooxygenase activities in PB- and PCN-induced fetal livers, whereas only BP hydroxylase activity was increased in the fetuses of non-induced rats. Several possible explanations are offered for the opposite effects produced by interferon inducing agents in maternal and fetal livers. Unlike maternally administered tilorone, which induced fetal cytocbrome P-450 and monooxygenase activities in the liver, intrauterine tilorone depressed cytochrome P-450 and had no effect on AP N-demethylase or BP hydroxylase activities in the fetal liver. Intrauterine poly IC was without effect on the cytochrome P-450 systems of the fetal liver. Treatment of pregnant rats with tilorone on days 17–20 of gestation inhibited normal maternal weight gain and produced overt signs of toxicity. A dose of 10 mg/kg of poly IC was very toxic in pregnant rats but produced no overt signs of toxicity in virgin female rats. Time courses of the depressant effects of a single injection of poly IC were observed in mid-term pregnant, late-term pregnant, lactating and adult virgin females. Maximum losses of cytochrome P-450 and ethyhnorphine (EM) N-demethylase activity were seen 48 hr after poly IC administration to pregnant and virgin rats, and recoveries were complete within 96 hr. Similar results were observed in lactating rats except that the nadir occurred at 24 rather than at 48 hr. The response of BP hydroxylase activity to poly IC was qualitatively similar except that this activity was not depressed in the mid-term pregnant rats.     

Inflammatory-mediated pathway in association with organochlorine pesticides levels in the etiology of idiopathic preterm birth

Elevated inflammation is a known risk factor in the pathogenesis of PTB. Despite intensive research, the etiology of idiopathic PTB is still unknown. The present study was designed to explore associations of blood concentrations of organochlorine pesticides (OCPs) with inflammatory/antioxidant gene expression, and cytokines and prostaglandin levels in PTB cases. Significantly high levels of α, β-hexachlorocyclohexane (α, β-HCH), dichlorodiphenyldichloroethane (o’p’-DDD), dichlorodiphenyldichloroethylene (p’p’-DDE), increased expression of cyclooxygenase-2 (COX-2), and decreased expression of manganese superoxide dismutase (Mn-SOD) and catalase (CAT) genes were seen in PTB cases. Also, increased protein levels of interleukin-6 (IL-6) and decreased protein levels of interleukin-4 (IL-4) and prostaglandin F2α (PGF2α) were found in maternal blood of PTB cases as compared to term controls. Elevated levels of β-HCH along with high expression of COX-2 gene or low expression of Mn-SOD or CAT genes were associated with the decrease in the period of gestation (POG).     

Effect of ehxachlorophene on hepatic drug-metabolizing enzymes in the rat

Hexachlorophene (HCP) at oral doses of 30–50 mg/kg causes significant increases in hexobarbital sleeping time in Wistar and Sprague-Dawley rats, with the maximum effect occurring 6 hr after administration of the bisphenol. Liver microsomal 0-demethylase activity is simultaneously reduced in rats receiving HCP. Incubation of rat liver microsomes with concentrations of HCP as low as 0.38 nmole/mg of microsomal protein in vitro inhibits the O-demethylase, nitroreductase and phenol UDP-glucuronyl transferase systems and also causes a reduction in the apparent content of cytochromes P-450 and b₅. The concentrations of HCP required to produce a 50 per cent inhibition or reduction in apparent cytochrome contents in vitro range between 4.7 and 98 nmoles HCP/mg of microsomal protein. Some evidence for a common inhibitory mechanism, perhaps involving interaction of HCP with the microsomal membrane, was obtained for the hepatic mixed function oxidase and cytochrome systems.     

Histopathological study of Poecilia reticulata (guppy) after long-term β-hexachlorocyclohexane exposure

Within the framework of toxicity studies in which several freshwater organisms were exposed to the β-isomer of hexachlorocyclohexane (β-HCH), histopathological examination was carried out on young Poecilia reticulata after 1- and 3-mth exposure to concentrations ranging from 0.0032–1.0 mg β-HCH/l water. Results showed a concentration-related increased hepatocellular basophilia caused by hypertrophy of the rough endoplasmic reticulum, an increase in staining intensity of the blood plasma, ascitic fluid and interstitial tissue fluid, hypertrophy of the endocardial lining cells caused by accumulation of hyalin droplets in lysosomes, and accumulation of hyalin droplets in glomerular and tubular epithelia of the kidney. These findings are attributed to an excessive production of the yolk precursor vitellogenin (Vg) by the liver, which was induced by an estrogen-like activity of β-HCH or its metabolite(s). Indeed, similar changes were found in liver, heart and body fluid in an additional short-term experiment, in which young fish were treated with 17β-estradiol. Vg was identified using slab-gel electrophoresis which demonstrated high concentrations of analogous, high molecular weight proteins in the body fluids both after exposure to β-HCH and to 17β-estradiol. Moreover, a histochemical staining method was developed for phosphoproteins such as Vg, by which it could be localized in routine tissue sections. The no-effect concentration of this complex of changes was 0.032 mg β-HCH/l after both 1 and 3 mth. In addition, after 3-mth exposure dysvitellogenesis was noted in the females, while in treated males the gonadotropic hormone producing cells in the pituitary gland appeared stimulated and testicular development was retarded at 0.32 mg/l and higher. From this study it is concluded that β-HCH exerts an estrogenic action on male and female guppies at 0.1 mg/l and higher, which is analogous to the effect reported in rodents.     

