Radioprotective effect of antioxidative flavonoids in {gamma}-ray irradiated mice

The anticlastogenic effect of 12 structurally different flavonoidswas investigated in whole body     

Base excision repair of DNA in {gamma}-irradiated human cells

Escherichia coli endonuclease IV was used to incise cellularDNA specifically at apurinic/apyrimidinic (AP) sites prior toalkaline elution to measure the resulting DNA strand breaks.-Irradiated HeLa cells initially contained DNA strand breaksand no AP sites. Upon incubation at 37?C the strand breaks wererapidly repaired and AP sites were generated and subsequentlyrepaired. The transient nature of the AP sites indicates thein vivo operation of a base excision repair pathway wherebydamaged bases are removed from DNA by DNA glycosylases to produceAP intermediates that are then substrates for AP endonucleases.     

Carcinogen treatment increases glutathione hydrolysis by {gamma}-glutamyl transpeptidase

The effect of carcinogen treatment on -glutamyl transpep-tidase(GGT)-mediated hydrolysis of GSH to glutamate and cysteinylglycinein the blood and bile compartments was investigated in liversperfused in situ. Treatment of rats with 40 p.p.m. diethylnitrosamlne(DEN) in the drinking water or 0.02% 2-acetylaminofhiorene (AAF)in the diet for 50–60 days increased GGT activity in liverhomogenates by 100 and 800% respectively. Bile flow and thesum of glutamate and glutathione (GSH) efflux into the bileof perfused livers was not affected by carcinogen treatment.However, the ratio of GSH to glutamate in bile was 2.1, 1.1and 0.2 in livers from control, DEN- and AAF-treated rats respectively.Pretreatment with L-(S,5S)--amino-3-chloro-4,5-dihydro-5-isox-azoleaceticacid (AT125) decreased GGT activity in liver homogenates byabout 85% and elevated the ratio of GSH to glutamate in thebile to 3.2 hi all groups. Thus, the hydrolysis of GSH to glutamatein the bile of perfused livers correlated with the degree ofinduction of GGT by DEN and AAF treatments. Exogenous GSH (10µM) infused into the portal vein of perfused livers fromcontrol, DEN- and AAF-treated rats was recovered completelyin the effluent perfusate. Pretreatment with AT125 had no effecton the recovery of exogenous GSH in the effluent perfusate.Thus, metabolism of GSH in the blood space was not detectedafter short-term carcinogen treatment. To increase the possiblehydrolysis of GSH in the perfusate, rats were treated for 130–180days with DEN and GSH ( n60 µM) was infused into the hepaticartery of livers perfused simultaneously via the hepatic arteryand portal vein. Only 50% of the infused GSH was recovered inthe effluent perfusate of perfused livers from DEN-treated rats.In contrast, significantly more GSH (80–90%) was recoveredfrom livers from control rats or DEN-treated rats that had receivedAT125 pretreatment. In addition AT125 pretreatment increasedthe basal rates of GSH efflux in livers from DEN-treated rats.Thus, DEN-induced GGT metabolizes GSH entering the liver viathe hepatic artery. Furthermore, GGT may act to decrease thenet efflux of GSH from perfused livers by causing the intra-organrecycling of GSH and its constituent amino acids.     

2{alpha} 3{alpha}-Epithio-5{alpha}-androstan-17{beta}-ol in Treatment of Gynecomastia

1. 1. The clinical effect of epitiostanol, a new anti-estrogenagent (2,3-epithio-5a-androstan-17ß-ol) against gynecomastiawas studied in comparison with dromostanolone propionate infifty-four patients ranging from twenty to fifty years in agewithout previous history of hormone therapy and with normalliver function. The experiment was performed for eight weeksby double blind methods in three dosage groups, epithiostanol10 mg, and 20 mg and dromostanolone propionate 50 mg.
2. 2. Epithiostanol20 mg was most effective with regards to effecton mass sizeand tenderness, (effective in 96%, 20/21), followedby 10 mgepitiostanol (effective in 89%, 16/18) and dromostanolonepropionate50 mg (effective in 89%, 16/18) in descending order.No sideeffects were observed in any of the three groups.
3. 3. Basedon the results of the present study, epitiostanol isconcludedto be at least as effective as dromostanolone propionateagainstgynecomastia and to be safe from the viewpoint of sideeffects.A satisfactory therapeutical effect on gynecomastiacan be expectedwith a weekly dosage of 20 mg of epitiostanolfor an administrationperiod of between five to eight weeks.

