Gondi S. Rao

Release of iron from ferritin by metabolites of benzene and superoxide radical generating agents

The release of iron from ferritin in the presence of benzene metabolites, viz. phenol (P), catechol (CT), hydroquinone (HQ) and superoxide radical generating compounds, viz. pyrogallol (PL), phloroglucinol (PG), phenylhydrazine (PH) or phenylenediamine (PD) was studied in acetate buffer, pH 5.6. Monitoring the formation of the iron-ferrozine complex quantitated the release of iron from ferritin. The presence of P (125 microM) did not result in the release of iron from ferritin, whereas the same concentration of CT, HQ, PL, PH or PD resulted in the release of significant amounts of iron from ferritin and a marginal amount of iron in the presence of PG, CT, HQ, PL, PH or PD concentration and time-dependent increase in iron release from ferritin were observed although the increase was not linear as a function of time and concentration of the compounds studied. The presence of superoxide dismutase inhibited significantly the release of iron from ferritin by CT, HQ, PL, PH or PD. The iron released from ferritin by CT, HQ, PL, PH or PD enhanced lipid peroxidation in rat brain homogenate and released aldehydic products from bleomycin-dependent degradation of DNA and also caused single strand nicks to pUC18 DNA. These studies indicate that CT and HQ, the two principal polyphenolic metabolites of benzene and PL, PH or PD, the superoxide radical generating compounds were capable of reducing ferric iron from ferritin and also mobilizing and releasing iron from ferritin core. The release of iron from ferritin by these compounds is a result of direct reduction of ferritin iron by electron transfer and also reduction via superoxide radical. The release of iron from ferritin by CT and HQ may have toxicological implications in relation to benzene toxicity. The release of iron by superoxide radical generating agents suggests that oxidative stress may play a role as this could lead to disruption of intracellular iron homeostasis.

Prooxidant and antioxidant properties of iron-hydroquinone and iron-1,2,4-benzenetriol complex. Implications for benzene toxicity

Bleomycin-dependent degradation of DNA in bone marrow cells was studied in vitro in the presence of iron or iron polyphenol chelates which are formed during biotransformation of benzene. Iron polyphenol chelates markedly enhanced bleomycin-dependent DNA degradation in comparison to iron alone. About 1.5 and 2.5-fold increase was observed in the presence of iron hydroquinone (HQ) chelate and iron 1,2,4-benzenetriol (BT) chelate, respectively. Endogenous iron chelators such as glutamate, citrate, aspartate, glycine, cysteine, dithiothreitol, AMP, ADP and ATP did not enhance iron-catalysed bleomycin-dependent degradation of DNA. By bleomycin assay, the recovery of iron polyphenol chelate added externally to bone marrow lysate was complete. However, the presence of iron polyphenol chelate resulted in less thiobarbituric acid reactive products from glutamate or brain homogenate than with iron alone. The optical spectra of BT were modified in the presence of ferrous sulphate, revealing a new absorption peak at 259 nm indicating complexation with iron. Thus, the iron polyphenol chelate, on one hand, is a more potent DNA cleaving agent in the presence of bleomycin, and on the other hand, it is a less effective free radical generator as compared to iron alone.

Glutathionyl hydroquinone: a potent pro-oxidant and a possible toxic metabolite of benzene

Iron catalysed bleomycin (an antitumor antibiotic)-dependent degradation of DNA was investigated in the presence of glutathionyl hydroquinone (GHQ). DNA degradation was enhanced twelve-fold in the presence of iron and GHQ and three-fold in the presence of iron and glutathione (GSH) as compared to iron alone. The degradation of DNA was linear with the increase in concentration of GHQ or GSH keeping the iron, bleomycin and other factors constant. The presence of oxyradical scavengers, viz., thiourea, mannitol, albumin, superoxide dismutase, catalase and dimethyl sulfoxide caused significant inhibition of degradation of DNA by GHQ and iron. All the externally added GHQ to bone marrow cell lysate was completely demonstrable by the assay of iron-catalyzed bleomycin-dependent degradation of DNA. Superoxide radical generation was demonstrable during the incubation of GHQ. Thus, the present study revealed that GHQ is a potent pro-oxidant and this observation is significant in understanding the mechanism of benzene toxicity with the possibility of GHQ as one of the toxic metabolites of benzene.

Subcutaneous kerosene toxicity in albino rats

Biochemical, histopathological, and hematological parameters were studied in male Wistar rats after repeated subcutaneous administration of commercial kerosene (0.5 ml/kg body wt, 6 days a week) for a period of 35 days. At necropsy, treatment-related increases in the weights of liver, spleen, and peripheral lymph nodes were noted. Correspondingly, there was an increase in DNA, RNA, protein, and lipid contents of liver and spleen. Histopathological examination of liver, spleen, thymus, kidney, adrenal, and lymph nodes revealed treatment-related lesions. Similarly, biochemical indices studied in liver revealed an increase in alkaline phosphatase and a decrease in benzo[a]pyrene hydroxylase levels. Furthermore serum cholinesterase, carboxylesterase, and albumin levels were significantly diminished while serum alkaline phosphatase levels were found to be greatly enhanced. The findings might be related as the likely systemic effects in workers upon percutaneous kerosene exposure during work.

