Ginsenoside Rg3 protects mouse leydig cells against triptolide by downregulation of miR-26a.

BACKGROUND: Ginsenoside Rg3 has been reported to exert protection function on germ cells. However, the mechanisms by which Rg3 regulates apoptosis in mouse Leydig cells remain unclear. In addition, triptolide (TP) has been reported to induce infertility in male rats. Thus, this study aimed to investigate the protective effect of Rg3 against TP-induced toxicity in MLTC-1 cells. METHODS: CCK-8, immunofluorescence assay, Western blotting and flow cytometry were used to detect cell proliferation and cell apoptosis, respectively. In addition, the dual luciferase reporter system assay was used to detect the interaction between miR-26a and GSK3ß in MLTC-1 cells. RESULTS: TP significantly inhibited the proliferation of MLTC-1 cells, while the inhibitory effect of TP was reversed by Rg3. In addition, TP markedly induced apoptosis in MLTC-1 cells via increasing the expressions of Bax, active caspase 3, Cyto c and active caspase 9, and decreasing the level of Bcl-2. However, Rg3 alleviated TP-induced apoptosis of MLTC-1 cells. Moreover, the level of miR-26a was obviously downregulated by Rg3 treatment. The protective effect of Rg3 against TP-induced toxicity in MLTC-1 cells was abolished by miR-26a upregulation. Meanwhile, dual-luciferase assay showed GSK3ß was the direct target of miR-26a in MLTC-1 cells. Overexpression of miR-26a markedly decreased the level of GSK3ß. As expected, upregulation of miR-26a could abrogate the protective effects of Rg3 against TP-induced cytotoxicity via inhibiting the expression of GSK3ß. CONCLUSION: These results indicated that Rg3 could protect MLTC-1 against TP by downregulation of miR-26a. Therefore, Rg3 might serve as a potential agent for the treatment of male hypogonadism.

Triptolide inhibits migration and proliferation of fibroblasts from ileocolonic anastomosis of patients with Crohn's disease via regulating the miR­16­1/HSP70 pathway.

Anastomotic fibrosis is highly likely to lead to reoperation in Crohn's disease (CD) patients. Triptolide (TPL) is considered to have anti­inflammatory and antifibrotic effects in a variety of autoimmune diseases, including CD. The present study aimed to investigate the effects of TPL on fibroblasts from strictured ileocolonic anastomosis of patients with CD and its underlying mechanism. Primary fibroblasts were obtained from strictured anastomosis tissue (SAT) samples and matched anastomosis­adjacent normal tissue (NT) samples which were collected from 10 CD patients who underwent reoperation because of anastomotic stricture. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) was used to measure miR­16­1 and heat shock protein 70 (HSP70) levels. Western blotting was conducted to determine expression of HSP70, collagen I (Col­I), collagen III (Col­III) and α­smooth muscle actin (α­SMA) proteins. Agomir­16­1 and antagomir­16­1 were used to up and downregulate the expression of miR­16­1, respectively. Small interfering RNA (siRNA) was employed to inhibit the expression of HSP70. A wound healing assay was performed to measure the migration of fibroblasts. Cell proliferation was evaluated by MTT and 5­bromo­2­deoxyrudidine assays. Cell apoptosis was determined by caspase­3 activity and TUNEL assays. The results demonstrated that the levels of Col­I, Col­III and α­SMA were all significantly upregulated in SAT compared with NT. miR­16­1 levels in the SAT group were significantly compared with the NT group; conversely, the expression levels of HSP70 mRNA and protein in the SAT group were significantly lower compared with the NT group. Next, fibroblasts were treated with TPL to examine its effect on the miR­16­1/HSP70 pathway. The results demonstrated that the elevated expression of miR­16­1 in the SAT group was effectively inhibited by TPL treatment. Compared with the NT group, both the mRNA and protein levels of HSP70 were significantly downregulated in the SAT group cells, while TPL exhibited a strong promoting effect on HSP70 synthesis. Furthermore, upregulation of miR­16­1 reversed the effect of TPL on the miR­16­1/HSP70 pathway in fibroblasts from SAT. Overexpression of miR­16­1 significantly reversed the inhibitory effects of TPL treatment on migration, proliferation and extracellular matrix (ECM)­associated protein expression of fibroblasts from SAT. Finally, downregulation of miR­16­1 caused similar effects to the fibroblasts as the TPL treatment; however, the inhibitory effects on cell biological functions induced by antagomir­16­1 were all significantly reversed by HSP70 silencing. The present findings indicated that TPL may be a potential therapeutic option for postoperative anastomosis fibrosis of patients with CD. The miR­16­1/HSP70 pathway had a substantial role in the inhibitory effects of TPL on migration, proliferation and ECM synthesis rate of fibroblasts from strictured anastomosis tissues.

