In vitro and in vivo inhibition by benserazide of clorgyline-resistant amine oxidases in rat cardiovascular tissues

Benserazide (d, l-serine 2-[2,3,4-trihydroxybenzyl]-hydrazide) has been shown to inhibit the clorgyline-resistant amine oxidase (CRAO) activities which metabolize benzylamine in homogenates of rat aorta, heart and brown adipose tissue. In vitro studies showed a concentration- and timedependent inhibition of CRAO in heart and aorta which was reversed by dialysis for 18hr. At high concentrations (10^?4?10^?3M) benserazide appeared to increase enzyme activity towards and occasionally above control value. These increases became more prominent after long periods of preincubation (especially in the presence of saturating benzylamine concentrations) and remained after dialysis of those homogenates preincubated with benserazide. The administration of benserazide for one or seven days in daily doses of 5–150 mg/kg also inhibited CRAO activity in vivo in a dose-dependent manner, with greater inhibition after seven days treatment. Reversal of inhibition, by dialysis of tissue homogenates from benserazide-treated rats, was much slower than was found with homogenates incubated in vitro with the drug. After benserazide administration to rats, MAO-A activity towards 5-hydroxytryptamine was generally not inhibited, and in fact was significantly increased in some cases. The administration of l-DOPA (250 mg/kg) together with benserazide (40 mg/kg) resulted in a similar degree of CRAO inhibition m aorta and heart to that seen after benserazide alone. These findings are discussed with regard to the use of these drugs in the therapy of Parkinson's Disease, although the paucity of information about the physiological function of CRAO makes the significance of its inhibition by benserazide unclear.     

Non-human primate species as metabolic models for the human situation: comparative studies on meperidine metabolism.

It is established that nonhuman primates in general provide better animal metabolic models of the human situation than do subprimate species. However, very little is known about interprimate variations in drug metabolism, and therefore meperidine metabolism was studied in the vervet, patas and mona monkeys and the mangabey after intramuscular injection of 5 mg/kg. Urinary metabolites were analyzed by gas-liquid chromatography, and in addition to unchanged meperidine, normeperidine, meperidine-N-oxide, meperidinic and normeperidinic acids (free and conjugated with glucuronic acid) and, in some cases, 4′-hydroxymeperidine were all detected and quantitated. The metabolic profile of meperidine in each species was compared with data from man and rat, which showed that there were considerable interprimate variations in the relative proportions of the metabolites found in its urine. The mangabey appeared to provide a good metabolic model for man, the mona and patas monkeys were less acceptable, and the vervet was unsuitable for this purpose. The metabolism of meperidine in the rat was markedly different from that in man and in the monkey species examined.     

Increased epidermal transglutaminase activity following 2,3,7,8-tetrachlorodibenzo-p-dioxin: In vivo and in vitro studies with mouse skin

In previous studies it has been shown that topical treatment of hairless mice with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) induces hyperproliferation and hyperkeratinization in the epidermis of hairless mice. The present investigation demonstrated that such TCDD-induced morphological changes in skin in vivo are accompanied by increased levels in activity of epidermal transglutaminase (ETG), the enzyme associated with terminal epidermal differentiation. Exposure of mouse epidermal cells in tissue culture to 10^?9m TCDD also resulted in a significant increase in ETG activity, despite the fact that morphologically these cultures (grown at 0.07 mm ionic calcium concentrations) exhibited no signs of terminal differentiation. Thus one mechanism of action of TCDD in inducing cutaneous changes appears to relate to the stimulation of increased ETG levels.     

Metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in chicken aortas: Monooxygenation,bioactivation to mutagens,and covalent binding to DNA in vitro

The capacity of enzymes present in chicken aortas to catalyze the conversion of polynuclear aromatic hydrocarbons to metabolites that covalently bind to DNA and produce mutations in Salmonella typhimurium auxotrophs is described. Chicken aortic homogenates contained active monooxygenases capable of catalyzing the conversion of benzo[a]pyrene (BaP) and 7,12-dimethylbenz[a]anthracene (DMBA) to oxidized products as measured by a highly sensitive radiometric assay and high-pressure liquid chromatography. BaP was converted primarily to phenols, while DMBA was converted to hydroxymethyl, phenolic, and diol metabolites. Pretreatment of chickens with Aroclor 1254, 3-methylcholanthrene (3-MC), or 5,6-benzoflavone (5,6-BF) resulted in significant (p < 0.05) increases in the aortic oxidative metabolism of both BaP and DMBA; phenobarbital (Pb) and pregnenolone 16α-carbonitrile (PCN) were ineffective as inducers of chicken aortic monooxygenation. Chicken aortic S-9 homogenates catalyzed the bioactivation of BaP and DMBA to mutagens for S. typhimurium strains TA-98 and TA-1538. Aroclor 1254, 3-MC, 5,6-BF, Pb, or PCN pretreatments did not significantly enhance detectable mutagenesis. When aortic homogenates from untreated chickens were utilized, low specific activities of bound products of BaP, DMBA, or 3-MC to DNA were observed. Pretreatment of chickens with either Aroclor 1254 or 3-MC resulted in 7- to 12-fold increases in specific binding activity. Inhibition of DMBA and BaP metabolism as well as DNA binding was observed when 7,8-benzoflavone (130 μm) or β-estradiol (100 μm) were added to incubation flasks. 1,2-Epoxy-3,3,3-trichloropropane (13 μm) enhanced binding of [³H]BaP and [³H]DMBA to DNA. Additions of glutathione to reaction flasks inhibited ³H binding to DNA, suggesting an important role of aortic nonoxidative metabolism of polycyclic aromatic hydrocarbons.     

Sulphinpyrazone prevents in vivo the inhibitory effect of aspirin on rat platelet cyclo-oxygenase activity

Sulphinpyrazone reportedly inhibits in vitro platelet cyclo-oxygenase activity. This study shows that Sulphinpyrazone administration (200 mg/kg) to rats was followed by long lasting inhibition of platelet cyclo-oxygenase, as measured by malondialdehyde generation by sodium arachidonate. The inhibition was apparently competitive and could in fact be ascribed to supposed metabolites of the drug. When given 30min–6 hours before aspirin (5–25mg/kg), Sulphinpyrazone and to a considerable extent its metabolites significantly prevented permanent inhibition of platelet cyclo-oxygenase activity normally produced in vivo by aspirin. Sulphinpyrazone at 100 mg/kg was unable by itself to modify platelet malondialdehyde or thromboxane B₂ generation, yet if effectively interfered with aspirin activity. This suggests that Sulphinpyrazone and its metabolites may interact with a binding site on cyclo-oxygenase not directly involved with the enzyme activity. Interaction of aspirin with this binding site would be a prerequisite for its inhibitory effect on the enzyme active site.The clinical implications of this study include a reappraisal of the pharmacological basis for the association of Sulphinpyrazone and aspirin in thrombosis prevention trials.     

Nicotyrine inhibits in vivo metabolism of nicotine without increasing its toxicity

The effect of pretreatment with β-nicotyrine on tissue concentrations of nicotine and metabolically formed cotinine was studied in male albino mice injected with [¹⁴C]nicotine. Acute toxicity of nicotine administered ip or iv was also compared in the pretreated and untreated animals. The pretreatment with nicotyrine resulted in a significant dose-dependent increase of nicotine concentrations in the liver, blood, and brain. Concomitantly, there was a significant decrease in the cotinine concentrations suggesting an inhibition of nicotine liver metabolism. Despite the higher concentrations of nicotine in the brain of the pretreated mice, the LD50 after an ip injection of nicotine was not different from the untreated animals. On the other hand there was complete protection against the lethal effect of iv-injected nicotine in the pretreated animals suggesting a direct protective interaction of nicotyrine with nicotine in CNS.     