Effects of phenobarbital and 3-methylcholanthrene administration on glutathione-S-epoxide transferase activity in rat liver

Glutathione-S-epoxide transferase activity in rat liver was studied with three epoxide substrates. The specific activities in the high-speed liver supernatant fraction for 3MC 11, 12 oxide, styrene oxide and 3,3,3-trichloro-1,2 epoxy propane were 5.7, 86.4 and 165 nmoles/5 min/mg protein, respectively. Phenobarbital (75 mg/kg body wt for 3 days) or 3-methylcholanthrene (20 mg/kg body wt for 2 days) administration to rats resulted in a 40–60% increase in enzyme activity. β-napthoflavone administration on the other hand was without any effect on glutathione-S-epoxide transferase activity.     

Differential effects of aging on hepatic microsomal monooxygenase induction by phenobarbital and β-naphthoflavone

The influence of aging on hepatic microsomal monooxygenase induction by phenobarbital (PB) or β-naphthoflavone (BNF) was investigated in male Fischer 344 rats maintained in a constant environment. PB-induced increases in microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity were similar in rats aged 3–5 months (young-adult) and 24–25 months (old), but increases in benzephetamine N-demethylase activity were markedly diminished in the old rats. Separation of hepatic microsomal proteins by sodium dodecylsulfate gel electrophoresis demonstrated that aging decreased the induction by PB of a polypeptide with a molecular weight of 52,500. BNF-induced increases in microsomal cytochrome P-450 and nitroanisole O-demethylase activity were greater in old than in young-adult rats, and BNF induction of 55,000 and 57,000 molecular weight microsomal polypeptides was increased slightly in livers from old rats. The results indicate that age-related effects on monooxygenase induction vary with different inducers of the hepatic microsomal enzyme system.     

Acute paracetamol intoxication of starved mice leads to lipid peroxidation in vivo.

Lipid peroxidation was monitored in female mice in vivo by the measurement of exhalated hydrocarbons. In liver homogenates in vitro lipid peroxidation as determined by malondialdehyde formation, and hepatic total glutathione levels were measured. After a dose of 500 mg/kg i.p. of paracetamol, the hepatic glutathione of fed mice decreased from 61 nmoles/mg liver protein to 30 nmoles/mg, while the animals expired 5 nmoles of ethane/kga · hr. The same dose in starved mice led to a glutathione level of 6 nmoles/mg and an exhalation rate of 125–150 nmoles ethane/kg · hr. In vivo determined and post-mortem in vitro determined lipid peroxidation correlated with a coefficient of 0.66. If hepatic glutathione was depleted to the same extent by administration of diethylmaleate, no significant lipid peroxidation was found. Our findings demonstrate that the drug-induced depletion of liver glutathione leads in vivo to lipid peroxidation, provided that the glutathione level has been diminished by starvation. The data indicate that glutathione depletion alone by other mechanisms does not account for lipid peroxidation. Hence the hepatoprotective role of liver glutathione against drug-induced liver injury has to be reconsidered in detail. This investigation shows a suitable model for such studies.     

