Oxidation of N-methylthiobenzamide and N-methylthiobenzamide S-oxide by liver and lung microsomes

The in vitro oxidation of N-[14C]methylthiobenzamide (NMTB) and N-[14C]methylthiobenzamide S-oxide (NMTBSO) by rat lung and liver microsomes was studied. In the presence of an NADPH-generating system, NMTB was rapidly converted to NMTBSO and small amounts of N-methylbenzamide (NMBA) and covalently bound metabolites (CVB). Under similar conditions, NMTBSO was converted to NMBA and CVB. Studies with metabolic inhibitors indicate that both the S-oxidation of NMTB and its further conversion to NMBA and CVB, probably via enzymatic oxidation to the S,S-dioxide, are catalyzed by both cytochromes P-450 and the FAD-containing monooxygenase (MFMO). Based on differential effects of inhibitors with lung vs liver microsomes, it would appear that in lung microsomes the MFMO plays a significantly greater role than cytochrome P-450 in the oxidation of NMTB and NMTBSO, whereas in the liver the contribution of these two pathways is more nearly equal. 1-Methyl-1-phenyl-3-benzoylthiourea, which blocks the in vivo pneumotoxicity of both NMTB and NMTBSO, also inhibited their in vitro microsomal metabolism and CVB, suggesting that these oxidations are obligatory steps in the expression of toxicity by these compounds.     

Accumulation of methylsulfonyl derivatives of some bronchial-seeking polychlorinated biphenyls in the respiratory tract of mice.

Seventeen polychlorobiphenyls (PCBs), ¹⁴C-labeled and/or unlabeled, were studied with regard to their deposition as such or as metabolites in the lung tissues after iv or ip injection in mice. Four of these compounds were known by autoradiography to possess affinity for bronchial tissues. Lung extracts of mice treated with the 11 labeled PCBs were analyzed in a partition scheme to indicate the presence of hydroxylated and/or methylsulfonyl metabolites. Extracts of lungs from mice treated with the unlabeled compounds were analyzed by gas chromatography. The extracts that appeared to contain methylsulfonyl metabolites were further analyzed by mass fragmentography. Five of the biphenyls studied were each found to appear in the lungs as two isomeric methylsulfonyl PCBs. The structures of the major metabolites were confirmed by comparison with synthetic reference compounds. One of the biphenyls previously known to accumulate in the bronchi was found not to occur in the lung as methylsulfonyl derivatives, but most probably only as the unchanged compound. None of the remaining 11 PCBs studied was found to occur as methylsulfonyl derivatives in the lung.     

The uptake and secretion of 2-acetylaminofluorene by the rat and dog prostate

In unanesthetized rats examined 4–140 hr after the ip administration of 1.8 mg/kg of [9-¹⁴C]2-acetylaminofluorene ([¹⁴C]2-AAF), detectable amounts of radioactivity were found in the plasma and in the ventral and dorsolateral lobes of the prostate. Radioactivity was also found in the prostatic fluid collected from anesthetized rats between 25 and 29 hr after doses of 2 mg/kg. When three unanesthetized dogs with surgically prepared fistulas allowing the collection of prostatic fluid were given intraperitoneal doses of 0.16–0.25 mg/kg of [¹⁴C]2-AAF and followed for 6 hr (two dogs) or 166 hr (one dog), there was a relatively rapid rise in the amount of radioactivity in plasma followed by a slow decline and an appearance of radioactivity in the prostatic fluid. Radioactivity was also present in the prostate glands of each of two dogs examined 6 hr after treatment. Thus 2-AAF and/or metabolites were found to enter both the prostate gland and the prostatic secretion of both the rat and dog.     

Evidence for a contribution by purines to the neurogenic response of the guinea-pig urinary bladder

In order to determine if ATP contributes as an excitatory transmitter in the guinea-pig bladder, experiments were conducted with ANAPP3, a photoaffinity analogue of ATP, which is an antagonist of adenine nucleotides in several other smooth muscles. With or without photoactivation with visible light, ANAPP3 antagonized contractile responses of in vitro strips of bladder to exogenous ATP. The antagonism was specific in that responses to acetylcholine and KCl were not affected by ANAPP3. Responses of strips of bladder to transmural electrical stimulation were not antagonized by ANAPP3 and were relatively insensitive to atropine. However, combined treatment with ANAPP3 and atropine produced a marked antagonism of the neurogenic response. In experiments with bladders obtained from animals pretreated with 6-hydroxydopamine, the ANAPP3-sensitive component of the neurogenic response was absent. These results suggest that acetylcholine, released from cholinergic nerves, and a purine, released from 6-hydroxy-dopamine-sensitive nerves, are both involved in motor transmission in this tissue.     