Zeranol: a review of the metabolism, toxicology, and analytical methods for detection of tissue residues

Zeranol, an anabolic agent produced commercially for use in cattle and sheep intended for human consumption, is noncarcinogenic, nonteratogenic, and nonmutagenic. Toxicity testing (acute, subacute, and chronic) in several species by various routes of administration reveals an extremely low toxicity, the oral rat LD50 exceeding 40 g/kg. Postmortem abnormalities of high-dose animals are attributed to the effects of the compound on the endocrine system. Both zeranol itself and zearalanone, the major Phase I metabolite in the seven species studied, are excreted in the feces and in the urine, either free or as sulfates/glucuronides. A minor urinary metabolite has been identified as taleranol, an epimer of zeranol. Both metabolites exhibit a very low order of toxicity (oral rat LD50 exceeding 10 g/kg in both cases), and both exhibit less biological activity than the parent compound. The four types of analytical methods which have been employed for the specific detection and quantitation of residues of zeranol in edible tissues are: (1) gas chromatography (detection limit = 20 ppb), (2) high-performance liquid chromatography (detection limit = 5 ppb), (3) thin-layer chromatography (detection limit = 1-3 ppb), and (4) radioimmunoassay methods (detection limit to be published). The following residue levels were determined radiometrically in tissue samples taken 45 days after implantation of cattle with 36 mg tritiated zeranol: less than or equal to 2 ppb in liver, less than or equal to 1 ppb in kidney and fat, and less than or equal to 0.2 ppb in muscle and plasma. A no-effect level (NEL) of 0.225 mg/kg was determined as the highest oral dosage of zeranol which produced no estrogenic effects in female monkeys. Based on the NEL, a tolerance level for tissue residues of zeranol was calculated as 315 ppb.     

Nitrogen metabolism in o,p′-DDT-fed rats

Urea synthesis from 5 mm NH₄Cl plus 10 mm ornithine was decreased in isolated hepatocytes from rats fed 1000 ppm o,p′-DDT for 2 weeks and starved for 48 hr. DDT feeding had no effect on urea synthesis from 10 mm alanine, serine, or threonine in the presence of 10 mm ornithine. When rats were not starved prior to hepatocyte isolation, rates of urea synthesis from 10 mm NH₄Cl plus 10 mm ornithine were similar in hepatocytes from control and DDT-fed rats. In vivo, DDT-fed and subsequently starved rats had the same survival rate as starved controls after injection of 5 mmol NH₄Cl/kg body weight. Plasma urea levels were also similar. A feeding study demonstrated that rats fed a 90% casein diet supplemented with 1000 ppm o,p′-DDT gained weight at the same rate as rats fed only the 90% casein diet. Hepatocytes were then isolated from these rats, and it was found that rats fed the 90% casein diet plus DDT exhibited decreased rates of urea synthesis from 10 mm NH₄Cl plus 10 mm ornithine. These data show that DDT feeding resulted in a decreased capacity for urea synthesis from NH₄Cl in hepatocytes from starved and high-protein-fed rats. This reduced capacity did not appear to decrease in vivo ammonia detoxification under the conditions studied.     

On the capacity of human placental enzymes to catalyze the formation of diols from benzo[a]pyrene

Studies were performed on the oxidative biotransformation of benzo[a]pyrene in fortified preparations of human placental microsomes by analysis with high-pressure liquid chromatography. These investigations revealed that the utilization of substrate concentrations (1–2 × 10^?4m) sufficiently high to assure zero-order reaction kinetics (in terms of the generation of phenolic metabolites) produced a marked inhibitory effect on the formation of dihydrodiols in the same reaction mixtures. Relative quantities of dihydrodiols generated increased with decreasing substrate concentrations between 200 and 2.7 μm. Additions of manganese or ferric ions to reaction mixtures altered the ratios of generated phenols to dihydrodiols but did not provide an explanation for the differences observed in the literature. Identical results were obtained with either ¹⁴C- or ³H-labeled benzo[a]pyrene as substrates. The data suggested the possibility that considerable quantities of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, a proximate mutagen/carcinogen, may be generated in vivo by placental tissues of women who smoke.     

Effect of 6-azauracil, and of certain structurally similar compounds, on three pyridoxal-phosphate requiring enzymes involved in neurotransmitter metabolism.