Present Address: Department of Surgery, Keio University Hospital,Shinanomachi, Shin-juku-ku, Tokyo, Japan.     

Detection by 32P-postlabeling of thymidine glycol in {gamma}-irradiated DNA

The ³²P-postlabeling method has been adapted for the analysisof thymidine-cis-glycol-3'-phosphate (cis-dTGp, cis-5,6-dihydroxy-5,6-dihydrothymidine-3'-phosphate).Cis-dTGp was isolated and purified from normal nucleotides byphenylboronate affinity chromatography and phosphorylated byT4 polynucleotide kinase in presence of 1 mM BeCl₂ at pH 7.5.These modifications of the postlabeling method resulted in a5'-phosphorylation of dTGp with a labeling efficiency of upto 20% whereas the natural nucleotides were almost completelydephosphorylated at the 3' position under these conditions.The reaction products, containing radio-labeled thymidine-cis-glycol-3',5'-bis-[5'-³²P]phosphate (cis-^*pdTGp), were separated by two-dimensionalanion-exchange TLC on polyethyleneimine cellulose sheets. Boricacid was added in the second dimension in order to selectivelyretard cis-glycols. The method was applied to -irradiated nucleotidesand calf thymus DNA. In the nucleotide mixture, 330–99000 thymine glycol (TG) moieties were detected per 10⁶ thymines(T) in a dose range of 14–1000 Gy respectively. In DNA,these values ranged from 400 to 2700 TG/10⁶ T. The data arein good agreement with methods using radiochemical and immunologicaltechniques. Non-irradiated DNA showed a background level of1OTG/10⁶ T. This practical limit of detection was higher thancan be achieved with the postlabeling technique, indicatingthat the present method might be a sensitive alternative fora determination of oxidative DNA damage.     

An examination of the relative resistances to aflatoxin B1 and susceptibilities to {gamma}-glutamyl p-phenylene diamine mustard of {gamma}-glutamyl transferase negative and positive cell lines

Two epithelial cell lines have been derived from rat liver.One, containing only low levels of glutamyl transferase (GGT)was obtained from normal liver and the other, containing highlevels of the enzyme was isolated from an aflatoxin B₁inducedhepatoma. The GGT levels present in the two cell lines havebeen examined by histochemical staining, by a fluorescence assayusing disrupted cells, and also by intact fluorescence-labelledcells in a laser flow cytofluori-meter. The cells containinghigh levels of GGT have been found to be less sensitive to microsomally-activatedaflatoxin B₁ than are the GGT negative cells, a feature of thein vivo situation. Evidence for activation of the -glutamylderivative of an alkylating mustard analogue of p-phenylenediamine by the GGT positive cells is presented. This findingcould be of relevance to the possible chemotherapy of GGT-richlesions in vivo.     

Antioxidants can delay liver cell maturation which in turn affects {gamma}-glutamyltranspeptidase expression

In normal rats just before weaning the majority of hepatocytesare mononucleated diploids, but within days the number of binucleatedcells reaches a peak (50%) before declining again and thereis a steady shift of diploid to tetraploid nuclei. When weanlingrats were exposed to ethoxyquin (EQ), the conversion of 2N nucleito 4N and 8N nuclei as measured by flow cytometry was sloweddown. The rapid rise in the number of binucleate cells was alsodelayed, although the long-term effect was an increased numbercompared with age-matched controls. It appeared that when EQwas present in the diet, significant numbers of diploid hepatocytesundergoing DNA synthesis also underwent mitosis and cytokinesisgiving rise to new diploid hepatocytes. However, many hepatocytesfrom animals maintained on a control diet did not undergo cytokinesis.Thus the slower ‘conversion’ of 2N to 4N nucleiin treated hepatocytes was due in part to promotion of cytokinesisin diploid cells undergoing DNA synthesis. The ploidy of a cellwould be expected to affect gene expression. EQ is a very potentinducer of -glutamyltranspeptidase (GGT), but expression dependedon the age of the animals, the length of treatment time andapparently the ploidy status of the liver. In weanling ratstreated with EQ for 7 days, >80% of the hepatocytes expressedGGT, while in 42 day old rats similarly treated <50% werepositive for this enzyme. GGT expression was closely correlatedwith the percentage of 2N nuclei present in hepatocytes, suggestingthat it was more easily induced in cells containing these nucleithan in those containing nuclei of higher ploidy. Although butylatedhydroxytoluene (BHT), at the same concentration in the diet,had a similar negative effect on weight gain as did EQ, it hadno effect on ploidy, nor did it induce GGT to the same extentas EQ.     