The penetration of N-hydroxyurethane into the prostate and prostatic secretion of the rat and dog

To further define the possible inteactions of carcinogenic chemicals and the prostate gland, the ability of N-hydroxyurethane to enter the prostate and its secretion has been assessed in rats and dogs. In anesthetized rats in which prostatic fluid was collected for up to 6 hr after treatment, N-hydroxyurethane rapidly entered the prostatic fluid following the intravenous administration of doses of 100 mg/kg. The mean gland to plasma ratios for the ventral and dorsolateral lobes of the prostates from these rats were 1.33 and 0.91, respectively, while the prostatic fluid concentrations averaged 0.97 times the mean plasma concentration over the periods of collection. In six unanesthetized dogs, four with prostatic fistulas, the intravenous administration of 100 mg/kg of N-hydroxyurethane resulted in the appearance of the chemical in both the gland and its secretion. The mean gland to plasma ratio for four dogs examined at 120 min after treatment was 2.60, and the mean prostatic fluid to plasma ratio for nine samples of fluid collected from three dogs over the first 120–180 min after treatment was 0.95. Thus, N-hydroxyurethane was found to readily penetrate from plasma into both the prostate gland and the prostatic secretion of the rat and dog.     

Inhibition of serine proteases by steroids

Proteolysis of ¹⁴C-labeled globin, as well as the hydrolysis of the specific substrate benzoyl tyrosine ethyl ester, by purified bovine chymotrypsin was found to be inhibited by several steroid hormones. The inhibition of chymotrypsin by the steroids was of a competitive nature, with K_i values of 9.9 × 10^?5 M for triamcinolone (9-fluoro-11β, 16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione), 1.6 × 10^?4 M for cortisol (11β,17α,21-trihydroxypregn-4-ene-3,20-dione), 3.7 × 10^?4 M for testosterone (17β-hydroxy-4-androsten-3-one), 5.0 × 10^?4 M for dexamethasone (9-fluoro-11β,17,21-trihydroxy-16α-methyl-1,4-pregnadiene-3,20-dione), and 1.0 × 10^?4 M for epicortisol (11α,17,21-trihydroxy-4-pregnene-3,20-dione). The activity of purified bovine trypsin on its specific substrate, TAME (tosyl arginine methyl ester), also showed a similar pattern of inhibition by steroids. Both chymotrypsin and trypsin were found to bind ³H-labeled dexamethasone and cortisol. This binding was markedly inhibited by the general protease inhibitor, PMSF (phenylmethanesulfonyl fluoride), whereas the chymotrypsin-specific inhibitor, TPCK (l-[1-tosyl-amido-2-phenyl]ethylchloromethyl ketone), inhibited only the steroid binding to chymotrypsin but not to trypsin. These observations indicate that serine proteases recognize steroid hormones in a fashion similar to the recognition of their specific substrates and that the steroids inhibit activity of these enzymes at their binding sites.     

Induction of ion-permeable channels by the venom of the fanged bloodworm Glycera dibranchiata

Venom from the poison glands of the polychaete annelid Glycera convoluta has been reported to dramatically increase the frequency of miniature end-plate potentials at the frog and crayfish neuromuscular junctions, without causing detectable ultrastructural changes. We report here that addition of venom from the related annelid Glycera dibranchiata to one side of a lipid bilayer results in the formation of ion-permeable channels in the membrane. The channel forming activity was found in the void volume of a Sephadex G-25 column (mol. wt. greater than 5000). The conductance of a single channel is about 350 pmho in 0.1 M NaCl and is ohmic. The channels exhibit moderate (but not ideal) cation selectivity in NaCl or KCl gradients. Other selectivity measurements suggest that Ca2+ and Mg2+ are also permeable. The channels show a slight voltage sensitivity. The steady state conductance at--70 mV (side opposite venom) is about 5 times the conductance at + 70 mV. We suggest that these channels in the venom may evoke transmitter release at neuromuscular junctions either by (1) depolarizing the pre-synaptic terminal and thus opening voltage-dependent Ca2+ channels, or (2) directly allowing Ca2+ to enter the terminal. Black widow spider venom is known to produce similar effects on neuromuscular junctions and lipid bilayers. The single channel conductances and ionic selectivities of the channels found in the venoms of Glycera and Latrodectus are strikingly similar. Taken together, these results suggest that channel formation can explain the electrophysiologic effects of these two different venoms.     

