Hepatic metabolism and pulmonary toxicity of monocrotaline using isolated perfused liver and lung

Monocrotaline is a pyrrolizidine alkaloid obtained from the seeds of Crotalaria spectabilis. When perfused through an isolated liver, monocrotaline is metabolized to Ehrlich reactive (E+) metabolites. Metabolism of monocrotaline was faster in livers from male rats than female rats, was inducible with phenobarbital pretreatment, and was inhibited by coperfusion with the P-450 mixed-function oxidase inhibitor SKF-525A, anoxic perfusion conditions, and low temperatures. When metabolites generated by an isolated liver were perfused through isolated lungs in a recirculatory manner, serotonin transport by the pulmonary endothelium was reduced in correlation with the amount of E+ material contained in the perfusion medium. When metabolism of monocrotaline by the liver was inhibited with SKF-525A, low temperature perfusions or anoxic conditions, serotonin transport by the pulmonary endothelium was unchanged from controls. Monocrotaline alone had no effect on the lung. Thus, isolated perfused livers metabolized monocrotaline to chemical species which produced pulmonary damage in vitro. This provides direct evidence that liver metabolites can cause one of the pneumotoxic effects of monocrotaline observed in vivo.     

Dietary modification of metabolism and toxicity of chemical substances—with special reference to carbohydrate

Rats were fed various test diets only on the day before sacrifice or every day for 3 weeks prior to sacrifice in order to assess the effects of protein (casein), fat (a mixture of olive and corn oils) and carbohydrate (sucrose) on the liver mixed-function oxidase activity. The activity was determined by measuring metabolic rates of 8 volatile hydrocarbons, i.e., benzene, toluene, styrene, chloroform, carbon tetrachloride, 1,2-dichloroethane, 1,1-dichloroethylene, and trichloroethylene. Contrary to the general belief, it was found that carbohydrate, not protein or fat, regulates the metabolism of these hydrocarbons: a diet which was deficient in carbohydrate remarkably enhanced the metabolism irrespective of protein and fat contents in the diet. This conclusion was confirmed by employing two types of diet, one in which the carbohydrate was replaced by an isocaloric amount of protein or fat (thus keeping total calories of each diet constant) and the other in which the carbohydrate content was varied with protein and fat contents fixed (total calories of each diet differed from others according to the carbohydrate content). In accordance with this, dietary carbohydrate intake also exerted a remarkable influence on the hepatotoxicity of carbon tetrachloride which needs to be metabolically activated to become cytotoxic: the smaller the intake, the more severe the liver injury.     

Effect of 2'-carboxymethoxy-4,4'-bis(3-methyl-2-butenyloxy)chalcone (SU-88) on prostaglandin metabolism in hog gastric mucosa

SU-88 [2'-carboxymethoxy-4,4'-bis(3-methyl-2-butenyloxy)chalcone] inhibited the activity of the prostaglandin (PG)-metabolizing enzyme, 15-hydroxy-PG-dehydrogenase (15-OH-PG-DH), in a cytoplasmic fraction of gastric mucosa. This compound had no effect on the PG synthetase of bovine seminal vesicle microsomes and lactate dehydrogenase in rat liver. The 15-OH-PG-DH activity of gastric mucosa was not influenced by a specific inhibitor of alcohol dehydrogenase, 4-methylpyrazole. Carbenoxolone (CBX) also inhibited 15-OH-PG-DH activity. The IC50 values for SU-88 and CBX were approximately 20 and 40 microM respectively. SU-88 inhibited 15-OH-PG-DH activity uncompetitively or competitively according to whether PGE1 or NAD was used as substrate. CBX inhibited competitively the activity of this enzyme for both substrates. After the addition of SU-88 or CBX to the incubation medium of gastric mucosa, the PGE2 level of the medium was increased significantly while that of the tissue remained unchanged. These results indicate that SU-88 specifically inhibited 15-OH-PG-DH activity and suggest that 15-OH-PG-DH activity regulates the level of PGs in gastric mucosa and may have an anti-ulcer influence.     