{gamma}-Glutamyltranspeptidase-conferred resistance to hydroquinone induced GSH depletion and toxicity in isolated hepatocytes

Hepatocyte resistance against glutathione (GSH) depleting xenobioticswas studied in an in vitro model. Hepatocytes were isolatedfrom carcinogen treated rats that had received phenobarbitalfor three weeks. Isolated cells were incubated in GSH containingbuffer with hydroquinone, which depleted GSH. Cells were thenseeded on collagen coated plates and cultured overnight in completemedium. Attached cells were stained and the proportion of -glutamyltranspeptidase(GGT)-positive cells was counted. It was found that toxicityrelated to GSH depletion increased the proportion of GGTpositivecells from 10–15% up to 40–60%, indicating thatthe toxicity mainly affected GGT-negative cells. GSH added tothe buffer was essential for this effect. It is concluded thatGGT may protect GGT-positive hepatocytes from GSH depletionand toxicity early during liver carcinogenesis.     

Mutagenicities of {gamma}-carboline derivatives related to potent mutagens found in tryptophan pyrolysates

The mutagenicities of ten synthetic 3-amino--carboline homologuesincluding the mutagens Trp-P-1 and Trp-P-2 found in tryptophanpyrolysate, were tested using Salmonella typhimurium TA98 andTA100 with S-9 mix. Among these ten compounds, Trp-P-2 was thestrongest mntagen to TA98 and TA100.     

Glutathione mutagenesis in Salmonella typhimurium is a {gamma}-glutamyltranspeptidase-enhanced process involving active oxygen species

Reduced glutathione (GSH) is mutagenic in Salmonella in thepresence of -glutamyltranspeptidase (GGT), with the highestresponse obtained in strain TA102. Reduced cysteinylglycine,one of the products of GGT metabolism of GSH, is mutagenic inthe absence of GGT. In strain TA102, GSH mutagenesis was dependenton molecular oxygen, enhanced by iron, inhibited by EDTA, desferrioxaminemesylate, mannitol, butylated hydroxyanisole, peroxidase andcatalase, but not by superoxide dismutase. Binding of GSH orits GGT-dependent metabolites to DNA in vitro was not detected.This is consistent with a model of an indirect mechanism ofmutagenesis, i.e. cleavage of GSH by GGT, followed by facileauto-oxidation of the resulting cysteinylglycine, with the productionof free radicals which lead to the (pen)ultimate mutagen, H₂O₂.     

Interactions of peroxisome proliferator-activated receptor {gamma} and diet in etiology of colorectal cancer.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is one of a group of ligand-activated nuclear receptors responsible for regulation of glucose, lipid homeostasis, cell differentiation, and apoptosis. The 12 proline-to-alanine (Pro12Ala) substitution polymorphism in PPARgamma produces proteins with lower activity. Variation in PPARgamma expression in the bowel and the role of dietary fatty acids as ligands for PPARgamma led investigation of whether the associations of diet with colon and rectal cancer risk were modified by PPARgamma genotype. Data (diet, lifestyle, and DNA) came from case-control studies of colon (1,577 cases and 1,971 controls) and rectal cancer (794 cases and 1,001 controls) conducted in Northern California, Utah, and the Twin City, Minnesota Metropolitan area (colon cancer study only). Unconditional logistic regression models were adjusted for age at selection, body mass index, physical activity, energy intake, dietary fiber, and calcium. We found no significant interactions between macronutrient (fat, protein, and carbohydrate) and colorectal cancer. High lutein intake [odds ratio (OR), 0.63; 95% confidence interval (95% CI), 0.44-0.89], low refined grain intake (OR, 0.70; 95% CI, 0.53-0.94), or a high prudent diet score (OR, 0.66; 95% CI, 0.49-0.89) and PA/AA PPARgamma genotype were associated with reduced colon cancer risk. Risk of rectal cancer was increased among those with the PA/AA PPARgamma genotype and a high mutagen index (OR, 1.63; 95% CI, 1.12, 2.36). Its unclear whether the alterations in risk in those with the less active phenotype for PPARgamma is related to activation of PPARgamma by nutrients or dietary patterns acting as ligands or direct influences of these nutrients on colon and rectal cancer processes that are important with lower PPARgamma activity.     

