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Biomedical subjects

D Ross

Publications and source records attributed to D Ross.

At least 289 records · Page 16Linked to original sources
PubMed1985

Metabolism of diethylstilbestrol by horseradish peroxidase and prostaglandin-H synthase. Generation of a free radical intermediate and its interaction with glutathione.

Diethylstilbestrol is carcinogenic in rodents and in humans and its peroxidatic oxidation in utero has been associated with its carcinogenic activity. Horseradish peroxidase-catalyzed oxidation of [14C]diethylstilbestrol and [14C]diethylstilbestrol analogs induced binding of radiolabel to DNA only when the compound contained a free hydroxy group (Metzler, M., and Epe, B. (1984) Chem. Biol. Interact. 50, 351-360). We have found that horseradish peroxidase or prostaglandin-H synthase-catalyzed oxidation of diethylstilbestrol in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide caused the generation of an ESR signal indicative of a free radical intermediate (aN = 14.9 G, aH = 18.3 G). The identity of the trapped radical could not be identified on the basis of published hyperfine coupling constants, but the observation that horseradish peroxidase-catalyzed oxidation of 1-naphthol produced an identical ESR signal suggests that the radical was either a phenoxy or phenoxy-derived radical. During horseradish peroxidase-catalyzed oxidation of diethylstilbestrol in the presence of glutathione the thiol reduced the diethylstilbestrol radical to generate a thiyl radical. This was shown by a thiol-dependent oxygen uptake during horseradish peroxidase-catalyzed oxidation of diethylstilbestrol and the observation of an ESR signal consistent with 5,5-dimethylpyrroline-N-oxide-glutathionyl radical adduct formation. A diethylstilbestrol analog devoid of free hydroxy groups, namely diethylstilbestrol dipropionate, did not produce an ESR signal above control levels during horseradish peroxidase-catalyzed metabolism in the presence of 5,5-dimethylpyrroline-N-oxide. Thus, free radicals are formed during peroxidatic oxidation of diethylstilbestrol and must be considered as possible determinants of the genotoxic activity of this compound.

Carbon Radioisotopes↗
PubMed1985

The generation and subsequent fate of glutathionyl radicals in biological systems.

Horseradish peroxidase-catalyzed oxidation of p-phenetidine in the presence of either glutathione (GSH), cysteine, or N-acetylcysteine led to the production of the appropriate thioyl radical which could be observed using EPR spectroscopy in conjunction with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide. This confirms earlier work using acetaminophen (Ross, D., Albano, E., Nilsson, U., and Moldéus, P. (1984) Biochem. Biophys. Res. Commun. 125, 109-115). The further reactions of glutathionyl radicals (GS.), generated during horseradish peroxidase-catalyzed oxidation of p-phenetidine and acetaminophen in the presence of GSH, were investigated by following kinetics of oxygen uptake and oxidized glutathione (GSSG) formation. Oxygen uptake and GSSG generation were dependent on the concentration of GSH but above that which was required for maximal interaction with the primary amine or phenoxy radical generated during peroxidatic oxidation of p-phenetidine or acetaminophen, suggesting that a secondary GSH-dependent process was responsible for oxygen uptake and GSSG production. GSSG was the only product of thiol oxidation detected during peroxidatic oxidation of p-phenetidine or acetaminophen in the presence of GSH, but under nitrogen saturation conditions its production was reduced to 8 and 33% of the corresponding amounts obtained under aerobic conditions in the cases of p-phenetidine and acetaminophen, respectively. Nitrogen saturation conditions did not affect horseradish peroxidase-catalyzed metabolism. This shows that the main route of GSSG generation in such reactions is not by dimerization of GS. but via mechanism(s) involving oxygen consumption such as via GSSG-. or via GSOOH.

Acetaminophen↗
PubMed1985

The interaction of reduced glutathione with active oxygen species generated by xanthine-oxidase-catalyzed metabolism of xanthine.