The role of lysosomes in the hepatic accumulation and release of beryllium

The toxic metal beryllium (Be) can produce hepatic necrosis and is known to be concentrated by the liver after i.v. injection of particulate and soluble Be compounds. In the present study the accumulation and release of Be by a hepatic lysosome fraction of the rat has been examined after i.v. administration of sublethal doses of particulate Be phosphate (12.5 and 150 μmoles/kg) or the more hepatotoxic soluble BeSO₄ (12.5 and 25 μmoles/kg). Maximal lysosomal Be content is produced within 2 hr or 5 hr after the injection of Be phosphate or BeSO₄ respectively and in both cases this is followed by a gradual decrease in the lysosomal Be concentration of the next 7 days to 30–40 per cent of the maximal values, which is consistent with the loss of Be observed for whole liver. The release of Be from lysosomes has been examined by measurement in vitro of the liberation of Be into a suitable incubation medium from lysosomes prepared from Be treated animals. The results indicate that the liberation of Be was maximal for lysosomes isolated 5 hr after injection of either form of Be and was accompanied by a significant release of lysosomal β-glucuronidase activity, particularly at the higher doses of Be used, and therefore indicates that some loss of lysosomal integrity occurred. Rupture of the lysosomal membrane could not be demonstrated in vitro by incubation of lysosomes from untreated animals with externally added Be compounds. It is concluded, however, that any release of hydrolytic enzymes into the cytosol occurring in vivo may not be the major cause of cell necrosis produced by Be and it is suggested, therefore, that the role of lysosomes in Be hepatotoxicity is primarily in the intracellular accumulation and subsequent release of Be and that the main cytotoxic target for Be is probably extralysosomal.     

Effect of selenium on glutathione metabolism. Induction of gamma-glutamylcysteine synthetase and glutathione reductase in the rat liver

The effect of sodium selenite (Na₂SeO₃, Se) on cellular glutathione metabolism was examined, particularly with respect to its ability to alter the activities of γ-glutamylcysteine synthetase and glutathione disulfide (GSSG) reductase. The treatment of rats with Se (5, 10 and 20 μmoles/kg) caused time- and dose-dependent increases in the activities of the synthetase and the reductase in the liver. The activity of γ-glutamylcysteine synthetase, the rate-limiting enzyme of the glutathione (GSH) biosynthesis, was particularly susceptible to Se treatment. The Se-mediated increases in the activities of the above enzymes were inhibited by puromycin and the increases could not be elicited in vitro. Selenium treatment caused time-dependent perturbations in the levels and ratio of GSSG and GSH in the liver. When compared to the control animals, rats treated for 3 hr with 10 and 20 μmoles Se/kg showed increased cellular levels of GSSG; in contrast, 24 hr after Se treatment the concentration of GSH was increased significantly. The activity of γ-glutamyl transpeptidase, which catalyzes the initial reaction in GSH breakdown, was unaltered by Se treatment. Repeated administration of low doses of Se (7.0 μmoles/kg, three times) also increased the activities of the reductase and the synthetase as well as the cellular levels of hepatic GSH and GSSG. It is suggested that the Se-mediated increases in the activities of γ-glutamylcysteine synthetase and GSSG-reductase represent cellular responses to Se-mediated perturbations in the levels and ratio of GSH and GSSG.     

Effects of polychlorinated biphenyls on the monooxygenase systems in fetal livers of rats

The ontogenetic development of monooxygenase activities in fetal livers of rats and inducing effects of PCB on them were investigated. The following results were obtained. 1. The activities of monooxygenase systems in fetal livers of rats were detected on Days 17 to 22 of gestation, associated with NADPH-cytochrome c reductase activity and cytochrome P-450 content. All activities and content increased during the development. 2. These activities and content on Days 17 to 22 of gestation were induced by PCB given orally to pregnant rats. 3. The inducibilities of hexobarbital- and aminopyrine-metabolizing activities were very different from that of the benzo[α]pyrene-metabolizing activity. The results would indicate the existence of the different monooxygenase systems in the fetal liver.     

Further studies concerning the effects of clofibrate on respiration and oxidative phosphorylation of rat liver mitochondria

Clofibrate, administered in vitro, inhibited rat liver mitochondrial respiration at two sites within the respiratory chain. One site was between the interaction of NADH with NADH dehydrogenase and the point at which electrons from succinate oxidation enter the electron transport chain; another, less sensitive site, was between the interaction of succinate with succinate dehydrogenase and cytochrome c. In addition to these specific sites, clofibrate inhibited respiration by causing a depletion of pyridine nucleotides that was accompanied or followed by large-amplitude, non-energy-linked swelling. Clofibrate uncoupled oxidative phosphorylation at coupling sites II and III but not at site I. The concentrations required to cause loss of pyridine nucleotides were lower than those required to inhibit at the specific sites. p-Chlorophenoxyisobutyrate (CPIB) also inhibited succinate and β-hydroxybutyrate-linked respiration, and uncoupled oxidative phosphorylation, but at much higher concentrations (50 per cent inhibition of β-hydroxybutyrate oxidation at about 3·7 μmoles/mg of protein) than were required of clofibrate (50 per cent inhibition of β-hydroxybutyrate oxidation at about 0·17 μmole/mg of protein). Clofibrate administration to rats (100 and 300 mg/kg p.o. daily for 1 week) lowered serum lipid levels and increased the liver size, the amount of mitochondrial protein/g of liver, and the oxygen consumption of liver slices. However, mitochondria, isolated from livers of the treated rats, respired normally. A single administration of clofibrate (100 or 300 mg/kg, p.o.) did not affect liver slice respiration.     

