Biomedical subjects

H J Segall

Publications and source records attributed to H J Segall.

At least 37 records · Page 2Linked to original sources

PubMed1996

Cell cycle alterations associated with covalent binding of monocrotaline pyrrole to pulmonary artery endothelial cell DNA.

In the monocrotaline (MCT) rat model of pulmonary hypertension, the pulmonary vascular endothelium is thought to be the early target of the bifunctionally reactive metabolite monocrotaline pyrrole (MCTP). In previous studies, bovine pulmonary arterial endothelial cells (BPAEC) exposed to MCTP exhibited inhibition of proliferation. Since other compounds that crosslink DNA lead to cell cycle alterations, we utilized BPAEC to correlate the effects of MCTP on the cell cycle with the extent of covalent binding of [14C]MCTP to BPAEC DNA. Dose response (0.0 to 50.0 micrograms MCTP/ ml) and 96-hr time course (5 micrograms MCTP/ml low dose or 34.5 micrograms MCTP/ml high dose) studies were carried out followed by flow cytometric cell cycle analysis. High concentrations of MCTP caused cell cycle arrest in S phase, beginning by 24 hr, while an S phase delay was observed at low concentrations, but progressed to a G2 + M phase arrest by 48 hr. Covalent DNA binding (34.5 micrograms/ml of [14C]MCTP incubated with BPAEC) occurred within 1 hr and progressively increased through 96 hr. In conclusion, covalent binding of MCTP to DNA is associated with cell cycle arrest; however, the position of cell cycle arrest is dependent on dose, with an S phase arrest at high concentrations and a G2 + M phase arrest at low concentrations of MCTP. The mechanism by which MCTP induces proliferative inhibition could be cell cycle arrest.

Animals↗

PubMed1995

Hydrolysis rates of pyrrolizidine alkaloids derived from Senecio jacobaea.

Many of the commonly studied pyrrolizidine alkaloids (PAs) are built upon the subgroup retronecine (RET), which is released from the parent molecule by either base catalyzed or enzymatic hydrolysis of the ester linkages. The rate of appearance of RET in a hydrolytic study would thus reflect the rate of hydrolysis for the PA being tested. We have developed a gas chromatographic (GC) method to measure the release of RET from incubations of PAs with the guinea pig carboxylesterase, GPH1. The PAs tested were the following: jacobine (JAB), jacozine (JAZ), retrorsine (RES), and seneciphylline (SNP). The KmS for SNP and JAZ were determined to be 64.9 and 349.2 microM, respectively. In addition, a qualitative assessment of hydrolytic activity toward a radiolabelled mixture of retrorsine/riddelliine (RES/RIL) was performed with HPLC and radiometric detection.

Animals↗

PubMed1995

Biliary excretion of pyrrolic metabolites of [14C]monocrotaline in the rat.

The biliary excretion of amphoteric pyrroles was studied using in situ isolated rat liver preparations perfused in a recirculatory manner for 60 to 90 min with 14C-monocrotaline (40 mumol/120 ml of Krebs-Henseleit buffer). After 90 min, 20% of the administered 14C and an amount of pyrrole equivalent to 9 mumol of dehydroretronecine was recovered in the bile. Bile collected between 15 and 30 min contained the highest levels of 14C and pyrrolic metabolites. Isoelectric focusing (IEF) and electrophoretic separations on matrices of polyacrylamide and silica were used to isolate and estimate levels of pyrrolic conjugates. IEF and electrophoretic separations on silica revealed the presence of six pyrrole-positive metabolites. Reactions of monocrotaline pyrrole with glutathione produced three conjugates that had the same pI values as components found in bile. Electrophoretic separations on silica and polyacrylamide followed by reversed phase chromatography removed sufficient biliary matrix contaminants to permit identification of glutathione and cysteinyl-glycine conjugates of 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine and 1-formyl-7-hydroxy-6,7-dihydro-5H-pyrrolizine with fast atom bombardment MS/MS. This study revealed a complexity in the biliary pyrrolic excretion profile that had not previously been realized.

Animals↗

PubMed1994

Glutathione conjugation with the pyrrolizidine alkaloid, jacobine.