[Monoside antagonizes triptolide-induced hepatocyte apoptosis via the anti-oxidative stress pathway].

OBJECTIVE: To investigate the protective effect of monoside against triptolide-induced liver injury and explore its molecular mechanism. METHODS: BALB/C mice treated with gastric lavage with triptolide and monoside, either alone or in combination, were examined for changes of hepatic biochemical parameters using the serological method. The growth inhibition rate of HepG2 cells treated with triptolide or monoside or both was assessed with MTT assay, and the cell morphological changes were observed using laser confocal microscopy; the expressions of the target proteins in the antioxidative stress pathway were detected using flow cytometry and Western blotting. RESULTS: In BALB/C mice, gastric lavage of triptolide induced obvious hepatic damage. In HepG2 cells, treatment with triptolide significantly inhibited the cell growth, resulting in a cell viability as low as 72.83% at 24 h; triptolide also induced obvious cell apoptosis and cell nucleus deformation, causing an apoptosis rate of 43.1% in the cells at 24 h. Triptolide significantly reduced the expressions of Nrf2 and HO-1 proteins related with the oxidative stress pathway. Combined treatment with morroniside obviously reversed these changes, resulting in significantly decreased hepatic biochemical parameters and the liver index in BALB/C mice and in significantly lowered cell apoptosis rate, improved cell morphology, and increased Nrf2 and HO-1 protein expressions in HepG2 cells. CONCLUSIONS: Monoside protects against triptolide-induced liver injury possibly by relieving oxidative stress.

Triptolide induces p53-dependent cardiotoxicity through mitochondrial membrane permeabilization in cardiomyocytes.

Triptolide (TP), a major active component of Tripterygium wilfordii Hook f., is widely used in the treatment of inflammation and autoimmune disorders. Its clinical application is limited by severe adverse effects, especially cardiotoxicity. Accumulative evidences indicate that TP induces DNA damage by inhibiting RNA polymerase. Considering the relationship among DNA damage, p53, and the role of p53 in mitochondria-dependent apoptosis, we speculate that TP-induced cardiotoxicity results from p53 activation. In this study, the role of p53 in TP-induced cardiotoxicity was investigated in H9c2 cells, primary cardiomyocytes, and C57BL/6 genetic background p53-/- mice. p53 protein level was elevated by TP in vitro and in acute heart injury models. With TP administration (1.2 mg/kg), p53 deficiency prevented heart histology injury and decreased serum cardiac troponin I (cTn-I) and apoptotic proteins. Mechanistically, immunoblotting and immunofluorescence staining identified that TP-induced toxicity is dependent on p53 nuclear translocation and transactivation of Bcl2 family genes, leading to mitochondrial outer membrane permeabilization (MOMP) and mitochondria dysfunction. Consistently, p53 antagonist PFTα counteracted TP-induced p53 overexpression and regulation of Bcl2 family transcription, which improved mitochondrial membrane integrity and prevented apoptosis. Moreover, Bax antagonist Bax inhibitor peptide (BIP) V5 ameliorated TP-induced apoptosis through suppressing membrane depolarization and ROS accumulation. These results suggest that TP-induced cardiotoxicity is p53-dependent by promoting Bax-induced mitochondria-mediated apoptosis.

Resveratrol protects against triptolide-induced cardiotoxicity through SIRT3 signaling pathway in vivo and in vitro.