On the mechanism of butylated hydroxytoluene-induced hepatic toxicity in rats

The relations between serum transaminase activity and the hepatic contents of glutathione and lipid peroxide were examined following oral administration to rats of butylated hydroxytoluene (BHT; 500 or 1000 mg/kg). The glutathione level rapidly diminished and reached a minimum at 6 hr after BHT administration. The period of depletion was dependent on dose: restoration of the glutathione level took longer in high-dose rats than in low-dose rats. The content of hepatic lipid peroxide was not markedly changed by BHT throughout the experimental period. The activity of glutathione S-transferase was not affected until 12 hr after BHT administration but, thereafter, it increased with time and was accompanied by elevation of the glutathione level. Though the activities of serum glutamate-oxaloacetate transsminase and glutamate-pyruvate transaminase were not affected by low-dose BHT, they increased rapidly in the high-dose rates after a lag period of about 6 hr and reached a maximum at 24 hr after administration; at that time, the livers of the high-dose rats showed centrilobular necrosis. The results indicate that acute hepatic injury was induced by the high-dose BHT. Pretreatment with cobaltous chloride inhibited the increase in the activities of the serum transaminases produced by the high-dose of BHT accompanying the depletion of microsomal cytochrome P-450 content and the induction of glutathione content. These observations suggest that hepatic damage was associated with prolonged depletion of glutathione rather than with lipid peroxidation in the liver, and that the activated metabolites of BHT rather than the parent compound induced the tissue damage.     

In vivo inhibition of hepatic lipogenesis in the rat by cyclandelate (3, 3′, 5-trimethylcyclohexanylmandelate)

Rates of hepatic lipogenesis were measured in vivo in rats by incorporation into lipids of [³H] from injected [³H]H₂O 17 hr after a single oral dose of cyclandelate (3, 3′,5-trimethylcyclohexanylmandelate, a vasoactive substance). Cyclandelate administration resulted in a significant inhibition (40–60%) of both sterol and fatty acid synthesis in the livers which was independent of the 3.2-fold diurnal variation in the rates of hepatic sterol and fatty acid synthesis. The inhibition of accumulation of newly synthesized fatty acid in intestine also reached statistical significance. The accumulation of newly synthesized sterol was significantly depressed in serum but did not result in any change in the concentration of serum total cholesterol. These results are interpreted in terms of the inhibitory effect of cyclandelate on hepatic 3-hydroxy-3-methylglutaryl-CoA reductase previously reported by us (Biochem. Pharmac. 32, 649, 1983).     

Sex and age dependence of the “selective” induction of rat hepatic microsomal epoxide hydratase following trans-stilbene oxide,l-α-acetylmethadol,or phenobarbital treatment

Conflicting reports exist regarding the “selective” induction of rat hepatic microsomal epoxide hydratase (EH) by transstilbene oxide (t-SO). We reasoned that rats of differing age and sex used in previous studies could be the source of the apparent discrepancy. To clarify the effect of t-SO on the microsomal enzymes, immature male, adult male and adult female rats were used. The rats were treated with t-SO, phenobarbital, or l-α-acetylmethadol (LAAM), all inducers of EH, and the effects of age and sex on the responses of selected microsomal enzymes were determined. t-SO was found to increase benzo[a]pyrene hydroxylase (AHH) activity in the adult females and immature males. However, it did not increase AHH activity in the adult male. t-SO increased aminopyrene N-demethylase activity in adult males and adult females, and it elevated EH activity in all rats studied. The effects of t-SO were similar to those of phenobarbital. LAAM was found to increase EH in adult male and adult female rats; it increased AHH in the females, but not in the males. In castrated male rats, t-SO and phenobarbital affected AHH in a fashion equivalent to that observed in normal adult males. In adult males, each of the agents studied raised EH activity without a simultaneous increase in AHH activity. This “selectivity”, however, did not hold for other cytochrome P-450-dependent monooxygenases.     

Effect of three calcium antagonists on platelet secretion and metabolism

Three compounds, 8-(N,N-diethylamino-octyl 3,4,5-trimethoxybenzoate, HCl (TMB-8), 2-propyl-3-dimethylamino-5,6-methylenedioxyindene HCl (2-PIA), and chlortetracycline, were investigated to determine whether their effects on washed human platelets were compatible with a suggested role as calcium antagonists. TMB-8 had little effect on levels of metabolic ATP and IMP, whereas chlortetracycline caused a decrease in metabolic ATP and an increase in IMP; both compounds inhibited thrombin-induced secretion and changed the platelets to spheres. At concentrations up to 0.5 mM, 2-PIA caused a decrease in ATP and an increase in IMP, an induction of secretion, and a centralization of electron-dense material in the platelets, all changes which suggest induction of secretion. The ultrastructural, functional and metabolic effects of TMB-8 and the type of interference by the drug with the effects of thrombin and 2-PIA on the same variables suggest that TMB-8 is mainly a membrane-active drug. Chlortetracycline on the other hand caused changes in platelet metabolism, ultrastructure, and function which, at least partly, indicate an effect on intracellular mechanisms. Both TMB-8 and 2-PIA, and to lesser degree chlortetracycline, caused loss of cytoplasmic nucleotides from the platelets.     