The reaction of glutathione with the epoxide containing pyrrolizidine alkaloid, jacobine, was catalyzed by guinea pig hepatic glutathione-S-transferase enzymes in in vitro experiments; the rate of the reaction in the presence of rat hepatic glutathione-S-transferases did not differ from the non-enzymatic rate. Using ion-pairing liquid chromatography we were able to isolate the conjugate and obtain a daughter ion spectrum using Fast Atom Bombardment Mass Spectrometry together with Collisionally Activated Dissociation/Mass Analyzed Kinetic Energy experiments.

Animals↗

PubMed1993

Strain differences in the response of Fischer 344 and Sprague-Dawley rats to monocrotaline induced pulmonary vascular disease.

The pyrrolizidine alkaloid (PA) monocrotaline (MCT) is thought to be activated in the liver to monocrotaline pyrrole (MCTP) which is then transported to the lungs where it causes a pulmonary vascular syndrome characterized by elevated pulmonary artery pressure and right ventricular hypertrophy. We have found that, as opposed to Sprague-Dawley (SD) rats, Fischer 344 (F344) strain rats are resistant to the ventricular hypertrophy and pressure changes induced by MCT. To determine whether this strain difference might be related to differences in hepatic activation of MCT to MCTP, we compared the response of SD and F344 rats to treatment with MCT or MCTP. We determined right ventricular pressure and ventricular weight ratios with each treatment for each strain. We also compared subjective lesion scores of histopathologic changes characteristic of MCT pneumotoxicity. Sprague-Dawley rats treated with either MCT or MCTP had elevated right ventricular pressures (Control 13 +/- 1 mmH2O, MCTP 29 +/- 3 mmH2O, MCT 24 +/- 2 mmH2O) and increased right ventricular weight ratio (RV/LV + S) of 0.30 +/- 0.01 (Control), 0.44 +/- 0.05 (MCTP), 0.44 +/- 0.02 (MCT). Histopathologic evaluation demonstrated that significant alveolar septal fibrosis, edema and type II cell hypertrophy was induced by both PAs in both rat strains but that F344 rats had significantly less vascular medial hypertrophy and adventitial inflammation than SD rats. MCTP treated SD rats had similar vascular and parenchymal alterations as those treated with MCT but with a lesser inflammatory component. We conclude that the strain differences in cardiac and pulmonary vascular responses to MCT also occur with MCTP treatment. This, combined with the similarity in alveolar parenchymal response to both PAs in both strains, suggests that these differences are related to the pulmonary vascular response rather than differences in hepatic metabolism.

Animals↗

PubMed1993

Analysis of mercapturic acid conjugates of xenobiotic compounds using negative ionization and tandem mass spectrometry.

Mass spectra of mercapturic acid conjugates of two xenobiotic products of lipid peroxidation (trans-4-hydroxy-2-hexenal and trans-4-hydroxy-2-nonenal) as well as conjugates of 1,3-dichloropropene, styrene oxide, 1,2-naphthalene oxide and alpha-chlorotoluene were obtained using fast atom bombardment or negative chemical ionization. Fragmentation pathways were investigated using linked scan and mass-analyzed ion kinetic energy spectrometric techniques. Characteristics of the spectra obtained using different ionization and sample introduction techniques are compared. Deprotonated molecular ions of mercapturic acids gave simple daughter ion spectra, with the dominant mode of decomposition involving cleavage of C-S bonds giving a characteristic neutral loss of 129 Da. Screening for mercapturates in urine samples was performed using neutral loss scanning and yielded limits of detection in the low nanogram per milliliter range. Quantitative analysis of the S-benzyl mercapturic acid at 1 p.p.b. in urine has been demonstrated using combined gas chromatography/electron capture mass spectrometry with d3-S-benzyl mercapturic acid as internal standard.

Acetylcysteine↗

PubMed1993

COR pulmonale is caused by monocrotaline and dehydromonocrotaline, but not by glutathione or cysteine conjugates of dihydropyrrolizine.