Targeting human {gamma}delta} T cells with zoledronate and interleukin-2 for immunotherapy of hormone-refractory prostate cancer

The increasing evidence that gammadelta T cells have potent antitumor activity suggests their value in immunotherapy, particularly in areas of unmet need such as metastatic carcinoma. To this end, we initiated a phase I clinical trial in metastatic hormone-refractory prostate cancer to examine the feasibility and consequences of using the gammadelta T-cell agonist zoledronate, either alone or in combination with low-dose interleukin 2 (IL-2), to activate peripheral blood gammadelta cells. Nine patients were enlisted to each arm. Neither treatment showed appreciable toxicity. Most patients were treated with zoledronate + IL-2, but conversely only two treated with zoledronate displayed a significant long-term shift of peripheral gammadelta cells toward an activated effector-memory-like state (T(EM)), producing IFN-gamma and perforin. These patients also maintained serum levels of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), consistent with a parallel microarray analysis showing that TRAIL is produced by gammadelta cells activated via the T-cell receptor and IL-2. Moreover, the numbers of T(EM) gammadelta cells showed a statistically significant correlation with declining prostate-specific antigen levels and objective clinical outcomes that comprised three instances of partial remission and five of stable disease. By contrast, most patients treated only with zoledronate failed to sustain either gammadelta cell numbers or serum TRAIL, and showed progressive clinical deterioration. Thus, zoledronate + IL-2 represents a novel, safe, and feasible approach to induce immunologic and clinical responses in patients with metastatic carcinomas, potentially providing a substantially increased window for specific approaches to be administered. Moreover, gammadelta cell phenotypes and possibly serum TRAIL may constitute novel biomarkers of prognosis upon therapy with zoledronate + IL-2 in metastatic carcinoma.     

Biphasic early changes in rat liver {gamma}-glutamyl transpeptidase in response to aflatoxin B1

The present study was designed to examine the earliest changesin -glutamyl transpeptidase (GGT) in response to administrationof the hepatocarcinogen aflatoxin B₁ (AFB₁). Male Fischer 344rats were either fed a diet containing 4 p.p.m., or receiveda single i.p. injection (0.72 or 1.73 mg/kg) of AFB₁. GGT levelswere determined histochemically and by quantitative fluorimetricassay. In livers of animals receiving 4 p.p.m. AFB₁, GGT activityshowed a biphasic response during a 15 week period. During thefirst 4 weeks the increasing activity was localised predominantlyin the bile duct epithelial cells and some periportal hepatocytes.At 4 weeks the first foci of GGT-positive hepatocytes, characteristicof the very early carcinogenic changes began to appear. As feedingcontinued there was an overall temporary decrease in GGT, duringwhich time biliary hyperplasia regressed but the number of focigradually increased. The second phase of increased activitycorresponded to increase in size and number of foci, so thatby 15 weeks most of GGT was present in hepatocytes. Single injectionsof AFB₁ produced dose-dependent increases in GGT which borea linear relationship to biliary hyperplasia.     

Induction of {gamma}-glutamyl transpeptidase mRNA by aflatoxin B1 and ethoxyquin in rat liver

We have studied the induction of rat liver -glutamyl transpeptidase(GGT) mRNA by the antioxidant ethoxyquin and during aflatoxinB₁-induced carcinogenesis. Using a rat kidney GGT cDNA probe,Northern blot analysis revealed that GGT mRNA induced in liverby either compounds was slightly larger than that found in untreatedkidney. GGT mRNA was not detected in untreated liver or freshlyisolated hepatocytes, but induction of the message in treatedtissues correlated with the increase in enzymic activity observedby histochemistry and quantitative assay. Slot-blot analysisof poly(A)⁺ mRNA indicated that constitutive GGT mRNA levelsin kidney were at least 5-fold greater than those in the mostGGT-positive liver-derived tissue examined.     

