Metabolism of melphalan by rat liver microsomal glutathione S-transferase

One of the major problems in the treatment of human cancer is the phenomenon of drug resistance. Increased glutathione (gamma-glutamylcysteinylglycine, GSH) conjugation (inactivation) due to elevated level of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) remains uncertain. In our experiments, a combination of liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between GSH and melphalan, one of the alkylating agents. The spontaneous reaction of 1mM melphalan with 5mM GSH at 37 degrees C in aqueous phosphate buffer for 1h gave primarily the monoglutathionyl and diglutathionyl melphalan derivatives, with small amounts of mono- and dihydroxy melphalan derivatives. We demonstrated that rat liver microsomal GST presented a strong catalytic effect on the reaction as determined by the increase of monoglutathionyl and diglutathionyl melphalan derivatives and the decrease of melphalan. We showed that microsomal GST was activated by melphalan in a concentration- and time-dependent manner. Microsomal GST which was stimulated approximately 1.5-fold with melphalan had a stronger catalytic effect. Thus microsomal GST may play a potential role in the metabolism of melphalan in biological membranes, and in the development of ADR.     

Metabolism of chlorambucil by rat liver microsomal glutathione S-transferase

Clinical efficacy of alkylating anticancer drugs, such as chlorambucil (4-[p-[bis [2-chloroethyl] amino] phenyl]-butanoic acid; CHB), is often limited by the emergence of drug resistant tumor cells. Increased glutathione (gamma-glutamylcysteinylglycine; GSH) conjugation (inactivation) of alkylating anticancer drugs due to overexpression of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance to alkylating agents. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) to CHB remains uncertain. In our experiments, a combination of lipid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between CHB and GSH. The spontaneous reaction of 1mM CHB with 5 mM GSH at 37 degrees C in aqueous phosphate buffer for 1 h gave primarily the monoglutathionyl derivative, 4-[p-[N-2-chloroethyl, N-2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG) and the diglutathionyl derivative, 4-[p-[2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG2) with small amounts of the hydroxy-derivative, 4-[p-[N-2-S-glutathionylethyl, N-2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBSGOH), 4-[p-[bis[2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBOH2), 4-[p-[N-2-chloroethyl, N-2-S-hydroxyethyl]amino]phenyl]-butanoic acid (CHBOH). We demonstrated that rat liver microsomal GST presented a strong catalytic effect on these reactions as determined by the increase of CHBSG2, CHBSGOH and CHBSG and the decrease of CHB. We showed that microsomal GST was activated by CHB in a concentration and time dependent manner. Microsomal GST which was stimulated approximately two-fold with CHB had a stronger catalytic effect. Thus, microsomal GST may play a potential role in the metabolism of CHB in biological membranes, and in the development of ADR.     

Reduced glutathione protection against rat liver microsomal injury by carbon tetrachloride. Dependence on O2.

Rat liver microsomal membranes contain a reduced-glutathione-dependent protein(s) that inhibits lipid peroxidation in the ascorbate/iron microsomal lipid peroxidation system. It appears to exert its protective effect by scavenging free radicals. The present work was carried out to assess the effect of this reduced-glutathione-dependent mechanism on carbon tetrachloride-induced microsomal injury and on carbon tetrachloride metabolism because they are known to involve free radicals. Rat liver microsomes were incubated at 37 degrees C with NADPH, EDTA and carbon tetrachloride. The addition of 1 mM-reduced glutathione (GSH) markedly inhibited lipid peroxidation and glucose 6-phosphatase inactivation and, to a lesser extent, inhibited cytochrome P-450 destruction. GSH also inhibited covalent binding of [14C]carbon tetrachloride-derived 14C to microsomal protein. These results indicate that a GSH-dependent mechanism functions to protect the microsomal membrane against free-radical injury in the carbon tetrachloride system as well as in the iron-based systems. Under anaerobic conditions, GSH had no effect on chloroform formation, carbon tetrachloride-induced destruction of cytochrome P-450 or covalent binding of [14C]carbon tetrachloride-derived 14C to microsomal protein. Thus, the GSH protective mechanism appears to be O2-dependent. This suggests that it may be specific for O2-based free radicals. This O2-dependent GSH protective mechanism may partly underlie the observed protection of hyperbaric O2 against carbon tetrachloride-induced lipid peroxidation and hepatotoxicity.     