Monocrotaline (MCT) produces pulmonary hypertension and right ventricular hypertrophy in rats. It is generally believed that MCT must undergo hepatic metabolism to reactive metabolites that are subsequently transported to the lungs to induce a pneumotoxic response. Several studies suggest that dehydromonocrotaline (MCTP) is the reactive intermediate that initiates pulmonary toxicity. We recently identified two other MCT metabolites, the glutathione and N-acetylcysteine conjugates of 6,7-dihydro-7-hydroxy-1-hydromethyl-5H-pyrrolizine (DHP). To determine the potential pulmonary toxicity of the glutathione conjugate (DHP-GSH) and the unacetylated cysteine conjugate precursor (DHP-Cys) of the N-acetylated excretion product, we conducted parallel in vivo toxicity studies with DHP-GSH, DHP-Cys, MCT, and MCTP. Relative pneumotoxicity was evaluated by measurements of right ventricular pressure (RVP), ventricular weight ratio (RV/LV+S), subjective histopathology, and measurements of components of the arteriolar wall. Animals given a single injection of MCT (60 mg/kg) developed pulmonary hypertension at the end of 3 weeks, as indicated by significant elevation in RVP when compared to the controls (22.1 +/- 2.4 mm Hg vs 13.2 +/- 0.8 mm Hg). A parallel and significant increase in RV/LV+S was also evident: 0.37 +/- 0.021 (MCT) vs 0.299 +/- 0.011 (control). Histopathology showed marked alterations in both pulmonary vasculature and parenchyma in MCT- and MCTP-treated animals. MCTP (1 mg/kg) caused a significantly elevated RVP (MCTP vs control: 28.1 +/- 3.4 mm Hg vs 16.8 +/- 0.97 mm Hg) and an increased RV/LV+S (MCTP vs control: 0.445 +/- 0.051 vs 0.284 +/- 0.026). Both MCT- and MCTP-treated rats had increased arteriolar medial thickness and decreased lumen diameter, but MCTP-treated rats had a milder vascular inflammatory response and less parenchymal lesions. Neither DHP-GSH (24 or 12 mg/kg) nor DHP-Cys (12 mg/kg) caused detectable changes in pulmonary circulation and no structural alteration in the lung was observed in these treatment groups. Although they are all pyrrolic metabolites of MCT, these studies demonstrate that only MCTP but not the glutathione or cysteine conjugates, is pneumotoxic at the doses tested.

Animals↗

PubMed1993

In vivo metabolism of retrorsine and retrorsine-N-oxide.

The in vivo metabolism and excretion of the urinary metabolites from the pyrrolizidine alkaloids (PAs), retrorsine (RET) and retrorsine-N-oxide (RET-NO) have been studied in rats. Isatinecic acid (INA), pyrrolic metabolites, N-oxides and retronecine accounted for 31.0, 10.3, 10.8 and 0.39% of the administered RET. Predosing rats with triorthocresyl phosphate (TOCP), had no effect on the excretion of pyrrolic metabolites and INA. Phenobarbital (PB) increased the excretion of both pyrrolic metabolites and INA with a corresponding decrease in the excretion of RET and N-oxides; the retronecine levels remained unaltered. When RET-NO was administered i.p., the urinary levels of pyrrolic metabolites, INA and RET were decreased relative to those treated with RET. The p.o. administration of RET-NO produced significantly higher levels of pyrrolic metabolites, INA and RET. These results suggest that esterase hydrolysis plays a minor role in the formation of INA and that a common metabolic pathway may exist between pyrrolic metabolites and INA formation.

Animals↗

PubMed1992

Quantification of cytochrome P450 reductase gene expression in human tissues.

We have isolated and sequenced cDNA clones that code for a variant of human cytochrome P450 reductase. An RNase protection assay was used to quantify the corresponding mRNA in adult and fetal tissues. The results demonstrate that, in the samples analyzed, the cytochrome P450 reductase gene displays very little inter-individual variation in its expression in adult liver and is subject to little developmental or tissue-specific regulation.

Aging↗

PubMed1992

Hydrolysis of pyrrolizidine alkaloids by guinea pig hepatic carboxylesterases.