Clinical application of triptolide (TP), a main active ingredient of the traditional Chinese herb Tripterygium wilfordii Hook f. (TWHF), is limited by a series of severe toxicities, including cardiotoxicity. In previous studies, we found the activation of sirtuin 3 (SIRT3) attenuated TP-induced toxicity in cardiomyocytes. Resveratrol (RSV), a polyphenol from the skins of grapes and red wine, is an activator of SIRT3. The current study aimed to investigate the protective effect of RSV against TP-induced cardiotoxicity and the underlying mechanisms. Mice were treated with a single dose of TP (2.5 mg/kg) via the intragastric (i.g.) route. After 24 h, TP induced abnormal changes of serum biochemistry, activity decrease of antioxidant enzymes and damage of heart tissue such as myocardial fiber rupture, cell swelling and interstitial congestion. In contrast, administration with RSV (50 mg/kg i.g. 12 h before and 2 h after the administration of TP) attenuated the detrimental effects induced by TP in BALB/c mice. Moreover, the cardiomyocyte protective effects of RSV on TP-induced heart injury were associated with the activation of SIRT3 and its downstream targets. In vitro study also indicated that RSV counteracted TP-induced cardiotoxicity through SIRT3-FOXO3 signaling pathway in H9c2 cells. Collectively, these findings suggest the potential of RSV as a promising agent in protecting heart from TP-induced damage.

Isoliquiritigenin protects against triptolide-induced hepatotoxicity in mice through Nrf2 activation.

Isoliquiritigenin, a flavonoid found in licorice, has been considered as an antioxidive and hepato-protective agent. Recent studies have shown that a possible mechanism for triptolide-induced hepatotoxicity is related to oxidative damage induced by reactive oxygen species. This study was done to investigate the protection effect of isoliquiritigenin against triptolide-induced hepatotoxicity and the mechanism involved. An acute liver injury model was established by intraperitoneal injection of triptolide (1.0 mg · kg-1) in mice. Different doses of isoliquiritigenin (12.5, 25 and 50 mg · kg-1) were employed as protection. The activities of AST, ALT, ALP and LDH in serum and levels of GSH, GPx, SOD, CAT and MDA in liver tissue were detected. The histopathological changes of liver tissues were observed after HE staining. The protein expression of Nrf2 was detected by western blot. Pretreatment with isoliquiritigenin significantly prevented the triptolide-induced hepatotoxicity indicated by reduced activities of AST, ALT, ALP and LDH. Moreover, isoliquiritigenin pretreatment also prevented from triptolide-induced hepatotoxicity by inhibiting MDA and restoring the levels of GSH, GPx, SOD and CAT. In addition, isoliquiritigenin could attenuate histopathological changes induced by triptolide. Furthermore, the results indicated that isoliquiritigenin pretreatment caused an increase in the protein expression of Nrf2. These results indicated that isoliquiritigenin could protect against triptolide-induced hepatotoxicity via activation of the Nrf2 pathway.

Estrogenic effects of fusarielins in human breast cancer cell lines.

The fusarielins are a group of metabolites found in several Aspergillus and Fusarium species that have been reported to have with weak antifungal, antibiotic and cytotoxic effects. This study identifies fusarielin A, F, G and H isolated from Fusarium as mycoestrogens. Mycoestrogens are compounds from fungi that bind to the estrogen receptors and induce an estrogenic response in targeted cells. All four tested fusarielins stimulate MCF-7 cell proliferation with fusarielin H as the most potent, able to stimulate cell proliferation 4-fold in a resazurin metabolism assay at 25µM. MDA-MB-231 cells without the estrogen receptor-α and MCF-10a cells without estrogen receptors were not stimulated by fusarielins. Furthermore, the stimulation was prevented in MCF-7 cells when fusarielins were incubated in the presence of the estrogen receptor antagonist fulvestrant. These observations suggest that fusarielins bind to the estrogen receptor and act as weak mycoestrogens.

Protective effect of acetyl-l-carnitine and α-lipoic acid against the acute toxicity of diepoxybutane to human lymphocytes.

The biotransformation and oxidative stress may contribute to 1,2:3,4-diepoxybutane (DEB)-induced toxicity to human lymphocytes of Fanconi Anemia (FA) patients. Thus, the identification of putative inhibitors of bioactivation, as well as the determination of the protective role of oxidant defenses, on DEB-induced toxicity, can help to understand what is failing in FA cells. In the present work we studied the contribution of several biochemical pathways for DEB-induced acute toxicity in human lymphocyte suspensions, by using inhibitors of epoxide hydrolases, inhibitors of protective enzymes as glutathione S-transferase and catalase, the depletion of glutathione (GSH), and the inhibition of protein synthesis; and a variety of putative protective compounds, including antioxidants, and mitochondrial protective agents. The present study reports two novel findings: (i) it was clearly evidenced, for the first time, that the acute exposure of freshly isolated human lymphocytes to DEB results in severe GSH depletion and loss of ATP, followed by cell death; (ii) acetyl-l-carnitine elicits a significant protective effect on DEB induced toxicity, which was potentiated by α-lipoic acid. Collectively, these findings contribute to increase our knowledge of DEB-induce toxicity and will be very useful when applied in studies with lymphocytes from FA patients, in order to find out a protective agent against spontaneous and DEB-induced chromosome instability.