Release of iron from ferritin by 1,2,4-benzenetriol

Release of iron from ferritin in the presence of polyhydroxy metabolites of benzene i.e., hydroquinone (HQ) or 1,2,4-benzenetriol (BT) was studied in acetate buffer, pH 5.6. The release of iron from ferritin was quantitated by monitoring the formation of iron-ferrozine complex. The presence of hydroquinone (330 microM) did not result in the release of iron from ferritin, whereas the same concentration of BT resulted in the release of significant amounts of iron (3.2 microM/min) from ferritin. BT concentration-dependent increase in iron release from ferritin was observed although the increase was not linear with the concentration of BT. Under a N2 atmosphere the presence of BT resulted in the release of iron (2.1 microM/min) from ferritin. The presence of oxyradical scavengers i.e., albumin, catalase or superoxide dismutase significantly inhibited iron release from ferritin by BT. The iron released from ferritin by BT enhanced lipid peroxidation in rat brain homogenate and released aldehydic products from bleomycin-dependent degradation of DNA. Addition of BT to bone marrow lysate resulted in an increase of iron release as a function of time. These studies indicate that BT is a potent reductant of ferric iron of ferritin and also mobilizes and releases iron from ferritin core. The release of iron from bone marrow lysate by BT may be of toxicological significance as this could lead to disruption of intracellular iron homeostasis in bone marrow cells.

Toxicity of petroleum products: effects on alkaline phosphatase and lipid peroxidation.

Abstract Liver lipid peroxidation was increased in the rat after intraperitoneal administration of benzene, Iomex, petroleum ether, or gasoline. Increases in lipid peroxidation and alkaline phosphatase activity were observed, while only a slight decrease in glucose- 6-phosphatase activity was noted. The effects of a single dose of benzene on alkaline phosphatase and lipid peroxidation in rat liver and kidney lasted up to 20 days.

Release of 2-thiobarbituric acid reactive products from glutamate or deoxyribonucleic acid by 1,2,4-benzenetriol or hydroquinone in the presence of copper ions

Cytotoxic effects of various quinone compounds are thought to be due to the formation of semiquinone free radicals. Hydroquinone and 1,2,4-benzenetriol in the presence of copper ions release from glutamate or DNA aldehydic products capable of reacting with 2-thiobarbituric acid (TBA). The formation of TBA reactive products (TBAR) was greater in the presence of 1,2,4-benzenetriol in comparison with hydroquinone. Complete inhibition of formation of TBAR from glutamate by 1,2,4-benzenetriol and copper was observed in the presence of catalase, thiourea and mannitol. Albumin and superoxide dismutase offered substantial protection. Complete protection of formation of TBAR from DNA was observed in the presence of catalase and thiourea. Presence of albumin, mannitol and superoxide dismutase caused only partial inhibition. The formation of TBAR from glutamate or DNA is dependent on copper ion concentration. The present data indicate that hydroquinone and 1,2,4-benzenetriol in the presence of copper ions can lead to the formation of reactive hydroxyl radicals which can release TBAR from glutamate or DNA.

Bioreactivity of glutathionyl hydroquinone with implications to benzene toxicity.

Glutathionyl hydroquinone (GHQ), a highly reactive metabolite of benzene, has been implicated as a causative intermediate of benzene toxicity. To substantiate, the bioreactivity of GHQ was investigated under in vitro and in vivo conditions using end points, characteristic of benzene toxicity. Under in vitro conditions, the presence of GHQ: (a) linearly increased the release of aldehydic products from L-glutamate or deoxyuridine at GHQ concentrations of 5-25 microM and from rat liver homogenates at GHQ concentrations of 50-250 microM; (b) cleaved plasmid pUC 18 supercoiled DNA through a single strand nick to yield open circular relaxed DNA, and through a double strand cut to give out linear DNA at GHQ concentrations of 25-200 microM, with evidence of protection by catalase and superoxide dismutase; and (c) induced cross-linking and polymerization of lymphocyte nuclear DNA through in situ generation of GHQ, which was protected by pretreatment of lymphocytes with N-ethylmaleimide. In vivo exposure of Swiss albino mice to GHQ (100 mg/kg, intraperitoneally once daily for 30 days) resulted in significant increase of liver weight and inhibition of mitotic index in the bone marrow. The other test parameters, namely spleen weight, hematological indices, hepatic sulphahydryl content and nonenzymatic lipid peroxidation, and chromosomal aberrations in the bone marrow were, however, unaffected by GHQ treatment. The observations indicate pro-oxidant and cytotoxic potential of GHQ, mediated by the reactive oxygen species generated during the course of its auto-oxidation. Bioreactivity of GHQ with cellular macromolecules in vitro and inhibition of mitotic index of bone marrow on in vivo exposure have relevance to benzene toxicity, although in situ generation of GHQ at the site of action appears critical in bringing about hematological and chromosomal effects that were probably spared due to rapid metabolic disposition and, consequently, poor bioavailability of intraperitoneally administered GHQ.

Hematin catalysed autooxidation of hydroquinone or 1,2,4-benzenetriol

Autooxidation of hydroquinone (HQ) or 1,2,4-benzenetriol (BT), catalysed by hemin in the presence of dithiothreitol was studied in phosphate buffered saline. Inclusion of glutamate in the above reaction mixture resulted in the formation of thiobarbituric acid reactive products (TBAR) only in an aerobic atmosphere and was linear up to 2 h. Oxygen consumption was noticed during the reaction process. The formation of TBAR was linear with the increase in concentration of heme (1-4 microM), dithiothreitol (0.2-2 mM) or BT (0.17-0.85 mM). Linearity of TBAR formation from glutamate for up to 2 h was observed during the autooxidation of BT in the presence of heme. Besides glutamate, heme concentration dependent formation of TBAR from deoxyuridine or DNA was also observed. Almost complete inhibition of TBAR formation from glutamate, deoxyuridine or DNA was observed in the presence of catalase or superoxide dismutase (SOD). The presence of thiourea or mannitol in the reaction mixture caused substantial diminution of TBAR formation. Albumin or dimethyl sulfoxide also caused partial inhibition. Complete to partial inhibition observed in the presence of oxyradical scavengers in this study indicates that hemin catalysed autooxidation of BT results in the formation of reactive oxygen radicals.