Effect of fraction V powder from bovine serum on rat liver microsomal glutathione S-transferase activity

When bovine serum fraction V powder, which contains 98-99% albumin, is incubated with rat liver microsomes, the glutathione S-transferase activity is stimulated 3-fold. The fraction V stimulates the transferase activity even in microsomes pretreated with N-ethylmaleimide. There is no such stimulating activity in protein fractions other than fraction V. The corresponding cytosolic enzyme activity is not changed by fraction V powder or by N-ethylmaleimide. Neither treatment with charcoal nor heating in boiling water for 5 min can remove the stimulating activity from fraction V.     

Activation of rat liver microsomal glutathione S-transferase by gallic acid

The effect of phenolic antioxidants on the rat liver microsomal glutathione S-transferase (MGST1) was investigated in vitro. When microsomes were incubated with various polyphenolic antioxidants, gallic acid (3,4,5-trihydroxybenzoic acid) markedly increased MGST1 activity and the increase was prevented in the presence of superoxide dismutase (SOD) or catalase. The MGST1 activity increased by gallic acid was decreased by further incubation with sodium arsenite, a sulfenic acid reducing agent, but was not with dithiothreitol, a disulfide bond reducing agent. The incubation of microsomes with gallic acid in the presence of the NADPH generating system which generates reactive oxygen species (ROS) through cytochrome P-450 system increased the MGST1activity in spite of scavenging the ROS and the increase was also depressed by SOD/catalase. The increase of MGST1 activity by gallic acid was prevented by co-incubation with a stable radical, 1,1-diphenyl-2-picrylhydrazyl or ferric chloride. These results suggest that the gallic acid acts as a pro-oxidant and activates MGST1 through oxidative modification of the enzyme.     

Binding of inositol trisphosphate by a liver microsomal fraction.

To test the hypothesis that inositol trisphosphate (InsP3) mediates adaptation and excitation in invertebrate photoreceptors, we measured its formation on a rapid time scale in squid retinas. For squid, excitation and adaption occurs within 0.1 and 1-2 s respectively. We could detect an elevation in InsP3 within 200 ms of a bright flash. This increase is about 240% over dark basal levels and is maintained for at least 2 min after a flash. The increase probably occurs in the photoreceptors, which are the only neurons in squid retinas. Analysis by h.p.l.c. indicates that the light-regulated isomer is Ins(1,4,5)P3, which is formed by the hydrolysis of phosphatidylinositol bisphosphate (PtdInsP2).     

Preferential transport of glutathione versus glutathione disulfide in rat liver microsomal vesicles

A bi-directional, saturable transport of glutathione (GSH) was found in rat liver microsomal vesicles. GSH transport could be inhibited by the anion transport blockers flufenamic acid and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid. A part of GSH taken up by the vesicles was metabolized to glutathione disulfide (GSSG) in the lumen. Microsomal membrane was virtually nonpermeable toward GSSG; accordingly, GSSG generated in the microsomal lumen could hardly exit. Therefore, GSH transport, contrary to previous assumptions, is preferred in the endoplasmic reticulum, and GSSG entrapped and accumulated in the lumen creates the oxidized state of its redox buffer.     

Activation of rat liver microsomal glutathione transferase by limited proteolysis.

The activity of rat liver microsomal glutathione transferase is increased by limited tryptic proteolysis; the membrane-bound and purified forms of the enzyme are activated about 5- and 10-fold respectively. The cleavage sites that correlate with this activation were determined by amino acid sequence analysis to be located after Lys-4 and Lys-41. Differences in the relative extent of cleavage at these two sites did not consistently affect the degree of activation. Thus the data support the conclusion that cleavage at either site results in activation. The trypsin-activated enzyme was compared with the form activated with N-ethylmaleimide, which modifies Cys-49. These two differently activated forms were found to have similar kinetic parameters, which differ from those of the unactivated enzyme. The relatedness of the two types of activation is also demonstrated by the observation that microsomal glutathione transferase fully activated by N-ethylmaleimide is virtually resistant to further activation by trypsin. This is the case despite the fact that the N-ethylmaleimide-activated enzyme is much more susceptible to trypsin cleavage at Lys-41 than is the untreated enzyme. The latter observation indicates that activation with N-ethylmaleimide is accompanied by a conformational change involving Lys-41.     