Developmental toxicity by exposure to bisphenol A diglycidyl ether during gestation and lactation period in Sprague-Dawley male rats.

OBJECTIVES: Bisphenol A diglycidyl ether (BADGE) is the major component in commercial liquid epoxy resins, which are manufactured by co-reacting bisphenol A with epichlorohydrin. This study was performed to show the developmental effects of prenatal and postnatal exposures to BADGE in male rat offspring. METHODS: Mated female rats were divided into four groups, each containing 12 rats. The dosing solutions were prepared by thoroughly mixing BADGE in corn oil at the 0, 375, 1500 and 3000 mg/kg/day concentrations. Mated females were dosed once daily by oral gavage on gestation day (GD) 6 - 20 and postnatal day (PND) 0 - 21. Pregnant female dams were observed general symptoms and body weight. Also, male pups were observed the general symptoms, body weight, developmental parameters (e.g. anogenital distance, pina detachment, incisor eruption, nipple retention, eye opening, testis descent), organ pathologic changes and hormone levels of plasma. RESULTS: Pregnant rats treated with BADGE died at a rate of about 70% in the 1500 mg/kg/day group and all rats treated with 3000 mg/kg/day died. Body weight, for male pups treated with doses of 375 mg/kg/day, was significantly lower than in the control group at PND 42, 56, and 63 (p<0.05). Evaluation of body characteristics including; separation of auricle, eruption of incisor, separation of eyelid, nipple retention, descent of testis, and separation of the prepuce in the BADGE treated group showed no difference in comparisons with the control group. AGD and adjusted AGD (mm/kg) for general developmental items in BADGE 375 mg/kg/day treated pups tended to be longer than in controls, however, these differences were not statistically significant. Relative weights of adrenal gland, lung (p<0.05), brain, epididymis, prostate, and testis (p<0.01) were heavier than in control in measures at PND 9 weeks. There were no significant changes in comparisons of histological findings of these organs. Loss of spermatids was observed in the seminiferous tubule at PND 9 weeks, but no weight changes were observed. The plasma estrogen levels were similar in the control and treatment groups at PND 3, 6 and 9 weeks. The plasma testosterone levels in the control group tended to increase with age. However, in the BADGE 375 mg/kg/day treated male pups it did not tend to increase. CONCLUSIONS: These findings suggest that BADGE is a chemical that has developmental effects consistent with it being an endocrine disruptor.

A potential role for triglyceride as an energy source during bovine oocyte maturation and early embryo development.

The potential role of endogenous triglyceride in bovine oocyte maturation and preimplantation development has been investigated. Bovine immature oocytes were recovered from abattoir-derived ovaries, matured and fertilised in vitro and the zygotes grown to the blastocyst stage in SOFaaBSA. Methyl palmoxirate (MP) blocks the oxidation of fatty acids by inhibiting mitochondrial carnitine palmitoyltransferase A. The development of zygotes exposed to MP during oocyte maturation, and of zygotes exposed to MP during embryo culture has been assessed in terms of oxygen consumption by oocytes and embryos during a 4-6 hr incubation period in the presence of MP and as blastocyst formation and cell number. Immature oocytes exposed to MP during maturation had reduced capacity to form blastocysts after fertilisation; the same effect was apparent, but to a lesser extent, in zygotes exposed to MP during embryo development. Oxygen consumption values of oocytes and blastocysts in the absence of exogenous substrates were similar to those in control medium containing nutrients. MP-inhibited oxygen consumption of immature oocytes, mature oocytes, cleavage stages embryos and blastocysts by 64, 45, 12 and 13%, respectively. The data are consistent with a role for triglyceride as a key energy source during bovine oocyte maturation and potentially, during preimplantation embryo development.