Fibrogenic structure-activity study of the bleomycin molecule

In the present study the fibrogenic potential of intact bleomycins as well as their acetyldipeptide and terminal polyamine constituents have been assessed. Administration of Blenoxane, bleomycin A2, or bleomycin B2 to rats produced histopathologic evidence of pulmonary fibrosis when tissues were examined 28 days following a single intratracheal dose. These compounds also produced a readily detectable increase in pulmonary collagen synthesis as evidenced by an approximate twofold increase over control values in the formation of [3H]hydroxyproline in an in vitro lung mince system. Lung collagen synthetic activity remained significantly elevated over control values for up to 2 weeks. However, neither the acetyldipeptides nor the polyamine constituents of bleomycin A2 and B2 produced detectable increases in lung collagen synthesis or in histopathologic evidence of pulmonary injury. Spermine and spermidine, the terminal amine components associated with bleomycin-A6 and with tallysomycin A, tallysomycin B, and bleomycin-A5, respectively, did produce significant pulmonary fibrotic injury in rats following intratracheal administration. Out of an extensive series of polyamines, bleomycin acetyldipeptides and intact bleomycin and tallysomycin analogs, only spermine and spermidine were found to produce hydrogen peroxide and acrolein upon incubation in vitro with amine oxidase, a common pulmonary enzyme. Conclusions regarding the relative toxicity of different bleomycin analogs based solely on the toxicity produced by administration of their terminal amine constituent must therefore be made with caution.     

The inhibition of catechol-O-methyltransferase by 2,3-dihydroxypyridine

Despite its structural similarity to catechol, 2,3-dihydroxypyridine is not a substrate but a “dead-end” inhibitor of purified pig liver catechol-O-methyltransferase. It inhibits the methylation of 3,4-dihydroxyphenylacetic acid competitively with an inhibitor constant of 15 μM. Against the methyl donor, $\text{S-}\text{adenosyl}\text{-}\text{l}\text{-}\text{methionine}$, it is an uncompetitive inhibitor (K′_i = 85 μM). Clearly, although 2,3-dihydroxypyridine interacts with the catechol-binding site of the enzyme, the presence of a nitrogen in the ring alters its susceptibility to O-methylation.     

Bleomycin toxicity: alterations in oxidative metabolism in lung and liver microsomal fractions

Separate groups of male rats received low doses (5 units) or high doses (15 units) of bleomycin i.p. twice weekly for 1.5, 3 or 6 weeks. The susceptibility of tissue lipid to peroxidation and the activities of mixed function oxidations were examined in microsomal fractions prepared from lung and liver. ADP-Fe (III)-initiated lipid peroxidation was stimulated in lung microsomal fractions only in animals treated with high-dose bleomycin for 1.5 weeks, whereas a 2- to 4-fold enhancement was observed in liver preparations from all bleomycin-treated animals. Microsomal ADP-Fe (III)-initiated lipid peroxidation was inhibited, however, by the in vitro addition of bleomycin in both lung and liver preparations, but this inhibition was an artifact resulting from the chelation of Fe (III) by bleomycin. Soybean lipoxygenase I-initiated microsomal lipid peroxidation, which does not require added iron, was unaffected by bleomycin in lung preparations but was inhibited in liver. Following in vivo treatment, lung microsomal hydrogen peroxide generation was inhibited by 1.5 weeks of high-dose bleomycin treatments, whereas benzphetamine N-demethylation was unchanged. These cytochrome P-450-dependent reactions were both suppressed, however, in liver microsomal fractions. In vitro, both reactions were also inhibited by bleomycin in liver but not in lung microsomal fractions. The lack of effect of in vitro bleomycin treatments on Superoxide generation in lung or liver preparations suggests that the NADPH cytochrome P-450 reductase component of the mixed function oxidase system was not affected. Minimal alterations in lipid peroxidizability and mixed function oxidase activities in lung microsomal fractions of bleomycin-treated animals suggest that the insensitivity could be due to: (1) the site of toxicity not being at the level of the endoplasmic reticulum; or (2) the target of bleomycin toxicity being limited to a small population of pulmonary cell types. Even though the liver is not susceptible to bleomycin toxicity, the hepatic microsomal mixed function oxidase system is highly sensitive to this chemical.     

