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R J Huxtable

Publications and source records attributed to R J Huxtable.

At least 37 records · Page 2Linked to original sources
PubMed1996

Effects of taurine and guanidinoethane sulfonate on toxicity of the pyrrolizidine alkaloid monocrotaline.

Monocrotaline (MONO), a pyrrolizidine alkaloid, causes pulmonary arterial hypertension and right ventricular hypertrophy due to hepatic metabolism to the alkylating pyrrole dehydromonocrotaline. Taurine a sulfonic amino acid, is hepato- and cardioprotective in a variety of conditions. We have examined the effects of taurine and its amidino analog, guanidinoethane sulfonate (GES), in rats injected i.p. with MONO (65 mg/kg). Taurine and GES were given as 1% solutions in drinking water beginning 14 days before administration of MONO and continuing for 14 days therafter, when the rats were killed. The MONO group had right ventricular hypertrophy and pulmonary hyperplasia. Compared with control, no significant changes in the right ventricle/left ventricle weight ratio, or the right ventricle/body weight ratio occurred in rats also given taurine of GES. Lung weights in these two groups were higher than in the control group, but below that of the MONO-alone group. The lethality of MONO over 14 days was decreased by taurine (LD50 for MONO alone 80 mg/kg; for MONO + taurine 121 mg/kg). Rats given only MONO had lower hepatic concentrations of GSH and cysteine (Cys), and higher activities of microsomal GSH transferase activity were no different from control. Gamma-Glutamylcysteine (Glu-Cys) synthetase and gamma-glutamyl transpeptidase activities were elevated. In MONO-injected rats given GES, hepatic GSH levels were higher and Cys levels were lower than in either the MONO alone or MONO + taurine groups. Gamma-Glu-Cys synthetase activity was depressed. Microsomal GSH transferase, GSH peroxidase and gamma-glutamyl transpeptidase activities were elevated. Livers of MONO-injected animals showed higher levels of serine (reversed by both taurine and GES) and glycine (Gly; reversed by GES) and lower levels of glutamine. Compared with control rats, the following changes occurred in serum amino acids: MONO alone: increased aspartate, taurine and lysine; taurine-supplemented: increased taurine, methionine (Met) and lysine, and decreased Gly; GES-supplemented: decreased asparagine, serine, Gly, arginine, taurine, and valine. Compared with the MONO-alone group, the taurine-supplemented group had higher glutamate (Glu), Met and alanine, and the GES-supplemented group higher alanine and lower serine, Gly, arginine and valine. We conclude that taurine protects against MONO-induced lethality and right ventricular hypertrophy. GES also protects against right ventricular hypertrophy. However, these agents act by different mechanisms, taurine preventing many of the biochemical changes induced by MONO, with GES inducing additional changes.

Amino Acids↗
PubMed1996

Effects of monocrotaline, a pyrrolizidine alkaloid, on glutathione metabolism in the rat.

Monocrotaline (MONO), a pyrrolizidine alkaloid, causes veno-occlusive disease of the liver, pulmonary arterial hypertension, and right ventricular hypertrophy. Toxicity is due to the hepatic formation of a pyrolic metabolite that can be detoxified by conjugation with glutathione (GSH). We have shown that the GSH content of the liver affects the quantity of the pyrrolic metabolite that is released from the liver. We have now examined whether MONO, in turn, affects GSH metabolism. Twenty-four hours after administration of MONO to rats (65 mg/kg, i.p.), the highest concentration of bound pyrrolic metabolites was found in the liver, followed by the lung and kidney. Heart and brain contained lower concentrations of these metabolites. Significantly higher levels of GSH were found in liver and lungs of MONO-treated rats than in saline-injected control animals. In the liver, activities of the following enzymes were elevated: gamma-glutamylcysteine synthetase, GSH synthetase, gamma-glutamyl transpeptidase, dipeptidase, and microsomal GSH transferase. The same changes were seen in the lung. In the heart, gamma-glutamyl transpeptidase activity was decreased markedly, and cytosolic GSH transferase activity was elevated. In the kidney, the activities of GSH synthetase, gamma-glutamyl transpeptidase, and cytosolic GSH transferase were increased. Our results establish a mutual interaction of MONO and sulfur metabolism. It appears that an early metabolic action of MONO is to modify sulfur amino acid metabolism, diverting cysteine metabolism from oxidation to taurine towards synthesis of GSH.

Animals↗
PubMed1996

Physicochemical and metabolic basis for the differing neurotoxicity of the pyrrolizidine alkaloids, trichodesmine and monocrotaline.