Interaction between ethanol and carbohydrate on the metabolism in rat liver of aromatic and chlorinated hydrocarbons

Metabolic rates of eight hydrocarbons (benzene, toluene, styrene, chloroform, carbon tetrachloride, 1,2-dichloroethane, 1,1-dichloroethylene, and trichloroethylene) were measured in vitro with the livers from rats that had consumed ethanol for 3 weeks in combination with various diets. Ethanol and carbohydrate antagonized each other in connection with activity of hepatic drug-metabolizing enzymes (mixed-function oxidases); the former increased and the latter decreased these enzymes. A decrease (increase) in carbohydrate intake augmented (suppressed) the action of ethanol in a dose-related manner. In particular, a combination of ethanol with a low-carbohydrate diet (DeCarli and Lieber diet) enhanced enzyme activity and potentiated carbon tetrachloride-induced hepatotoxicity. Enhancement of metabolism and potentiation of toxicity were due to the combination rather than to ethanol per se.     

Elevation of conjugation capacity in isolated hepatocytes from BHA-treated mice

Using harmol and paracetamol as the substrates, the elevation of conjugation reactions by BHA feeding and their significance towards the protective effects of this antioxidant has been studied with hepatocytes obtained from mice. With both substrates, an almost five fold elevation of the glucuronidation was observed. However, there was no change in the rate of sulfate conjugation. The rate of glutathione conjugate formation with paracetamol was also not enhanced, even though both the GSH level and glutathione S-transferase activities were increased. Finally, BHA administration afforded no protective effect against the hepatotoxic effects of paracetamol, even when the production of reactive paracetamol metabolites was increased by 3-methylcholanthrene pretreatment.     

The prophylactic use of l-methyl, 2-hydroxyiminomethylpyridinium methanesulfonate (P2S) in the treatment of organophosphate poisoning.

Studies have been carried out to determine the effects of prophylactic treatment with P2S (1-methyl,2-hydroxyinimomethylpyridinium methanesulfonate) on the survival of animals poisoned by lethal doses of sarin, soman, tabun, or VX (O-ethyl S-diisopropylaminoethyl methylphosphonothiolate). The influence of such prophylaxis on the efficacy of therapy with atropine and P2S after poisoning by the same organophosphates has also been investigated. Following pretreatment with P2S there was a significant increase, compared with control animals, in the survival time of rabbits and guinea pigs poisoned by 2.3 LD50 of sarin and VX. With soman and tabun, the increase in the time of survival was slight, but consistent. In every case, the time taken for signs of poisoning to occur was unaffected by the pretreatment. P2S prophylaxis enhanced the effectiveness of therapy with atropine, and P2S given after poisoning with organophosphates had a similar effect, although the extent of the protection against soman poisoning was considerably less than that achieved against poisoning by sarin or VX.     

Ribo- and deoxyribonucleoside effect on 3'-amino-2',3'-dideoxycytidine-induced cytotoxicity in cultured L1210 cells

3'Amino-2',3'-dideoxycytidine (3'-NH2-dCyd) is a potent inhibitor of the replication of cultured L1210 cells, with an IC50 of 1 microM. When ribo- and deoxyribonucleosides were examined for their effects on 3'-NH2-dCyd-induced cytotoxicity, only dCyd could both prevent and reverse these effects. Furthermore, even when the maximum increase in modal cell volume was allowed to develop (24 hr) in the presence of 2.5 microM 3'-NH2-dCyd, the addition of 25 microM dCyd to the medium containing 3'-NH2-dCyd reduced the modal cell volume nearly to control levels within 24 hr. Examination of the viability of these cells by colony formation in soft agar, following as much as a 9.5-hr exposure of 1, 2.5 and 10 microM 3'-NH2-dCyd, revealed that the lethal effects of the 3'-NH2-dCyd treatment were not observed only when 25 microM dCyd was added to the medium during this time. However, the lethality of a 24-hr exposure of 2.5 and 10 microM 3'-NH2-dCyd could not be prevented either by removal of the drug from the medium or by a 24-hr exposure of the medium containing 3'-NH2-dCyd to 25 microM dCyd. When the effect of 3'-NH2-dCyd on DNA biosynthesis in L1210 cells was examined, it was found that radiolabeled dAdo incorporation decreased by approximately 60, 80 or 90% following a 2.5-hr exposure to 2.5, 10 or 20 microM 3'-NH2-dCyd respectively. The addition of 25 microM dCyd under the same conditions resulted in a greater amount of dAdo incorporation compared to the unrescued cultures. Deamination of 3'-NH2-dCyd by partially purified human cytidine-deoxycytidine deaminase was about 2.5% that of either Cyd or dCyd deamination. The deaminated derivative, 3'-amino-2',3'-dideoxyuridine, was significantly less cytotoxic even at 50 microM.     