General pharmacological studies of fusarenon-X, a trichothecene mycotoxin from Fusarium species.

Fusarenon-X (F-X) is one of the trichothecene mycotoxins. In the present work, pharmacological properties of F-X were examined. (1) F-X induced hypothermia but did not produce appreciable behavioral changes of mice. (2) In anesthetized rats, F-X caused a rise in the blood pressure and a decrease in the respiratory rate but induced no significant change in the heart rate. (3) In isolated tissues such as fundus muscle, vas deferens, tracheal muscle, ileum, and atrium, F-X had no detectable effects on spontaneous activity and the responsiveness to agonists. (4) Subplantar administration of F-X into rat hindpaw induced edema. F-X had little influence on the histamine release from mast cells and the membrane stability of erythrocytes. (5) F-X decreased the spontaneous peristalsis of intestine but increased the intestinal propulsion of charcoal meal. (6) F-X induced vomiting in dogs which was suppressed by preliminary administration of chlorpromazine and metoclopramide. F-X may induce vomiting by stimulating the chemoreceptor trigger zone in dogs.     

Clearance of polonium-210-enriched cigarette smoke from the rat trachea and lung

The distribution and clearance of alpha radioactivity in the lungs of rats were measured after inhalation of smoke from cigarettes highly enriched in 210Po. Female Fischer rats were exposed daily for 6 months to smoke from cigarettes with 500 times the normal content of 210Po. Control rats were exposed to standard cigarette smoke. Animals were serially withdrawn and killed. After necropsy the trachea, major bronchi, larynx, and nasopharynx were examined for surface alpha activity by an etched track technique utilizing cellulose nitrate detectors. Areas of accumulated activity were seen on samples of larynx from rats exposed to the 210Po-enriched cigarettes. No other local accumulations were seen on the airways. The lower lungs were analyzed radiochemically for 210Po. Both radiochemical analysis and track measurements showed highly elevated activity concentrations in rats exposed to the 210Po-enriched cigarettes. Following withdrawal from smoking, both short- and long-term clearance components were seen. The parameters which fit the postexposure data for clearance of the lung burden cannot fit the buildup during the exposure period.     

Morphological changes induced by crotoxin in murine nerve and neuromuscular junction

The sequence and timing of structural changes induced by crotoxin were examined by electron microscopy following either a single systemic injection of a lethal dose or a single i.m. injection of a sub-lethal dose in mice. Changes in the diaphragm motor nerve terminals, including a reduction in synaptic vesicle population, the appearance of omega (Ω) shaped identations in the axolemma and swelling of mitochondria, were associated with clinical signs of developing muscular paralysis during systemic intoxication. No post-synaptic or myofibrillar changes were observed at the stage of cessation of respiration. Subsequent events observed following i.m. injection included the onset of myonecrosis, disorganization of the endplate, envelopment of axon terminals by Schwann cells and changes in the preterminal motor nerve. A ‘Wallerian-type’ degeneration of small myelinated axons supplying the soleus muscle was apparent by 24 hr. Re-innervation of the regenerated muscle was rapid.     

A high-performance liquid chromatographic method for the determination and control of the composition of gentamicin sulphate

A high-performance liquid chromatographic method for routine control of the composition of gentamicin sulphate is described. The method utilizes pre-column derivatization followed by reversed-phase chromatography with fluorescence detection and uses an internal standard for quantification. Analysis of 19 samples of gentamicin from various sources indicates that the ratio of the major components and the content of minor constituents varies with the geographical origin of the sample. The results were compared with those of a microbiological assay and the B.P. nuclear magnetic resonance (nmr) spectroscopic limit test of the same samples. The microbiological assay may be influenced by biologically active impurities whilst the nmr assay was insensitive to the presence of minor components. The method described offers a more discriminating and flexible means of monitoring the composition of gentamicin and hence of providing data upon which a realistic specification for this valuable antibiotic mixture can be prepared.     