Glomerular cytochrome P-450 and cyclooxygenase metabolites regulate efferent arteriole resistance.

Bradykinin dilates efferent arterioles via release of efferent arteriole epoxyeicosatrienoic acids when perfused retrograde (no glomerular autacoids). However, when efferent arterioles are perfused orthograde through the glomerulus, bradykinin-induced dilatation is caused by a balance between: (1) the glomerular vasoconstrictor 20-hydroxyeicosatetraenoic acid and vasodilator prostaglandins, and (2) epoxyeicosatrienoic acids from the efferent arteriole and possibly the glomerulus. However, the role of 20-hydroxyeicosatetraenoic acid has only been studied with a cyclooxygenase inhibitor, which may artificially enhance its production by shunting arachidonic acid into the cytochrome P450 pathway. We hypothesized that in the absence of cyclooxygenase inhibition, bradykinin induces release of 20-hydroxyeicosatetraenoic acid from the glomerulus, which blunts the vasodilator effect of bradykinin; and that prostaglandins released from glomeruli in response to bradykinin are generated by cyclooxygenase-1. Rabbit efferent arterioles preconstricted with norepinephrine were perfused orthograde from the end of the afferent arteriole. Bradykinin was added to the perfusate with or without a 20-hydroxyeicosatetraenoic acid antagonist (20-HEDE), epoxyeicosatrienoic acid synthesis inhibitor (MS-PPOH), and/or cyclooxygenase-1 (SC-58560) or cyclooxygenase-2 inhibitor (NS-398). Bradykinin-dependent dilatation was enhanced by 20-HEDE but blunted by MS-PPOH. When the inhibitors were present, bradykinin-induced dilatation was abolished by blockade of cyclooxygenase-1 but not cyclooxygenase-2. We concluded that: (1) in the absence of cyclooxygenase inhibitors, bradykinin causes the release of a glomerular vasoconstrictor (20-hydroxyeicosatetraenoic acid) that antagonizes the vasodilator effect of epoxyeicosatrienoic acids released from the efferent arteriole and perhaps from the glomerulus, and (2) bradykinin-induced vasodilatation is caused by the release of epoxyeicosatrienoic acids from the efferent arteriole and glomerular metabolites of cyclooxygenase-1.

Mutations induced by (-)-anti-11R,12S-dihydrodiol 13S,14R-epoxide of dibenzo[a,l]pyrene in the coding region of the hypoxanthine phosphoribosyltransferase (Hprt) gene in Chinese hamster V79 cells.

The polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) is an exceptionally potent carcinogen. Its direct DNA-reactive metabolite, the fjord region (-)-anti-11R,12S-dihydrodiol 13S,14R-epoxide [(-)-anti-DB[a,l]PDE], was used to investigate induction of mutations in the coding region of the hypoxanthine phosphoribosyltransferase (Hprt) gene in Chinese hamster V79 cells. Cells exposed to 1-10 nM (-)-anti-DB[a,l]PDE exhibited a close dose-responsive increase in the frequency of mutant clones resistant to 6-thioguanine. RNA was isolated from mutant clones and cDNAs were prepared by reverse transcription. The coding region of the cDNA of the Hprt gene was amplified by polymerase chain reaction and sequenced. Analysis of the DNA base sequence changes induced by (-)-anti-DB[a,l]PDE indicated that base substitutions were the most prevalent mutations, followed by exon deletions. Among the groups of V79 cells treated with low concentrations of (-)-anti-DB[a,l]PDE, most displayed high selectivity for both A:T-->T:A transversions and A:T-->G:C transitions, while cells exposed to a higher dose (10 nM) formed predominantly G:C-->T:A transversions. Also, the number of base substitutions per mutant clone increased with dose. In general, the mutation profiles induced by (-)-anti-DB[a,l]PDE exhibited a wide spectrum; in addition to base substitutions, deletions, insertions, frameshift mutations, as well as tandem mutations were detected. Analysis of the DNA adduct levels induced by (-)-anti-DB[a,l]PDE revealed that a concentration-dependent increase in the level of adduct formation preceded the concentration-dependent increase in mutational events in these cells and that an increasing proportion of DNA adducts at deoxyadenosine were formed with dose. The results of this study demonstrate a correspondence between the concentration and types of DNA adducts and the frequency and types of mutations induced by (-)-anti-DB[a,l]PDE in V79 cells.