The interaction of reduced glutathione (GSH) with active oxygen species generated during xanthine-oxidase-catalyzed metabolism of xanthine was investigated. The only GSH-derived product detected in this system was oxidized glutathione (GSSG). Catalase inhibited the oxidation of GSH to GSSG by more than 80%, whereas superoxide dismutase exerted a smaller but significant inhibition of GSSG formation. Hydroxyl radical (OH) scavengers or desferrioxamine (1 mM) had no effect on GSSG formation. Using EPR spectroscopy and the spin trap 5,5-dimethylpyrroline-N-oxide (DMPO), the production of superoxide was observed by the detection of a DMPO-OOH radical adduct. This spectrum was altered by the inclusion of GSH (5 - 20 mM) in the reaction mixture, indicating the generation of a different radical species consistent with DMPO-glutathionyl radical adduct generation.

Catalase↗
PubMed1985

The oxidation of p-phenetidine by horseradish peroxidase and prostaglandin synthase and the fate of glutathione during such oxidations.

The oxidation of p-phenetidine by horseradish peroxidase and prostaglandin synthase was investigated. The existence of a free radical intermediate formed during enzymatic oxidation was supported by a ratio of hydrogen peroxide: p-phenetidine consumed of 1:2 in the horseradish peroxidase system. Furthermore in both enzyme systems a rapid oxidation of added glutathione was observed and in the presence of the thiol there was a decreased removal of p-phenetidine. This suggests the reduction of a p-phenetidine radical by glutathione generating p-phenetidine and a thiyl radical. The latter react with oxygen and a rapid oxygen uptake was observed during enzymic oxidation in the presence of thiols. That p-phenetidine radicals were produced during horseradish peroxidase catalyzed oxidation of p-phenetidine was supported by experiments using the spin probe OXANOH. This was oxidized to its stable free radical form (OXANO.) in an enzyme- and substrate-dependent reaction and the EPR signal obtained was not decreased by SOD (80 micrograms/ml) or benzoate (10-100 mM). TLC characteristics of the products of the oxidation of p-phenetidine by both enzymes were almost identical inferring a similar mechanism of oxidation. Two of the metabolites were characterized by mass spectrometry and by comparison with reference compounds prepared by chemical oxidation. One metabolite was identified as 4,4'-diethoxyazobenzene, which further supports a radical mechanism, and the other was a p-phenetidine trimer which could exist in both oxidized and reduced forms. On the basis of these observations a mechanism for the oxidation of p-phenetidine and the fate of glutathione during such oxidations is proposed.

Aminophenols↗
PubMed1985

Stability-indicating liquid chromatographic determination of etoposide and benzyl alcohol in injectable formulations.

Simple and specific assays for etoposide, related impurities, and benzyl alcohol, as well as its degradation product, benzaldehyde, in injectable formulations were developed using high-performance liquid chromatography (HPLC). Etoposide, benzyl alcohol, benzaldehyde, and four etoposide impurities were determined with a mobile phase containing 26% acetonitrile in pH 4.0 acetate buffer with methyl p-aminobenzoate as the internal standard. Two etoposide precursors were additionally determined using 60% acetonitrile in the mobile phase with biphenyl as the internal standard. The assays were run sequentially. The etoposide assay had a total variability of less than 1% (RSD) and a recovery from placebo of 99.4%. Mean recovery of impurity standards spiked at the 2% level relative to the etoposide level was 98%. Recoveries for benzyl alcohol and benzaldehyde were 100.5% and 102.2%, respectively. Absorbance ratios generated from samples and compared with undegraded standards demonstrated specificity for etoposide. Similar response regression equations with correlation coefficients greater than 0.99 were obtained for etoposide and four impurities over the 2-8 micrograms/mL concentration range, indicating that an etoposide standard could be used to quantitate low impurity levels down to 0.2% relative to the etoposide injectable concentration. Benzaldehyde could be reliably detected at concentrations down to 0.5 micrograms/mL, equivalent to 0.16% of the benzyl alcohol level.