6-Azauracil, which is reported to have an hypnotic effect in man, rat and mouse, is a noncompetitive inhibitor of gamma-aminobutyric acid (GABA)-transaminase activity in rodent and human brain extracts. The K_i for the rat brain activity is 5 × 10^?4 M. GABA-transaminase is also non-competitively inhibited by a number of other compounds-both drugs and natural substances—which are reported to affect arousal and which, like 6-azauracil, include within their structure a pyrimidine or pyrimidine-like ring. None of these compounds, however, is as potent an inhibitor of the transaminase as 6-azauracil. Glutamic acid decarboxylase is inhibited only weakly (and non-competitively) by 6-azauracil and, to an even lesser extent, by the other substances tested. None of the compounds under study caused appreciable inhibition of DOPA decarboxylase except under conditions where the enzyme proved highly unstable, and even under these conditions the inhibition was weak.     

Lethal potencies and immunoelectrophoretic profiles of venoms of Vipera bornmulleri and Vipera latifii

Lethality determinations are reported for Vipera bornmulleri and V. latifii. V. latifii venom is significantly more toxic than V. bornmulleri by the i.v. route; by the i.p. and s.c. routes there is little difference between the species. Immunoelectrophoretic profiles indicate close antigenic relationship between venoms of these species and V. palaestinae and Bitis spp.; a more remote relationship with V. russelli and Echis carinatus. Protease activity of V. latifii venom is stronger than that of V. bornmulleri.     

Inhibitory effect of a broad-spectrum antiviral egent, (S)-9-(2,3-dihydroxypropyl)adenine,on spermatogenesis in mice

In subacute toxicity studies in NMRI mice, the broad-spectrum antiviral agent (S)-DHPA [(S-9-(2,3-dihydroxypropy)adenine] did not exert any specific organ toxicity, even when administered at doses up to 1 mg/g body wt, except for testicular germinal aplasia. This effect was obviously accompanied by sterility and could be established in both young (3-week-old) and adult (6-week-old) mice which had received the drug (at 1 mg/g/day) in their drinking water for a period of 21 days. Under these conditions, the testicular dysfunction generated by (S)-DHPA was apparently fully reversible: fertility was restored within 5–6 weeks after the end of drug treatment, and the resulting offspring did not show an increased death or malformation rate as compared to the offspring of untreated males. (S)-DHPA did not affect the fertility of female mice and its deleterious effects on male fertility were only observed at a dosage of 1 mg/g, and not at a dosage of 0.3 mg/g or lower.     

Hepatic microsomal metabolism and covalent binding of 2,4-dinitrotoluene

The effects of 2,4-dinitrotoluene (2,4-DNT) on xenobiotic metabolizing enzymes and the hepatic metabolism and covalent binding of this compound to microsomal proteins in vitro were studied. Male Fischer-344 rats received po doses of DNT daily for 5 days at 14, 35, and 70 mg/kg/day. Hepatic oxygen-insensitive cytosolic azoreductase activity was increased and microsomal nitroreductase was decreased by DNT treatments. A small but significant increase in liver/body weight ratio and in hepatic cytochromes P-450 and b₅ occurred in the absence of changes in microsomal biphenyl hydroxylase or aryl hydrocarbon hydroxylase activities. The patterns of in vitro microsomal metabolism of DNT were dependent on oxygen tension: under aerobic conditions, 2,4-dinitrobenzyl alcohol (DNBAlc) was the major metabolite whereas under anaerobic conditions no DNBAlc was detected; 2-amino-4-nitrotoluene (2A4NT) and 4-amino-2-nitrotoluene (4A2NT) were the major metabolites. Pretreatment of rats with phenobarbital or Aroclor 1254 increased the metabolism of 2,4-DNT to DNBAlc by six- to sevenfold. Metabolism to the alcohol was inhibited by SKF-525A. These data suggested that oxidative metabolism of 2,4-DNT to DNBAlc was mediated by cytochrome P-450-dependent mixed-function oxidases. Covalent binding studies showed that a maximum of only 7 pmol of 2,4-DNT-derived radioactivity was bound per milligram of microsomal protein per hour; this binding was increased to 1.0 nmol bound/mg protein/hr in microsomes from phenobarbital of Aroclor 1254-pretreated rats. It is concluded that 2,4-DNT treatment had little effect on the activity of some hepatic xenobiotic metabolizing enzymes and was readily metabolized by liver preparations in vitro. The pathways of in vitro metabolism were dependent on oxygen tension. This in vitro metabolism produced mostly polar metabolites which did not bind appreciably to microsomal macromolecules.     