Activation of rat liver microsomal glutathione S-transferase by reduced oxygen species

The effect of enzymatically generated reduced oxygen metabolites on the activity of hepatic microsomal glutathione S-transferase activity was studied to explore possible physiological regulatory mechanisms of the enzyme. Noradrenaline and the microsomal cytochrome P-450-dependent monooxygenase system were used to generate reduced oxygen species. When noradrenaline (greater than 0.1 mM) was incubated with rat liver microsomes in phosphate buffer (pH 7.4), an increase in microsomal glutathione S-transferase activity was observed, and this activation was potentiated in the presence of a NADPH-generating system; the glutathione S-transferase activity was increased to 180% of the control with 1 mM noradrenaline and to 400% with both noradrenaline and NADPH. Superoxide dismutase and catalase inhibited partially the noradrenaline-dependent activation of the enzyme. In the presence of dithiothreitol and glutathione, the activation of the glutathione S-transferase by noradrenaline, with or without NADPH, was not observed. In addition, the activation of glutathione S-transferase activity by noradrenaline and glutathione disulfide was not additive when both compounds were incubated together. These results indicate that the microsomal glutathione S-transferase is activated by reduced oxygen species, such as superoxide anion and hydrogen peroxide. Thus, metabolic processes that generate high concentrations of reduced oxygen species may activate the microsomal glutathione S-transferase, presumably by the oxidation of the sulfhydryl group of the enzyme, and this increased catalytic activity may help protect cells from oxidant-induced damage.     

Increase in glutathione disulfide level regulates the activity of microsomal glutathione S-transferase in rat liver

Glutathione disulfide stimulates the activity of rat liver microsomal glutathione S-transferase 2-fold after incubation at 25 degrees C for 10 min. When the microsomes were incubated with the disulfide for over 20 min, the transferase activity increased to the same extent as in the case of N-ethylmaleimide (6-fold). Even in the presence of reduced glutathione, some enhancement of the transferase activity was observed. The data presented here are evidence that increase in glutathione disulfide level, e.g. by lipid peroxidation, on endoplasmic reticulum causes the upregulation of microsomal glutathione S-transferase activity.     

In vivo study on alkylation site in DNA by the bifunctional dianhydrogalactitol

In vivo alkylation of Yoshida sarcoma cell DNA by ³H-labelled 1,2:5,6-dianhydrogalactitol (DAG) yielded N-7 monogalactitylguanines and 1,6-di-(guanin-7-yl)-galactitol, similar to the alkylated products obtained by in vitro reaction of DNA with dianhydrogalactitol in neutral solution. The ratio between monoalkylguanines and diguaninyl product was 2–2.5, slightly increasing with doses. Persistence of alkylated products in DNA was followed in function of time. There was no significant loss of either monoalkylated bases or diguaninyl derivative during the observation period i.e. 7–24 h after treatment. In contrast, the physical measurements of the amount of renaturable DNA showed a rapid opening of cross-links in the same period. Taking the presence of diguaninyl moiety as an indicator of cross-links in DNA, these two latter findings show an apparent contradiction which could be reconciled however by the mechanism proposed by Reid and Walker (Biochim. Biophys. Acta, 179 (1969) 179) for the removal of cross-linkage induced by HN₂. Accordingly, one arm of the cross-links is removed, probably enzymically, leaving the DNA non-renaturable, while the other arm of cross-link is still covalently attached to the DNA molecule rendering possible the detection of diguaninyl moiety in DNA at some later time. This concept for the removal of cross-links from DNA seems to be supported by our results too.     

In vitro DNA and nuclear proteins alkylation by 1,2-dimethylhydrazine

The methylating carcinogen 1,2-dimethylhydrazine (DMH) CAS 540.73.8 is highly organ-specific and, under certain experimental conditions, produces a high incidence of adenocarcinoma in the colon of rodents. We have tried to assess the possibility that part of the organ-specifity in the carcinogenic effect of DMH could be attributed to its metabolism by specific microsomal enzymes. In particular, we compared the in vitro effects of DMH in the presence of either colon or liver microsomes from animals that had been treated with microsomal enzyme inducers. V79 Chinese hamster cells were used as the target to evaluate the damage to the genetic material, as judged by (1) formation of adducts of DNA bases and (2) amino acid modifications in nuclear proteins using [Me-14C]DMH and appropriate analytical detection systems. Our results tend to support the above postulated hypothesis.     