Two carboxylesterases (GPL1 and GPH1) were isolated from guinea pig hepatic microsomes and assayed for activity using the following pyrrolizidine alkaloids (PAs): seneciphylline (SNP), monocrotaline (MCT), and a mixture of senecionine (SEN) and integerrimine (INT) referred to as SEN-INT. GPH1 was able to effect the hydrolysis of all PAs, however, only minimal activity was seen for SEN-INT. The specific activity of GPL1 for p-nitrophenyl acetate was four times that of GPH1, but the former showed no activity toward PAs. The molecular weights and pIs were determined for both enzymes, and the Michaelis-Menten constants for two PAs, SNP and MCT were obtained using GPH1. The response to inhibitors confirmed GPH1 as a type B serine hydrolase although it was also inhibited by HgCl2. The isolation of a PA active esterase from the guinea pig may help to explain the resistance of this animal to PA intoxication, while enzyme substrate specificity may explain how the guinea pig's susceptibility to PA intoxication can differ toward various PAs.

Animals↗

PubMed1992

Mechanisms and pathology of monocrotaline pulmonary toxicity.

Monocrotaline (MCT) is an 11-membered macrocyclic pyrrolizidine alkaloid (PA) that causes a pulmonary vascular syndrome in rats characterized by proliferative pulmonary vasculitis, pulmonary hypertension, and cor pulmonale. Current hypotheses of the pathogenesis of MCT-induced pneumotoxicity suggest that MCT is activated to a reactive metabolite(s) in the liver and is then transported by red blood cells (RBCs) to the lung, where it initiates endothelial injury. While several lines of evidence support the requirement of hepatic metabolism for pneumotoxicity, the mechanism and relative importance of RBC transport remain undetermined. The endothelial injury does not appear to be acute cell death but rather a delayed functional alteration that leads to disease of the pulmonary arterial walls by unknown mechanisms. The selectivity of MCT for the lung, as opposed to that of other primarily hepatotoxic PAs, appears likely to be a consequence of the differences in hepatic metabolism and blood kinetics of MCT. A likely candidate for a reactive metabolite of MCT is the dehydrogenation product monocrotaline pyrrole (MCTP). Secondary or phase II metabolism of MCT through glutathione (GSH) conjugation has been characterized recently and appears to represent a detoxification pathway. The role of inflammation in the progression of MCT-induced pulmonary vascular disease is uncertain. Both perivascular inflammation and platelet activation have been proposed as processes contributing to the response of the vascular media. This review presents the experimental evidence supporting these hypotheses and outlines additional questions that arise from them.

Animals↗

PubMed1991

Red blood cells augment transport of reactive metabolites of monocrotaline from liver to lung in isolated and tandem liver and lung preparations.

Monocrotaline (MCT) is a pyrrolizidine alkaloid that causes pulmonary hypertension in rats by mechanisms which remain largely unknown. MCT is thought to be activated in the liver to a reactive intermediate that is transported to the lung where it causes endothelial injury. Our previous pharmacokinetic work demonstrated significant sequestration of radioactivity in red blood cells (RBCs) of rats treated with [14C]MCT. To determine whether this RBC sequestration might be important in the transport of reactive MCT metabolites, we compared the effect of inclusion of RBCs in the perfusion buffer on the extent of covalent binding of [14C]MCT to rat lungs in tandem liver-lung preparations. The potential effect of RBCs in stabilizing reactive intermediates was evaluated by preperfusion of isolated liver preparations with [14C]MCT with and without RBCs, separation and washing of the RBC fraction, and subsequent (90 min later) perfusion of washed RBCs or buffer alone in isolated perfused lungs. Covalent binding to lung tissues was determined by exhaustive methanol/chloroform extractions of unbound label from homogenized lung tissue followed by scintillation counting of residual 14C. Covalent binding was expressed as picomole MCT molecular weight equivalents/mg protein. Comparison of the relative capability of these isolated organ preparations for conversion of MCT to polar metabolites was done by extraction and HPLC analysis of perfusate at the end of the experiment. Isolated livers converted 65-85% of MCT to polar metabolites compared with less than 5% conversion in the isolated lungs. Inclusion of RBCs in the buffer of tandem lung liver preparations perfused with 400 microM [14C]MCT increased the covalent binding to the lung from 97 +/- 25 (buffer alone) to 182 +/- 36 (buffer + RBC) pmol/mg protein. At the end of these perfusions, RBCs contained 1552 +/- 429 pmol/mg hemoglobin of which 333 +/- 98 pmol/mg hemoglobin resisted exhaustive solvent extraction. After 90 min at room temperature, buffer with 400 microM [14C]MCT preperfused in isolated livers resulted in covalent binding to isolated perfused lung of 0.8 +/- 0.4 pmol/mg protein while washed RBCs isolated from buffer of similar liver preperfusions preparations resulted in 53 +/- 7 pmol/mg protein bound to lung. Control groups perfused with 400 microM [14C]MCT in buffer or buffer + RBCs through isolated lungs only resulted in covalent binding of 2 +/- 1 or 1 +/- 0.6 pmol/mg protein respectively. We conclude: (1) RBCs significantly augment the transport of lung reactive MCT metabolites from the liver to the lung.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