Participation of carnitine palmitoyltransferase in the synthesis of dipalmitoylphosphatidylcholine in rat alveolar type II cells.

We have investigated the role of carnitine palmitoyltransferase (EC 2.3.1.21) in pulmonar type II pneumocyte, a lung cell responsible for the synthesis of surface active lipids. Adult type II pneumocytes were isolated from rat lung and purified by differential adherence. When these lung cells were incubated with radioactive palmitate, the percentage of radioactivity recovered into dipalmitoylphosphatidylcholine (DPPC), a major surface active lipid, was almost 60% with respect to total phosphatidylcholine (PC) molecular species. Cellular lysates from type II pneumocytes contained detectable amount of carnitine palmitoyltransferase (CPT) activity (1 nmol/min/mg). Most of the CPT activity found in these cells could be inhibited by incubating them for 60 min with 5 microM tetradecylglycidic acid (TDGA), a specific and irreversible CPT inhibitor of the malonyl-CoA sensitive CPT isoform (CPT I). TDGA treatment of adult type II pneumocytes caused a significant reduction in the incorporation of radioactive palmitate into PC, though this effect did not seem to be specific for DPPC. TDGA affected the incorporation of radioactive palmitate at the sn2 rather than the sn1 position of the glycerol backbone of PC. The incorporation of radioactive palmitate into DPPC was also observed when these lung cells were incubated with palmitate-labeled palmitoyl-L-carnitine. Our data suggest that type II pneumocyte CPT may play an important role in remodelling PC fatty acid composition and hence DPPC synthesis.

Glutamyl substrate-induced exposure of a free cysteine residue in the vitamin K-dependent gamma-glutamyl carboxylase is critical for vitamin K epoxidation.

The vitamin K-dependent carboxylase catalyzes the posttranslational modification of glutamic acid to gamma-carboxyglutamic acid in the vitamin K-dependent proteins of blood and bone. The vitamin K-dependent carboxylase also catalyzes the epoxidation of vitamin K hydroquinone, an obligatory step in gamma-carboxylation. Using recombinant vitamin K-dependent carboxylase, purified in the absence of propeptide and glutamic acid-containing substrate using a FLAG epitope tag, the role of free cysteine residues in these reactions was examined. Incubation of the vitamin K-dependent carboxylase with the sulfhydryl-reactive reagent N-ethylmaleimide inhibited both the carboxylase and epoxidase activities of the enzyme. This inhibition was proportional to the incorporation of radiolabeled N-ethylmaleimide. Stoichiometric analyses using [(3)H]-N-ethylmaleimide indicated that the vitamin K-dependent carboxylase contains two or three free cysteine residues. Incubation with propeptide, glutamic acid-containing substrate, and vitamin K hydroquinone, alone or in combination, indicated that the binding of a glutamic acid-containing substrate to the carboxylase makes accessible a free cysteine residue that is important for interaction with vitamin K hydroquinone. This is consistent with our previous observation that binding of a glutamic acid-containing substrate activates vitamin K epoxidation and supports the hypothesis that binding of the carboxylatable substrate to the enzyme results in a conformational change which renders the enzyme catalytically competent.

Overexpression of thioredoxin in Fanconi anemia fibroblasts prevents the cytotoxic and DNA damaging effect of mitomycin C and diepoxybutane.

Adult T cell leukemia derived factor (ADF)/thioredoxin (Trx) is known to be an important intracellular antioxidant involved in a number of redox reactions such as ribonucleotide reductase (RNR) as well as of tyrosinase. Since RNR is a key enzyme of nucleotide metabolism and DNA synthesis, a reduced Trx level would result in reduced enzymatic activity and cause DNA damage. Furthermore, Trx is considered to be an effective regulator of redox sensitive gene expression. The role of Trx in nucleotide metabolism and gene expression may be an explanation for increased chromosomal instability as well as hypersensitivity towards oxygen, ROI and ROI generating agents. The activity of tyrosinase, the key enzyme of melanin biosynthesis, is influenced by the thioredoxin level and by superoxide radicals. Low thioredoxin levels and high superoxide concentrations activate tyrosinase causing hyperpigmentation of the skin. In addition to the observed high superoxide concentration in Fanconi anemia (FA) patients, a low thioredoxin level might be responsible for the hyperpigmentation (café-au-lait spots) in this disease. We observed that overexpression of the thioredoxin cDNA in FA fibroblasts completely abolished the DNA damaging effects of mitomycin C and diepoxybutane and inhibited the constitutive activity of the nuclear factor kappaB (NF-kappaB) in SV40 transformed FA fibroblasts. However, spontaneous chromosomal breakage was not affected.