Monocrotaline and trichodesmine are structurally closely related pyrrolizidine alkaloids (PAs) exhibiting different extrahepatic toxicities, trichodesmine being neurotoxic (LD(50) 57 mu mol/kg) and monocrotaline pneumotoxic (LD(50) 335 mu mol/kg). We have compared certain physicochemical properties and metabolic activities of these two PAs in order to understand the quantitative and qualitative differences in toxicity. Both PAs were metabolized in the isolated, perfused rat liver to highly reactive pyrrolic dehydroalkaloids that appear to be responsible for the toxicity of PAs. More dehydrotrichodesmine (468 nmol/g liver) than dehydromonocrotaline (116 nmol/g liver) was released from liver into perfusate on perfusion for 1 hr with 0.5 mM of the parent PA. Dehydrotrichodesmine had a significantly longer aqueous half-life (5.4 sec) than that of dehydromonocrotaline (3.4 sec). In vivo, significantly higher levels of bound pyrroles were found in the brain 18 hr after injection of trichodesmine (25 mg/kg; i.p.) than were seen following either an equal dose (25 mg/kg; i.p.) or an equitoxic dose (90 mg/kg; i.p.) of monocrotaline. Trichodesmine had a higher partition coefficient than monocrotaline for both chloroform and heptane, indicating its greater lipophilicity. The pK(a) of trichodesmine (7.07) was only slightly higher than that of monocrotaline (pK(a¿ 6.83), suggesting that a difference in degree of ionization was not a major factor affecting the relative ability of the dehydroalkaloids to cross the blood-brain barrier. We conclude that the greater lethality and neurotoxicity of trichodesmine compared to monocrotaline is due to two structural characteristics: (i) steric hindrance at position 14 of dehydrotrichodesmine results in greater resistance to hydrolysis, allowing more to be released from the liver and to be delivered to the brain; (ii) the larger isopropyl substituent at position 14 of dehydrotrichodesmine renders the molecule more lipophilic, leading to greater penetration of the brain.

Alkaloids↗
PubMed1996

A simple procedure for determining the aqueous half-lives of pyrrolic metabolites of pyrrolizidine alkaloids.

We report a simple and rapid procedure for estimating the aqueous half-lives of the reactive metabolites of pyrrolizidine alkaloids that are responsible for toxicity. The metabolites (dehydroalkaloids; DHAs) were rapidly added to a 0.5 mM HEPES solution, pH 8.0. The subsequent fall in pH, due to ester hydrolysis, was followed potentiometrically. The change in pH was well described by single-component exponential decay, allowing the derivation of rate constants and half-lives of hydrolysis. Half-lives varied from 0.31 sec for dehydro-7-acetyllycopsamine to 5.36 sec for dehydrotrichodesmine. The results support the view that alkaloids whose DHA metabolites have longer half-lives produce greater extrahepatic toxicity.

Animals↗
PubMed1995

Fluorimetric determination of monobromobimane and o-phthalaldehyde adducts of gamma-glutamylcysteine and glutathione: application to assay of gamma-glutamylcysteinyl synthetase activity and glutathione concentration in liver.

The reversed-phase HPLC separation of fluorescent o-phthalaldehyde (OPA) derivatives has been applied to the assay of hepatic gamma-glutamylcysteine and glutathione (GSH) levels and the enzymes producing these peptides. The method has been compared to the assay using monobromobimane (MB) as the derivatizing agent. The OPA method has the advantage of faster derivatization, the lack of need to adjust the pH, isocratic separation and selectivity for GSH and gamma-glutamylcysteine. The MB method requires pH adjustment following derivatization and gradient elution chromatography. MB is also non-selective, yielding fluorescent derivatives of all biological thiols and more interfering peaks on the chromatogram. MB-based analyses are also approximately sixty times more expensive per sample. MB yields fluorescent degradation products on exposure to light. OPA adducts are stable for up to ten days when stored at -20 degrees C. OPA detection is sensitive to 12.5 pmol in the sample, at a signal-to-noise ratio of 2.5. The two methods correlate well. Hepatic gamma-glutamylcysteine synthetase in the same liver preparation was found to be 4.85 +/- 0.47 nmol min-1 mg-1 protein by the OPA method and 4.42 +/- 0.52 nmol min-1 mg-1 protein by the MB method. GSH concentrations were found to be 90.4 +/- 6.5 nmol/mg protein for the OPA method and 92.5 +/- 3.4 for the MB method.

Animals↗
PubMed1995

The relationship between the concentration of the pyrrolizidine alkaloid monocrotaline and the pattern of metabolites released from the isolated liver.