DNA cross-linking in mammalian cells by pyrrolizidine alkaloids: structure-activity relationships.

Pyrrolizidine alkaloids (PAs) are common constituents of many species of flowering plants which possess carcinogenic as well as anticarcinogenic activity in vivo. Pyrrolizidine alkaloids are genotoxic in various short-term assays. The mechanisms by which these compounds exert these effects is still unclear. In this study, we characterized the ability of eight bifunctional PAs, with differing stereochemistry and functional groups, to cross-link cellular DNA in cultured bovine kidney epithelial cells. PAs representative of three major structural classes, the macrocycles (seneciphylline, riddelline, retrorsine, senecionine, monocrotaline), the open diesters (heliosupine, latifoline), and pyrrolizidine base (retronecine) were cultured for 2 hr with cells and an external metabolizing system. Every PA induced DNA cross-links which consisted primarily of proteinase-sensitive cross-links (DPC), but also to a smaller extent, DNA interstrand cross-links (ISC). None of the PAs induced detectable amounts of DNA single-strand breaks. The PAs which produced DPC and/or ISC (ranked from most potent to least) were: seneciphylline (DPC greater than ISC); riddelline (DPC greater than ISC); retrorsine (DPC greater than ISC); senecionine (DPC greater than ISC); heliosupine (DPC greater than ISC); monocrotaline (ISC = DPC); latifoline (DPC greater than ISC); and retronecine (ISC greater than DPC). Although the PAs induced DNA cross-linking to varying degrees, cell viabilities for all treatment groups were greater than 90% as determined by trypan blue dye exclusion. Since the cross-linking ability of these PAs paralleled their ability to inhibit colony formation, cross-link formation may be involved in the biological activity of these compounds. Two structural determinants of biological activity appear to be the presence of both a macrocyclic necic acid ester and an alpha,beta-unsaturated ester function since the cross-linking ability of seneciphylline, riddelline, retrorsine, and senecionine far exceeded that of monocrotaline, heliosupine, latifoline, and retronecine. In addition, the stereochemical orientation of the ester linkage was found to have no effect on biological activity.

Animals↗

PubMed1991

Species difference in the urinary excretion of isatinecic acid from the pyrrolizidine alkaloid retrorsine.

1. The urinary excretion of the metabolites, isatinecic acid and pyrrolic metabolites from the pyrrolizidine alkaloid retrosine were lower in the resistant species, guinea-pigs, than the susceptible species, mice, hamsters and rats. 2. The urinary N-oxides levels, however, were higher in guinea-pigs relative to mice, hamsters and rats. 3. These results conform to the postulate that a common metabolic pathway exists between the formation of isatinecic acid and pyrrolic metabolites. 4. The resistance of guinea-pigs to PA poisoning is attributed to the high metabolism of PAs to N-oxides combined with a corresponding low conversion to pyrrolic metabolites.

Animals↗

PubMed1990

Excretion and blood radioactivity levels following [14C]senecionine administration in the rat.