Chemoprotective properties of chlorophyllin against vinyl carbamate, p-nitrophenyl vinyl ether and their electrophilic epoxides.

Chlorophyllin (CHL), a water-soluble sodium and copper derivative of chlorophyll, has been shown to be a strong antimutagen in several test systems, but its mechanism of antimutagenic action is largely unknown. In the present study, we have found the protective properties of CHL against vinyl carbamate, p-nitrophenyl vinyl ether and their electrophilic epoxides. CHL exhibited dose-related inhibition of his+ reversion in Salmonella typhimurium TA 1535 induced by these mutagens. Formation of DNA adducts from vinyl carbamate epoxide (VCO) and 2'-(4-nitrophenoxy)oxirane (NPO) was also markedly attenuated in the presence of CHL. Oral administration of CHL prior to the topical application of each of the above carcinogens resulted in significant reduction in both incidence and multiplicity of skin tumors in mice. The effective protection by CHL against VCO and NPO suggest that its formation of inactive complexes with these carcinogens is mediated by mechanisms other than pi-pi interactions.

Glutathione conjugation of styrene 7,8-oxide enantiomers by major glutathione transferase isoenzymes isolated from rat livers.

Male Sprague-Dawley rat liver cytosol mediated regioselective conjugation of styrene 7,8-oxide (STO) enantiomers with glutathione in completely trans-ring-opening manner to afford (1S)-S-(1-phenyl-2-hydroxyethyl)glutathione and (2R)-S-(2-phenyl-2-hydroxyethyl)glutathione in the ratio 22:1 for (R)-STO and also to afford (1R)-S-(1-phenyl-2-hydroxyethyl)glutathione and (2S)-S-(2-phenyl-2-hydroxyethyl)glutathione in the ratio 12:1 for (S)-STO. In the above cytosolic reactions, (R)-STO was conjugated 1.8 times faster than (S)-STO, while the (R)- to (S)-ratio in rate of the conjugation was 2.7 when racemic STO was used as a substrate. A kinetic study, carried out by using six major glutathione transferase (GST) isoenzymes isolated from the cytosol, indicated that GSTs 3-3, 3-4 and 4-4 (class mu enzymes) had much higher Kcat/Km values towards both STO enantiomers than the other three major isoenzymes, GSTs 1-1, 1-2 and 2-2 (class alpha enzymes). All the class mu enzymes mediated preferential glutathione conjugation of (R)-STO to (S)-STO. On the contrary, the class alpha enzymes catalysed the conjugation of (S)-STO preferentially to (R)-STO. The kinetic study strongly suggested that GSTs determining the higher enantioselectivity towards (R)-STO in the rat liver cytosol were the class mu enzymes, especially GST 3-3, which had the highest Kcat/Km value towards (R)-STO as well as the highest (R) to (S) ratio in the enantioselectivity among the six isoenzymes examined. GST 7-7, isolated as a major enzyme from the liver cytosol of the animals bearing hepatic hyperplastic nodules which were induced by chemical carcinogens, catalysed preferential GSH conjugation of (S)-STO to (R)-STO.

Partial correction of chromosome instability in Fanconi anemia by desferrioxamine.

The action of the iron chelator desferrioxamine (DFO) on the cytogenetic pattern of cultured lymphocytes from Fanconi anemia (FA) patients was investigated. The addition of 10(-4) M DFO throughout the culture time resulted in a 50% reduction of the spontaneous chromosome breakage of FA cells. In addition, the clastogenic action of diepoxybutane on FA lymphocytes was also partly counteracted by DFO. The above findings support the assumption that one of the mechanisms involved in the pathogenesis of FA might be an impaired capacity of the cells from such patients to remove active oxygen species. The relationship between intraleukocyte chelatable iron pool and free radical formation in FA subjects is discussed.