Benzaldehydes↗
PubMed1985

Mechanism of allyl alcohol toxicity and protective effects of low-molecular-weight thiols studied with isolated rat hepatocytes.

Freshly isolated hepatocytes from male rats were incubated with allyl alcohol at concentrations up to 2 mM. Allyl alcohol exerted a dose-dependent toxicity on the cells which was inversely related to cellular glutathione (GSH) content and accordingly influenced by stimulation as well as inhibition of GSH synthesis. The toxicity was prevented by inhibitors of alcohol dehydrogenase and augmented by the aldehyde dehydrogenase inhibitor disulfiram, suggesting that the toxic metabolite was the reactive aldehyde acrolein. The pattern of hepatocellular metabolism of allyl alcohol was monitored by high-pressure liquid chromatography (HPLC) analysis. The results suggest that acrolein, which is formed by the activity of alcohol dehydrogenase, preferentially reacts with cellular GSH to form an aldehyde-GSH adduct which subsequently is metabolized to the corresponding acid. In addition, a thiohemiacetal may be produced and subsequently degraded. In cells depleted of GSH, acrolein may react with essential macromolecules and thereby lead to structural and functional derangement and, eventually, irreversible injury.

1-Propanol↗
PubMed1985

Interaction of menadione (2-methyl-1,4-naphthoquinone) with glutathione.

The interaction of menadione with reduced glutathione (GSH) led to a removal of menadione and formation of menadione-GSH conjugate and glutathione disulfide (GSSG). The changes in thiol level were essentially biphasic with an initial rapid decrease in GSH and appearance of GSSG (less than 1 min) followed by secondary less pronounced changes. The interaction of menadione and GSH caused an oxygen uptake and both superoxide anion radical and hydrogen peroxide were produced during the reaction, the amount dependent on the GSH/menadione ratio. Catalase did not protect against the initial decrease in GSH level but markedly inhibited the secondary changes while superoxide dismutase had little effect. These results suggest that the initial changes in thiol level are the result in part of a redox reaction between menadione and GSH as well as conjugate formation, whilst the secondary changes reflect conjugate formation and the activity of other oxidants such as hydrogen peroxide. The potential biological significance of this reaction was investigated using hepatocytes depleted of reduced pyridine nucleotides and thus not able to perform enzyme-catalyzed reduction of menadione. In these cells menadione induced GSSG formation at a rate similar to that observed in control cells. This suggests that quinone-induced oxidative challenge caused by the chemical interactions of a quinone and glutathione may have biological relevance.

Animals↗
PubMed1985

Two cases of "pure" or "primary" erotomania successfully treated with pimozide.

Erotomania is a mono-delusional disorder which, in its "pure" or "primary" form, should probably belong to the diagnostic category of Paranoia. Traditionally, the prognosis and response to treatment are very poor, but one of the present authors has previously proposed that the condition should respond to pimozide. Case histories of two patients successfully treated with this drug are presented.

Adult↗
PubMed1985

Generation of reactive species and fate of thiols during peroxidase-catalyzed metabolic activation of aromatic amines and phenols.

The horseradish peroxidase (HRP)-catalyzed oxidation of p-phenetidine and acetaminophen was investigated. Studies using the spin probe 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) suggested these oxidations involve the generation of substrate-derived free radicals. This was confirmed by using glutathione (GSH) in these incubations in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). DMPO-glutathionyl radical adducts were observed using EPR spectroscopy during HRP-catalyzed oxidation of both p-phenetidine and acetaminophen. Investigations of oxygen uptake and oxidized glutathione (GSSG) formation during HRP-catalyzed oxidations of p-phenetidine and acetaminophen suggested that further reactions of the glutathionyl radical involve glutathione peroxysulfenyl radical and glutathione sulfenyl hydroperoxide production. Quinonoid products of the peroxidatic oxidations of p-phenetidine and acetaminophen, and their interaction with GSH via both conjugation and redox mechanisms are described. The relevance of these reactions of GSH with reactive species as detoxification mechanisms is discussed.