Hepatic metabolism of the pyrrolizidine alkaloid monocrotaline results in extrahepatic toxicity caused by the release of metabolites from the liver. We have quantified the release of pyrrolic metabolites into the perfusate and bile of isolated rat livers perfused with monocrotaline over the concentration range of 0.125-1.5 mM. Over a 1-hr perfusion period, the amount of dehydromonocrotaline released from the liver varied from 60 nmol/g liver at 0.125 mM monocrotaline to 460 nmol/g liver at 1.5 mM monocrotaline. As a percentage of total pyrrole release, this is a monotonic increase from 30 to 41%. The percentage of pyrroles released into the bile, representing mainly 7-glutathionyl-6,7-dihydro- 1-hydroxymethyl-5H-pyrrolizine (GSDHP), increased over the monocrotaline concentration range 0.125-1.0 mM, but fell sharply from 38% of total at the latter concentration to 21% of total at 1.5 mM monocrotaline. This is probably a reflection of glutathione depletion. Nonalkylating pyrrole released into the perfusate, represents largely 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP). Pyrrole released into perfusate showed an opposite pattern. The percentage of pyrroles released as DHP into the perfusate fell from 38% at 125 microM monocrotaline to 27% at 1.0 mM monocrotaline, but increased sharply to 38% at 1.5 mM monocrotaline. When calculated on a body weight basis, concentrations of monocrotaline of 500 microM result in the release from the liver of 5.3 mumol/kg of dehydromonocrotaline. This is comparable to the amount of dehydromonocrotaline, given in vivo, required for pneumotoxicity. The amounts of other pyrrolic metabolites released over a 1-hr period of perfusion are insufficient to produce pneumotoxicity in vivo. Based on the body weight of the donor rat, pyrrole release on perfusion of the isolated liver with 1,500 microM monocrotaline can be calculated as mumol/kg body weight. These amounts can then be compared to acute doses producing pneumotoxicity in vivo (given in parentheses): DHP, 13 mumol/kg body weight released (350 mumol/kg); GSDHP, 8 mumol/kg (300 mumol/kg); and dehydromonocrotaline, 14 mumol/kg (15 mumol/kg). This suggests, therefore, that dehydromonocrotaline is the pyrrolic metabolite contributing the most to the extrahepatic toxicity of monocrotaline.

Analysis of Variance↗
PubMed1995

Relationship between glutathione concentration and metabolism of the pyrrolizidine alkaloid, monocrotaline, in the isolated, perfused liver.

The influence of GSH concentration on metabolism of monocrotaline was examined in the isolated, perfused rat liver. Chloroethanol (0.37 mmol/kg), diethyl maleate (5.6 mmol/kg), and buthionine sulfoximine (72.9 mmol/kg) given in vivo reduced hepatic GSH from 3.7 mumol/g wet weight to 1.5, 0.6 and 0.9 mumol/g, respectively. Livers were then perfused in vitro for 1 hr with monocrotaline (0.5 mM). GSH depletion had no effect on the total release of pyrrolic metabolites of monocrotaline. Depletion, however, markedly affected the pattern of pyrrole release. Biliary release of 7-glutathionyl-6,7-dihydro-1-hydroxy-methyl-5H-pyrrolizine (GSDHP) was reduced by up to 72%. Pretreatment with diethyl maleate or buthionine sulfoximine increased the level of protein-bound pyrroles in the liver by 107 and 84%, respectively. Such pyrroles are probably responsible for liver toxicity. GSH depletion also led to a doubling of dehydromonocrotaline release into the perfusate. This metabolite is probably responsible for the extrahepatic toxicity of monocrotaline. Release into perfusate of the relatively nontoxic metabolite, 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) was correspondingly decreased. Hepatic GSH content was increased to 4.4 mumol/g by pretreatment with oxo-4-thiazolidine carboxylate (4.76 mmol/kg). This agent increased total pyrrolic metabolites by 54%. Biliary release of GSDHP and perfusate release of dehydromonocrotaline and DHP were all increased. Thus, hepatic GSH levels regulate the metabolism of monocrotaline and dehydromonocrotaline and, consequently, the hepatic and extrahepatic toxicity of monocrotaline. GSH depletion leads to a switch from the biliary release of the midly toxic GSDHP to the perfusate release of the highly toxic dehydromonocrotaline. GSH depletion also permits more dehydromonocrotaline in the liver to become available for macromolecular alkylation. These findings suggest that nutritional intake of sulfur-containing amino acids can influence the severity of pyrrolizidine poisoning.

Analysis of Variance↗
PubMed1995

The comparative metabolism of the four pyrrolizidine alkaloids, seneciphylline, retrorsine, monocrotaline, and trichodesmine in the isolated, perfused rat liver.