Effect of alpha-methyldopa on dopaminergic transmission in the corpus striatum.

α-Methyldopa (200 mg kg^?1 i.p.) administration to rats caused a decline in dopamine and a rise in α-methyldopamine levels in the corpus striatum which were maximal between 4–6 hr after injection. Subcellular fractionation of the striatum 4 hr after α-methyldopa administration demonstrated that α-methyldopamine had accumulated within synaptosomes in approximately equal quantities to dopamine and the subcellular distribution of both amines was similar. Ca²⁺-dependent release of both dopamine and α-methyldopamine could be stimulated by exposing the synaptosomes to 60 mmol l^?1 K⁺. α-Methyldopamine was substantially less potent than dopamine in stimulating the dopamine-sensitive adenyl cyclase in striatal homogenates, and, at concentrations higher than those normally present in the brain following α-methyldopa administration, did not inhibit the stimulation of adenyl cyclase by dopamine. These results indicate that α-methyldopamine can act as a “false transmitter” in the corpus striatum and this may be the mechanism by which it impairs dopaminergic transmission.     

Differential effects of disulfiram and diethyldithiocarbamate on small intestinal and liver microsomal benzo[a]pyrene metabolism

Microsomes isolated from rat small intestinal mucosa and liver were used to study the effects of disulfiram and diethyldithiocarbamate on benzo[a]pyrene monooxygenase activity. This activity was decreased in the intestinal microsomes to 25 per cent of control 24 hr after a single oral dose of disulfiram. In contrast, daily administration of disulfiram for 5 days produced a dose related increase of benzo[a]pyrene monooxygenase activity, above control level. The elevated activities were accompanied by a concomitant increase in the concentration of cytochrome P-450. This benzo[a]pyrene monooxygenase activity was further stimulated by addition of α-naphthoflavone to the incubation medium. Furthermore, the absorption maximum of this cytochrome was at 450 nm in the CO bound reduced difference spectrum. These observations indicate that the disulfiram induced cytochrome P-450 was of the control type. Daily pretreatment with diethyldithiocarbamate impaired both intestinal and liver microsomes at benzo[a]pyrene monooxygenase activities. Pretreatment with a single dose of 3-methylcholanthrene resulted in a more than 10-fold increase of intestinal benzo[a]pyrene monooxygenase activity after 24 hr. Administration of disulfiram 24 hr before treatment appeared to potentiate the 3-methylcholanthrene induced increase of intestinal benzo[a]pyrene monooxygenase activity. In vitro addition of disulfiram and diethyldithiocarbamate to incubates of intestinal or liver microsomes inhibited benzo[a]pyrene metabolism to various extents; the liver being more sensitive. Disulfiram was approximately 50-fold more potent as an inhibitor than diethyldithiocarbamate. The in vitro inhibition of intestinal benzo[a]pyrene monooxygenase activity obtained with disulfiram appeared to be caused both by direct interaction with the monooxygenase system and through NADPH dependent metabolic activation of disulfiram, while the inhibition of diethyldithiocarbamate may be a result of the latter process only.     

The effect of gamma-hexachlorcyclohexane on rat liver lysosomes.

(1). An intraperitoneal injection of gamma-HCH (40 mg/kg, ld₅₀) was shown to induce a significant release of three lysosomal enzymes—acid phosphatase, β-glucosidase and cathepsin D—in rat livers already 1 hr after administration of the agent. From 1 hr up to 24 hr of the observation period the release of selected lysosomal hydrolases consistently increased. In contrast to the biochemical findings, the morphological changes including, hepatic necrosis and fatty infiltration, were established only 24 hr after gamma-HCH treatment. (2). The in vitro studies on the latency of two lysosomal enzymes—acid phosphatase and β-glucosidase—in the rat liver lysosome-rich suspension treated with gamma-HCH, showed that the concentrations of 10^?5 M, 10^?4 M and 10^?3 M significantly decreased latency of both enzymes. However, acid phosphatase and β-glucosidase, exhibited differential responses to the in vitro effect of gamma-HCH, which might be due to the differential affinities of these enzymes for the lysosomal matrix. The above mentioned concentrations of gamma-HCH did not significantly change the activities of acid phosphatase, β-glucosidase and cathepsin D in vitro. (3). No significant alterations were observed in the total activities of enzymes studied after in vivo and in vitro administered gamma-HCH. (4). The present study suggests that gamma-HCH exerts its effect on rat liver lysosomes by modifying the structural properties of the lysosomal membrane.