Macrocyclic pyrrolizidine alkaloids (PAs) are a mixed group of phytotoxins with similar chemical structures and varying biological effects. A commonly studied member of this group is senecionine (SEN) which causes hepatotoxicity. We have undertaken metabolism and excretion studies of SEN in rats to provide data for comparison between PAs and to evaluate the potential role of metabolism and excretion in toxicity. Following intravenous administration of [14C]SEN (60 mg/kg, 10 microCi/kg), bile, urine and blood were collected over a 7-h period. Of the total administered radioactivity, 44% and 43% were excreted in the bile and urine, respectively. Using mass spectroscopy, senecionine N-oxide (SENNOX) was identified as the major metabolite in bile (52% of 44%) and urine (30% of 43%). For the total 7 h, less than 5% in bile and 18% in urine was excreted as parent alkaloid. The plasma concentration of Senecionine-equivalents/g (SEN-EQ/g) decreased from 107 to 12 nmol, while red blood cell (RBC) concentrations declined from 109 to 26 nmol/g. Without bile collection, the plasma levels of SEN-EQ were similar, while the final RBC level was almost double (47 vs. 26 nmol/g) and total radioactivity excreted in the urine was increased (59% vs. 43%). Biliary pyrrolic metabolites were estimated to be 1.43 mg, using a dehydroretronectin standard.

Animals↗

PubMed1990

N-acetylcysteine-conjugated pyrrole identified in rat urine following administration of two pyrrolizidine alkaloids, monocrotaline and senecionine.

This report demonstrates that an Ehrlich-reagent-positive metabolite of monocrotaline and senecionine is excreted in the urine of male rats as an N-acetylcysteine conjugate of (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (NAC-DHP). Isolation of the metabolite employed an initial organic extraction followed by HPLC separation of remaining urinary components using a reverse-phase, polymer-based, PRP-1 column. Fast-atom-bombardment tandem mass spectrometry was used to identify the metabolite. This finding suggests that reactive metabolites of pyrrolizidine alkaloids generated in the liver can survive the aqueous environment of the circulatory system as glutathione conjugates or mercapturic acids.

Acetylcysteine↗

PubMed1990

Isolation and identification of a pyrrolic glutathione conjugate metabolite of the pyrrolizidine alkaloid monocrotaline.

This report describes the isolation and identification of a monocrotaline-derived, glutathione-conjugated pyrrole obtained from the bile of male Sprague-Dawley rats. Bile obtained from rats given an intravenous bolus of 14C-monocrotaline was fractionated using a series of chromatographic separations. Initial purification with cholestyramine resin removed bile acid and pigment contaminants. Subsequent anion exchange and reversed-phase HPLC separations yielded several fractions that contained the 14C label and tested positive for pyrroles using Ehrlich's reagent. These fractions were analyzed using fast-atom-bombardment tandem mass spectrometry (FAB MS/MS). In addition to glutathione-conjugated dehydroretronecine, at least one other pyrrole present had similar ionic properties. The latter was not present in amounts sufficient for positive identification.

Animals↗

PubMed1990

Changes in type II cell populations in monocrotaline pneumotoxicity.

Unlike many other systemically administered pneumotoxicants affecting alveolar septa, Monocrotaline (MCT) does not cause a proliferative type II cell response. To determine whether MCT has an effect that might alter the type II cell response, we determined the numerical density and volume of type II cells in lungs from MCT-treated rats. Morphometric parameters were derived from estimates of nuclear volume based on point and intercept ratios counted in electron micrographs of type II cell profiles. These were combined with point and intercept counts of alveolar septal structures and counts of nuclear profiles per unit area to calculate average cell volume and numerical density of type II cells. Areal densities of type II cell profiles were determined by light microscopic counts of alveolar parenchyma and normalized to the volume of lung parenchyma. Cell volume of type II cells was markedly increased in MCT-treated animals (1.25 X 10(3) microns3) compared with controls (3.4 X 102) microns3). Nuclear diameter of type II cells was similarly increased (8.53 microns in treated animals versus 5.81 microns in controls). The number of type II cells was markedly decreased in treated animals (1.36 X 10(6] cells per cm3 lung parenchyma compared with 7.65 X 10(6) cells per cm3 parenchyma in controls). We conclude that MCT causes marked cellular hypertrophy of type II alveolar epithelial cells and that this hypertrophy is somehow related to a failure to maintain normal cell populations in the lung of MCT-treated rats. We hypothesize that this change is analogous to the hypertrophy and mitotic inhibition that occurs in the liver of animals treated with hepatotoxic pyrrolizidine alkaloids.

Animals↗