Despite their similarity in structure, pyrrolizidine alkaloids (PAs) vary in their LD50s and in the organs in which toxicity is expressed. We have examined whether there are differences in the metabolism of certain PAs that are associated with these quantitative and qualitative differences in toxicity. Isolated rat livers were perfused with one of four PAs (seneciphylline, retrorsine, monocrotaline, and trichodesmine) at 0.5 mM for 1 hr, and the pyrrolic metabolites determined that were released into perfusate and bile or bound in the liver. The proportion of the PA removed by the liver varied from 93% for retrorsine to 55% for trichodesmine. However, trichodesmine-perfused livers released the greatest amount of the dehydroalkaloid into the perfusate. These reactive pyrrolic metabolites appear to be largely responsible for the toxicity of PAs. Over the course of a 1-hr perfusion, dehydroalkaloid release varied fourfold among the PAs examined. Seneciphylline and retrorsine significantly increased bile flow. Highest concentrations of PAs in bile were achieved at 30-40 min perfusion. Conversion of dehydroalkaloid to the conjugate 7-glutathionyl-6,7-dihydro-1-hydroxymethyl-5H-pyrrolizine (GSDHP) is a detoxification reaction. GSDHP release into bile varied from 80 nmol/g liver for trichodesmine to 880 nmol/g for retrorsine. Release of the less toxic hydrolytic product of dehydroalkaloids, 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine, was also determined. Bound pyrroles in liver are probably an indication of heptatoxicity. At the end of perfusion these varied from 55 nmol/g for monocrotaline to 195 nmol/g for retrorsine. The chemical form of the bound pyrroles is a 7-thioether conjugate of 6,7-dihydro-1-hydroxymethyl-5H-pyrrolizine. No 7,9-dithio conjugate was detected, indicating that only monoalkylation has been found. These differences in metabolic pattern reflect differences in reactivity of the initially formed dehydroalkaloid and can account for the toxicological differences between the parent PAs.

Alkaloids↗
PubMed1995

Effect of the pyrrolizidine alkaloid, monocrotaline, on bile composition of the isolated, perfused rat liver.

Monocrotaline is a hepatotoxic pyrrolizidine alkaloid, releasing high levels of metabolites into bile of isolated, perfused liver. Although perfusion of rat liver with 0.5 mM monocrotaline does not affect bile flow over a 1 hr study period, it markedly affects bile composition. Biliary release of conjugated and free GSH increases 30-fold. Marked increases are also observed in the biliary concentration of the related sulfur-containing substances, cysteine and cysteinylglycine. However, biliary release of the sulfur amino acids, taurine and methionine, is unaffected. Only two amino acids show mildly increased releases, 23% for glycine and 46% for aspartate. Release of bile acids, cholesterol and phospholipids also decrease, both in terms of mM concentration in bile and in terms of nmol secreted per g liver. Thus, exposure to monocrotaline causes disturbances in sulfur metabolism in the liver and in the composition of bile. The consequences of the digestive properties of bile and gastrointestinal toxicity remain to be established. As sulfhydryl compounds are involved in detoxification of monocrotaline metabolites, these findings indicate a mutual interaction of pyrrolizidine toxicity and sulfur metabolism. This suggests that dietary sulfur amino acid intake may influence susceptibility to pyrrolizidine poisoning.

Amino Acids↗
PubMed1995

The effect of the pyrrolizidine alkaloids, monocrotaline and trichodesmine, on tissue pyrrole binding and glutathione metabolism in the rat.

One day after in vivo administration of equitoxic doses of the hepatotoxic and pneumotoxic pyrrolizidine alkaloid, monocrotaline (65 mg/kg, i. p.) or the related hepatotoxic and neurotoxic alkaloid trichodesmine (15 mg/kg, i. p.) hepatic GSH levels are increased by more than 50%. These doses of alkaloids represent 60% of the LD50 values. Accompanying these changes in GSH levels is an increase in the overall rate of GSH synthesis in supernatants of alkaloid-exposed livers. The ability of the rat to metabolize the two alkaloids was shown by the appearance of tissuebound pyrrolic metabolites of pyrrolizidines in various organs. The levels of these metabolites appear to correlate with organ toxicity. For the hepatic and pneumotoxic alkaloid, monocrotaline, higher levels are found in liver (17 nmoles/g tissue) and lung (10 nmoles/g) than for trichodesmine (7 nmoles/g and 8 nmoles/g, respectively). For the neurotoxic alkaloid, trichodesmine, higher levels are found in brain (3.8 nmoles/g tissue) than for monocrotaline (1.7 nmoles/g tissue).

Alkaloids↗