Asiatic acid inhibits liver fibrosis by blocking TGF-beta/Smad signaling in vivo and in vitro

Liver fibrosis is a major cause of liver failure, but treatment remains ineffective. In the present study, we investigated the mechanisms and anti-hepatofibrotic activities of asiatic acid (AA) in a rat model of liver fibrosis induced by carbon tetrachloride (CCl(4)) and in vitro in TGF-beta1-stimulated rat hepatic stellate cell line (HSC-T6). Treatment with AA significantly attenuated CCl(4)-induced liver fibrosis and functional impairment in a dosage-dependent manner, including blockade of the activation of HSC as determined by inhibiting de novo alpha smooth muscle actin (a-SMA) and collagen matrix expression, and an increase in ALT and AST (all p<0.01). The hepatoprotective effects of AA on fibrosis were associated with upregulation of hepatic Smad7, an inhibitor of TGF-beta signaling, thereby blocking upregulation of TGF-beta1 and CTGF and the activation of TGF-beta/Smad signaling. The anti-fibrosis activity and mechanisms of AA were further detected in vitro in HSC-T6. Addition of AA significantly induced Smad7 expression by HSC-T6 cells, thereby inhibiting TGF-beta1-induced Smad2/3 activation, myofibroblast transformation, and collagen matrix expression in a dosage-dependent manner. In contrast, knockdown of Smad7 in HSC-T6 cells prevented AA-induced inhibition of HSC-T6 cell activation and fibrosis in response to TGF-beta1, revealing an essential role for Smad7 in AA-induced anti-fibrotic activities during liver fibrosis in vivo and in vitro. In conclusion, AA may be a novel therapeutic agent for liver fibrosis. Induction of Smad7-dependent inhibition of TGF-beta/Smad-mediated fibrogenesis may be a central mechanism by which AA protects liver from injury.     

Persistent and heritable structural damage induced in heterochromatic DNA from rat liver by N-nitrosodimethylamine

Analysis, by benzoylated DEAE-cellulose chromatography, has been made of structural change in eu- and heterochromatic DNA from rat liver following administration of the carcinogen N-nitrosodimethylamine (10 mg/kg body weight). Either hepatic DNA was prelabeled with [3H]thymidine administered 2-3 weeks before injection of the carcinogen or the labeled precursor was given during regenerative hyperplasia in rats treated earlier with N-nitrosodimethylamine. Following phenol extraction of either whole liver homogenate or nuclease-fractionated eu- and heterochromatin, carcinogen-modified DNA was examined by stepwise or caffeine gradient elution from benzoylated DEAE-cellulose. In whole DNA, nitrosamine-induced single-stranded character was maximal 4-24 h after treatment, declining rapidly thereafter; gradient elution of these DNA preparations also provided short-term evidence of structural change. Following incubation of purified nuclei with micrococcal nuclease, 10-12% of labeled DNA was solubilized (eu-chromatin) by 1.0 unit of micrococcal nuclease (5 mg of DNA)-1 mL-1 after 9 min. In prelabeled animals, administration of N-nitrosodimethylamine caused a marked fall in the specific radioactivity of solubilized DNA, while that of sedimenting DNA was not affected. Caffeine gradient chromatography suggested short-term nitrosamine-induced structural change in euchromatic DNA, while increased binding of heterochromatic DNA was evident for up to 3 months after carcinogen treatment. Preparations of newly synthesized heterochromatic DNA from animals subjected to hepatectomy up to 2 months after carcinogen treatment provided evidence of heritable structural damage. Carcinogen-induced binding of heterochromatic DNA to benzoylated DEAE-cellulose was indicative of specific structural lesions whose affinity equalled that of single-stranded DNA up to 1.0 kilobase in length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational specificity of N-nitrosodimethylamine: Comparison between in vivo and in vitro assays

We have determined the DNA alterations recovered after treatment with Aroclor 1254-treated rat S9-activated N-nitrosodimethylamine (NDMA) in the N-terminal region of the LacI gene of E. coli. A total of 125 independent Lacl^-d mutants of E. Coli were characterized by DNA sequencing. Consistent with the known methylating ability of this compound, the predominant mutation was the G:C → A:T transition, which accounted for 90% of all the mutations recovered. Non G:C → A:T events include 2 G:C → T:A 2 G:C → C:G, 2 A:T → G:C, 3 A:T → T:A and 3 frameshifts. Contingency analysis reveals that NDMA-induced mutation recovered after in vitro activation (S9) have a spectrum very similar to that previously obtained after in vivo activation employing a mouse host-mediated assay. In both systems, G:C → A:T events clearly dominate and their distribution reveals similar site-specificity Moreover, the proportion and kind of non-G:C → A::T events are also similar