Biotransformation↗
PubMed1985

Prostaglandin synthase catalyzed metabolic activation of p-phenetidine and acetaminophen by microsomes isolated from rabbit and human kidney.

The metabolism of p-phenetidine in microsomes from rabbit kidney and the metabolism of acetaminophen and p-phenetidine in human kidney microsomes to protein binding metabolites were examined. Microsomal preparations from rabbit kidney medulla catalyzed the irreversible arachidonic acid-dependent binding of p-[14C]phenetidine to tissue protein. This was not observed in similar preparations from kidney cortex or if the microsomal protein was denatured. The Km (60 microM) of the binding reaction indicated that the enzymatic processes responsible for the binding have very high affinity for p-phenetidine. Indomethacin inhibited the binding to medullary microsomal protein whereas the inclusion of catalase and superoxide dismutase did not affect protein binding. Linolenic acid hydroperoxide was very effective in supporting binding whereas tertiary butylhydroperoxide and H2O2 were less effective. The binding in the presence of hydroperoxides was not sensitive to indomethacin or metyrapone. The binding ratio of 14C-ring to 14C-ethyl labeled p-phenetidine using rabbit kidney medulla microsomal protein was 2:1 suggesting that the binding species may be p-phenetidine quinone-imine and quinone-diimine dimers which have been shown previously to be products of the peroxidatic oxidation of p-phenetidine. The inclusion of reduced glutathione in incubations containing p-[14C] phenetidine, rabbit kidney medulla microsomes and arachidonic acid resulted in a decrease in radioactivity bound to protein and an increase in radioactivity in the aqueous phase after extraction. Thin-layer chromatography of the aqueous phase revealed the presence of reduced glutathione conjugates of the previously identified reactive dimers of p-phenetidine.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaminophen↗
PubMed1985

Characterization and mechanism of formation of reactive products formed during peroxidase-catalyzed oxidation of p-phenetidine. Trapping of reactive species by reduced glutathione and butylated hydroxyanisole.

OFF products of horseradish peroxidase (EC 1.11.1.7)-catalyzed oxidation of p-phenetidine were isolated and reactive species were trapped with reduced glutathione (GSH) and butylated hydroxyanisole (BHA). When BHA was added to a reaction mixture after 5 min, subsequent TLC and mass spectrometric analysis revealed the formation of an adduct of BHA and 4-(ethoxyphenyl)-p-benzoquinone diimine (A). The diimine derivative (A) was unstable and its expected degradation products, 4-(ethoxyphenyl)-p-benzoquinone imine (B) and ammonia, were recovered from the reaction in stoichiometric amounts. Ethanol was an early product of the reaction presumably resulting from radical coupling reactions and its formation agreed with the combined production of A and B, suggesting that this was its sole route of formation. The addition of GSH to a reaction at various times and subsequent TLC and high performance liquid chromatographic analysis revealed the presence of at least seven conjugates. Two conjugates were identified by fast atom bombardment mass spectrometry, one as a mono-GSH conjugate of A and another as a mono-GSH conjugate of B. When purified [14C]B was mixed with [3H]GSH, three conjugates were isolated by high performance liquid chromatography, two of which were tentatively identified as di-GSH conjugates. The conjugates isolated existed in both oxidized and reduced forms which could be easily interconverted by redox processes. The production of such reactive species and their conjugates in vivo may be a useful indicator of peroxidase-catalyzed metabolism.

Aminophenols↗
PubMed1984

Thiyl radicals--formation during peroxidase-catalyzed metabolism of acetaminophen in the presence of thiols.