Metabolism of bromobenzene to glutathione adducts in lung slices from mice treated with pneumotoxicants

Recent studies showing that the bronchiolar Clara cell and alveolar Type II cell are major loci of cytochrome P-450 monooxygenases in the lung suggested that measurement of xenobiotic metabolizing enzyme activity might provide a useful and sensitive index of injury to these cell types. Accordingly, an assay has been developed for quantitating the rate of formation of reactive bromobenzene metabolites in lung slices which is based upon measuring the rate of formation of bromobenzene glutathione adducts. To demonstrate that monitoring adduct formation would yield quantitatively similar data to the traditional covalent binding assay for measuring the formation of reactive bromobenzene intermediates, covalent binding and conjugate formation were assayed in incubations of phenobarbital-induced hepatic microsomes conducted in the presence of various cytochrome P-450 monooxygenase inhibitors. Incubation conditions which decreased the rate of covalent binding (incubations done in the absence of glutathione) resulted in similar decreases in conjugate formation (incubations done in the presence of glutathione). In lung slices, the metabolism of bromobenzene to glutathione conjugates was linear for 20 min and continued to increase with time over the entire 160 min of the study. The formation of bromobenzene glutathione adducts in lung slices from piperonyl butoxide-treated animals occurred at a significantly lower rate than control. Likewise, lung slices from animals treated with butylated hydroxytoluene or carbon tetrachloride, agents known to injure alveolar epithelial cells, metabolized bromobenzene to glutathione conjugates at significantly slower rates than control. In contrast, treatment with naphthalene or dichloroethylene, agents which damage the bronchiolar epithelial cells, had little or no effect on conjugate formation. Similarly, there were no significant differences in the rate of bromobenzene glutathione conjugate formation between lungs of air- and ozone-exposed (1.0 ppm × 4 hr) mice killed 2,24,48,72, or 120 hr after exposure. These studies suggest that monitoring the rate of bromobenzene glutathione conjugate formation in lung slices may be a useful and sensitive biochemical index of injury to certain cells of the lung but that severe damage to the nonciliated bronchiolar epithelial cells has little effect on the rate of metabolic activation of this aromatic hydrocarbon.     

The 1,2-dichloroethylenes: Their metabolism by hepatic cytochrome P-450 in vitro

Cis- and trans-1,1-dichloroethylene bound to the active site of hepatic microsomal cytochrome P-450 with the production of a Type I difference spectrum and stimulated CO-inhibitable hepatic microsomal NADPH oxidation. Incubation of cis- and trans-1,2-dichloroethylene plus hepatic microsomes, NADPH-generating system-EDTA resulted in the production of measurable levels of 2,2-dichloroethanol and dichloroacetaldehyde but not of 2-chloroethanol, chloroacetaldehyde or chloroacetic acid and, also, resulted in decreased levels of hepatic microsomal cytochrome P-450 and heme. In addition, dichloroacetic acid was produced from trans-dichloroethylene under these experimental conditions. The omission of any component of the incubation mixture eliminated the above effects, while the inclusion of SKF-525A, metyrapone or CO: O₂ (80, v/v) diminished these effects. The effects of β-naphthoflavone and phenobarbital pretreatment on the values of K_s, ΔA_max, K_m and V_mam for the binding and metabolism of the 1,2-dichloroethylenes are reported. The binding and metabolism of the 1,2-dichloroethylenes and the 1,2-dichloroethylene-mediated inactivation of cytochrome P-450 were enhanced per mg of microsomal protein, but generally not per nmole of cytochrome P-450 by prior induction with β-naphthoflavone or phenobarbital. It is concluded that multiple forms of hepatic microsomal cytochrome P-450 bind and metabolize the 1,2-dichloroethylenes. The role of cytochrome P-450 in the metabolic activation of the dichloroethylenes is considered.     

2-Hydroxylaminoimidazoles--unstable intermediates in the reduction of 2-nitroimidazoles

An unstable 2-hydroxylaminoimidazole (2-hydroxylamino-1-methylimidazole) was prepared by the reaction of 2-fluoro-1-methylimidazole with hydroxylamine. This substance was sufficiently stable (half-life of 1-2 days) in acid solutions to be observed and characterized by NMR spectroscopy; decomposition at neutrality was, however, rapid (half-life of 1-10 min). Radiochemical and electrochemical reduction experiments were carried out at pH 4 and pH 7 with 2-nitro-1-methylimidazole and misonidazole [1-(3'-methoxy-2'-hydroxypropyl)-2-nitroimidazole]. A four electron stoichiometry was found in every case. The pH 4 reduced product was identified as the 2-hydroxylamino derivative (greater than 80% yield). The pH 7 reduced solutions, on the other hand, showed no aromatic 1H NMR signals, suggesting that a simple imidazole ring was no longer present. A shift to pH 7 of the hydroxylamine produced at pH 4, however, resulted in very similar NMR spectra. The conclusion, therefore, is that the hydroxylamine was produced initially on reduction of the nitroimidazole, but it was not stable.