Modulation of pulmonary p-nitroanisole O-demethylase by intratracheally instilled heavy metal salts

Isolated, ventilated, and perfused rat lung preparations were used to investigate the acute effects of intratracheally applied heavy metal salts on the O-demethylation of p-nitroanisole (NA). The NA O-demethylase activity of the lung was characterized by induction (104%) following pretreatment of rats with β-naphthoflavone (BNF, 80 mg/kg), and by inhibition with SKF-525A (50 μm), metyrapone (0.1 mm), or ventilation of the lungs with CO. Pretreatment of rats with indomethacin (25 mg/kg × 3 days, po) or 3-amino-1,2,4-triazole (1 g/kg, ip) did not alter this activity. The NA O-demethylase was, therefore, suggested to be mixed function oxidase (MFO). Intratracheal instillation of NiCl₂ (0.1 or 1.0 μmol/lung) inhibited the O-demethylase activity (30 or 54%, respectively). The apparent K_m of the reaction (0.138 mm) was doubled by NiCl₂ treatment (0.252 mm), and the V_max was decreased from 26.8 to 20.9 nmol p-nitrophenol/min/g. Cadmium chloride (1.0 μmol/lung) increased the activity by 80%, but the K_m was unchanged. No effect was observed at a dose of 0.1 μmol CdCl₂/lung. At doses of 0.1 or 1.0 μmol/lung, CoCl₂ was without measurable effect on this activity. These data suggest that heavy metals present in the atmosphere can interact with pulmonary MFO in low concentrations to alter the metabolism of xenobiotic compounds.     

The metabolism of dibenzo(a, h)pyrene and dibenzo(a, i)pyrene and related compounds by liver preparations and their enzyme-induced binding to cellular macromolecules

Dibenzo[a, h]pyrene and dibenzo[a, i]pyrene are both metabolized to unidentified phenols and dihydrodiols by homogenates and microsomal fractions from the livers of rats that had been pretreated with 3-methylcholanthrene: some properties of these metabolites are described. In comparative experiments, in which the two ³H-labelled dibenzopyrenes and ³H-labelled benzo [a]pyrene were metabolized by microsomal fractions from the livers of normal rats, mice, hamsters and guinea-pigs, the dihydrodiols formed initially by the fractions from guinea-pigs, and to a lesser extent those formed by fractions from hamsters were further metabolized to unidentified products. Further metabolism of the dihydrodiols formed by microsomal fractions from the livers of rats and mice did not occur. Higher levels of enzyme-induced binding of all three hydrocarbons to the microsomal proteins were found in the preparations from guinea-pig livers than in the preparations from the livers of animals of the other species and in all preparations maximum levels of binding were reached after 30 min incubation. Enzyme-induced binding of the two dibenzopyrenes to DNA was observed when the ³H-labelled hydrocarbons and DNA were incubated with microsomal fractions from the livers of rats that had been pretreated with 3-methylcholanthrene, but the levels of binding were low as compared with those obtained with dibenz[a, h]anthracene.     

The action of equinatoxin,a peptide from the venom of the sea anemone, Actinia equina, on the isolated lung