We confirm using EPR spectroscopy in conjunction with the spin probe 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) that horseradish peroxidase catalyzed metabolism of the analgesic acetaminophen occurs via a one electron mechanism. When either glutathione cysteine or N-acetylcysteine were included in the reaction the thiols reduced the acetaminophen-derived radicals to generate thiyl radicals which were trapped with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and observed using EPR spectroscopy. Similarly, DMPO-thiyl radical adducts were observed during prostaglandin synthase catalyzed oxidation of acetaminophen in the presence of either glutathione or N-acetylcysteine. This is a mechanism of removal of reactive xenobiotic free radicals generated in metabolic systems but whether it represents a true detoxification reaction depends on the subsequent fate of the thiyl radicals generated.

Acetaminophen↗
PubMed1984

Studies of the mode of action of antitumour triazenes and triazines-V. The correlation of the in vitro cytotoxicity and in vivo antitumour activity of hexamethylmelamine analogues with their metabolism.

Experiments were conducted to ascertain whether the antitumour activity of hexamethylmelamine analogues correlated with their in vitro cytotoxicity and metabolism. Two analogues, namely pentamethylmelamine (PMM) and 2,2,4,4-tetramethylmelamine (TMM), and hexamethylmelamine (HMM) itself were shown to be active towards the murine ADJ/PC6A (PC6) plasmacytoma; another three, 2-chloro-4,6-bis(dimethylamino)-1,3,5-triazine (CBDT), 2,4-bis-(dimethylamino)-6-hydrazino-1, 3,5-triazine (HBDT) and 2,4,6-trimethylmelamine (TriMM) were inactive against the same tumour. The cytotoxicity of these compounds was examined against a PC6 tumour cell line in vitro. In the absence of liver microsomal activation only CBDT proved to be significantly cytotoxic at a concentration of 5 mM. In the presence of murine liver microsomes the three active antitumour agents were all cytotoxic at this concentration whereas HBDT and TriMM remained non-toxic. The degree of cytotoxicity correlated with the extent of metabolism for these analogues. The products of biotransformation of these compounds were stable precursors of formaldehyde (presumably N-hydroxymethyl intermediates) (FP) rather than formaldehyde itself. After injection of these 6 compounds to Balb/c mice the levels of FP generated in the plasma were markedly greater for the three active antitumour agents than for the inactive analogs. No free formaldehyde was detected in the plasma after administration of any of the compounds. These results suggest that for these compounds in vitro cytotoxicity correlates with in vitro biotransformation and their antitumour activity correlates with plasma levels of FP generated by metabolism in vivo.

Altretamine↗
PubMed1984

Effects of tumor growth on interleukins and circulating immune complexes. Mechanisms of immune unresponsiveness.

This study delineates the temporal relationship between immune complex formation and tumor growth, and provides one possible explanation for host immunosuppression during tumor growth. The authors have studied serial circulating immune complex (CIC) levels and interleukin (IL) elaboration by peripheral blood cells (IL-1 production by adherent mononuclear cells [AMC]; and IL-2 generation by peripheral blood mononuclear cells [PBMC]) during the growth of syngeneic tumor isografts in an inbred rat model. Male Wistar/Furth (W/Fu) rats were injected, subcutaneously (SC) with 2 X 10(6) W163 ( a dimethylhydrazine [DMH]-induced colon adenocarcinoma) cells into their hind limbs. Serial CIC levels, (measured by the antigen nonspecific polyethylene glycol turbidity assay) and IL-1 and IL-2 production were measured before isografting and weekly thereafter. Progressive local tumor growth occurred for 3 weeks followed by regional lymph node metastases during the fourth week. During local tumor growth, there was a progressive rise in CIC levels (123% rise compared with baseline value; P less than 0.05) which correlated with a fall in both IL-1 and IL-2 generation (r = -0.768). At the time of regional metastasis, the mean CIC levels declined, and there was a further significant decrease in IL production (IL-1 = 0.9% and IL-2 = 10% of controls in tumor bearers). These results show that progressive tumor growth results in decreased IL production by host PBC, and suggest that CIC may be involved in regulating IL generation.

Adenocarcinoma↗