Stimulation of nonbiliary, intestinal excretion of hexachlorobenzene in rhesus monkeys by mineral oil

Four rhesus monkeys were administered various doses of hexachlorobenzene (HCB) po, to achieve widely varying adipose tissue levels. One month later, each animal was provided with a bile duct bypass allowing for interruption of the enterohepatic circulation (EHC). Effects of mineral oil-supplemented diet and/or interruption of the EHC on urinary, biliary, and fecal excretion of HCB and its metabolites were quantified. Urinary excretion of HCB was not affected by mineral oil but was reduced 20 to 60% by interruption of the EHC. Similarly, biliary excretion of HCB was also reduced 25 to 60% by interruption of the EHC and was not altered by mineral oil. Fecal excretion was increased about fivefold by mineral oil, whereas interruption of the EHC had no effect on the amount of HCB in feces. Results demonstrate that interruption of the EHC reduced urinary and biliary excretion of HCB metabolites, whereas mineral oil specifically stimulated intestinal excretion of the parent compound.     

Effects of 3-methylcholanthrene and phenobarbital on the capacity of embryos to bioactivate teratogens during organogenesis

Pregnant Sprague-Dawley rats were divided into four groups and given ip injections of 3-methylcholanthrene (MC) in corn oil, corn oil only, phenobarbital (PB) in Hank's balanced salt solution (HBSS), or HBSS only. Maternal animals were killed on Day 10 of gestation, and embryos from each group were explanted in medium containing cyclophosphamide (CP), 2-acetylaminofluorene (AAF), or dimethylsulfoxide vehicle. After a 24-hr culture period, embryos from dams treated with HBSS, corn oil, or PB/HBSS exhibited no increase in abnormalities (as compared with controls) when either CP or AAF were added to the media. However, embryos transplacentally preexposed to MC and subsequently treated during culturing with AAF (but not CP) exhibited striking increases in malformation incidence. Commonly observed malformations included abnormally open neural tubes, abnormal flexure rotation, and prosencephalic defects. Homogenates of Day 10 embryos transplacentally preexposed to MC exhibited readily measurable oxidative biotransformation of AAF as assessed with HPLC. Biotransformation of AAF by embryos from the other three groups was virtually undetectable. Incorporation of exogenously supplemented bioactivating systems from livers of mature animals indicated that postmitochondrial supernatant fractions (S-9) from male, MC-pretreated rats effectively catalyzed the conversion of AAF (but not CP) to embryotoxic metabolites. Conversely, hepatic S-9 from adult, male, PB-pretreated rats was highly effective in converting CP (but not AAF) to embryotoxic metabolites. The results indicated the inducerspecific occurrence of embryonic bioconversion of AAF to embryotoxic metabolites via MC-inducible, P-450-dependent, embryonic enzyme systems.     

Endotoxin and tryptophan-induced hypoglycaemia in rats

Pretreatment of rats with increasing, but non-lethal, doses of endotoxin was associated with a parallel increase in sensitivity to induction of hypoglycaemia by tryptophan. Acutely streptozocin-diabetic animals became hypoglycaemic with endotoxin alone, and this was increased further by tryptophan. Variations in tryptophan sensitivity between rat populations cannot be explained by previous history of exposure to endotoxin. Endotoxin abolished the increase in tryptophan dioxygenase activity caused by triamcinolone, but not that caused by tryptophan. Triamcinolone was effective, however, when given together with tryptophan to endotoxin-treated rats. The activity of tryptophan dioxygenase in vivo and in liver cells in vitro is unchanged by exposure to endotoxin at 1 mg/kg body wt. Turnover studies indicated that hypoglycaemia resulted from inhibition of gluconeogenesis. There was no evidence to support a role for insulin in this process and results were consistent with an endotoxin-mediated hepatic insensitivity to glucagon. They also suggested that quinolinate, rather than 5-hydroxytruptamine, may be the intracellular agent responsible for inhibition of gluconeogenesis.