On perfusion through isolated lungs from male Sprague-Dawley rats, equinatoxin caused a dose-dependent increase in the wet to dry weight ratio. Ratios were significantly elevated above control values at equinatoxin concentrations of 80–200 ng/ml. The increased ratios were accompanied by an increase in the permeability of the lung vasculature. When equinatoxin was perfused through isolated lungs at concentrations of 100 ng/ml or greater, significantly more [³H]polyethylene glycol (PEG; approximately 900 mol. wt) was retained in the extravascular space as compared to controls. Perfusion pressures of the lung were significantly elevated above controls at equinatoxin concentrations greater than 100 ng/ml. These effects of equinatoxin were not mediated by degranulation of mast cells, as preperfusion of the lung with 100 or 200 μM Na cromolyn or 1 μM lanthanum chloride did not modify the pulmonary response to equinatoxin. At concentrations of equinatoxin below 150 ng/ml the fluid movement appears to be restricted primarily to intracellular, or possibly interstitial, spaces, as no significant amounts of [³H]polyethylene glycol were recovered by tracheal lavage. At concentrations of equinatoxin equal to or greater than 150 ng/ml, significant amounts of PEG were washed from the trachea. As it is a potent inducer of pulmonary edema, equinatoxin may become an important probe to study fluid regulation in the lung.     

The purification and characterization of an antihemorrhagic factor in woodrat (Neotoma micropus) serum

A protein antihemorrhagic factor was purified from the serum of the southern plains woodrat (Neotoma micropus) by gel filtration and ion exchange chromatography. The homogeneity of the purified factor was measured by disc electrophoresis in polyacrylamide gel. The factor had an isoelectric pH of 4.1, a molecular weight of 54,000, and migrated with α-globulins in disc electrophoresis. The factor failed to form a precipitate with crude western diamondback rattlesnake (Crotalus atrox) venom and did not show proteolytic activity with gelatin or hide powder. N. micropus has an antihemorrhagic factor in the serum which appears similar to that found in hispid cotton rat (Sigmodon hispidus) and in opossum (Didelphis virginiana) sera.     

Effects of drug metabolism inhibitors on butylated hydroxytoluene-induced pulmonary toxicity in mice

The administration of butylated hydroxytoluene (BHT) to mice results in lung cell damage followed by cellular proliferation which was quantitated by measuring the increase in thymidine incorporation into pulmonary DNA. Administration of SKF 525-A or piperonyl butoxide to mice treated with BHT prevented the increase in thymidine incorporation into pulmonary DNA. This effect was dose dependent, with complete protection from 400 mg/kg BHT achieved with 10 mg/kg SKF 525-A or 400 mg/kg piperonyl butoxide. SKF 525-A and piperonyl butoxide completely prevented the BHT-induced increase in pulmonary DNA synthesis even when given 1–2 hr after BHT and a partial protective effect was evident when they were given 6–12 hr after BHT. Pretreatment of mice with cobaltous chloride diminished the BHT-induced increase in thymidine incorporation into pulmonary DNA. Following the in vivo administration of [¹⁴C]BHT, radioactivity was covalently bound to lung, liver, and kidney macromolecules of both mice, which exhibited BHT-induced lung damage, and rats, which did not. The greatest amount of radioactivity was bound to lung tissue from mice. This binding was prevented by the administration of SKF 525-A and was a linear function of the BHT dose within a range of 50–600 mg/kg. Binding to other tissues from the mouse and all tissues examined in the rat was minimal and unaffected by SKF 525-A. These data suggest that a reactive metabolite of BHT rather than the parent compound produces lung damage in mice.     

Pyrrolizidine alkaloid-induced alterations in benzo[alpha]pyrene metabolism and binding of benzo[alpha]pyrene metabolites to deoxyribonucleic acid

Pretreatment of rats by oral administration of jacobine, a pyrrolizidine alkaloid and inducer of epoxide hydrolase, produced a marked shift in hepatic microsomal metabolism in vitro of benzo[alpha]pyrene. The formation of 9-hydroxybenzo[alpha]pyrene and 7,8-dihydroxy-7,8-dihydrobenzo[alpha]pyrene was decreased whereas the formation of 4,5-dihydroxy-4,5-dihydrobenzo[alpha]pyrene was increased following jacobine treatment. This shift in the ratio of benzo[alpha]pyrene metabolites was accompanied by a significant reduction in DNA binding. Addition of purified epoxide hydrolase to control or jacobine microsomes produced a similar decrease in total DNA binding. Chromatography of benzo[alpha]pyrene metabolite-DNA nucleoside adducts showed a marked reduction in four peaks and the elimination of one peak with microsomes from jacobine-treated rats.