The PPAR{gamma} Pro12Ala polymorphism and risk for incident sporadic colorectal adenomas

Peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear hormone receptor superfamily initially shown to be a key regulator of fat cell differentiation, can inhibit cell growth and induce apoptosis in colon cell lines. There are heterozygous loss of function mutations in the gene encoding PPARgamma in tumors from approximately 10% of human colon cancer patients. A common structural polymorphism has been detected in the PPARgamma gene at codon 12 (Pro12Ala). We investigated the hypothesis that the PPARgamma Pro12Ala polymorphism is associated with colorectal adenoma risk in a recently concluded case-control study of incident sporadic colorectal adenomas (163 cases and 212 controls). The multivariate-adjusted odds ratio (OR) for incident sporadic colorectal adenoma was 0.65 (95% CI 0.39-1.09) for those with the Pro12Ala or Ala12Ala genotype compared with those with the Pro12Pro genotype. Multivariate-adjusted inverse associations with the Ala12 variant were more pronounced among those who were female (OR 0.36, 95% CI 0.18-0.75) or did not take non-steroidal anti-inflammatory drugs (OR 0.38, 95% CI 0.14-1.00). Marginally significant results were observed among those with a lower waist:hip ratio (OR 0.52, 95% CI 0.24-1.12) or a lower body mass index (OR 0.46, 95% 0.20-1.05). Smoking was a very strong risk factor (OR 2.34, 95%CI 1.37-4.02) for colorectal adenoma among those with the wild-type (Pro12Ala) genotype, but not those with the Ala12 variant (OR 0.86, 95%CI 0.35-2.09). Larger studies are needed to validate these results, which suggest that the PPARgamma Pro12Ala polymorphism may interact with other factors to protect against colorectal adenoma.     

Isolation of {gamma}-glutamyl transpeptidase positive hepatocytes during the early stages of hepatocarcinogenesis in the rat

-Glutamyl transpeptidase (GT) positive hepatocytes were isolatedfrom F344 male rats fed 2-acetylaminofluorene. The isolationprocedure is rapid and highly selective for cells exhibitingGT on their surface. Suspensions of liver cells obtained fromperfusion in situ with a collagenase solution were incubatedon Petri dishes coated with affinity purified rabbit anti-GTantibody. GT-positive cells bound to the dish within fifteenminutes and could be recovered as viable cells. Approximately15% of the GT-positive cells are isolated using this procedure.Novikoff hepatoma cells, a GT-positive cell line, were usedto define the parameters of the assay. The binding was bothtime and temperature dependent. Binding of cells to the anti-GTantibody coated dishes was 50% inhibited by 2.8 mM sodium azideand 86% inhibited by 4.6 mM.     

2{alpha},3{alpha}-Epithio-5{alpha}-androstan-17{beta}-yl 1-Methoxycyclopentyl Ether in the Treatment of Advanced Breast Cancer --A Preliminary Clinical Trial--

A new orally active anti-estrogenic steroid, 2,3-epithio-5-androstan-17ß-yl1-methoxycyclopentyl ether (10364-S) was given to 41 advancedbreast cancer patients. Most patients were given a daily doseof 20 mg. The study was preliminary and not a controlled trialusing an already proven androgenic steroid. The remission rateon giving this compound to advanced breast cancer was 11/41or 26.8% and the average duration of the remission was 10.5months. Hoarseness (8/41, 19.5%) and hirsutism (5/41, 12.2%) were relativelyoften seen as virilizing side effects. No unfavorable effectson the hematopoietic organs, the liver or on calcium metabolismwere recognized in the study.     

In situ analysis of transforming growth factor-{beta}s (TGF-{beta}1, TGF-{beta}2, TGF-{beta}3and TGF-3 type II receptor expression in malignant melanoma

We have analysed, by in situ hybridization, mRNA expressionof TGF-ß1, TGF-ß2, TGF-ß3, andof TGF-ß type II receptor in benign melanocytic naevi,primary melanomas, and in skin metastases of malignant melanomas.Our results show that melanoma progression correlates with overexpressionof TGF-ß. All skin metastases and most primary melanomasinvasive to Clark's level IV-V revealed specific TGF-ß2mRNA and protein expression. However, expression of this cytokinewas not observed in benign melanocytic lesions and was detectedonly in one of five early primary melanomas investigated. Someprimary melanomas and skin metastases also revealed specificTGF-ß1 mRNA signals although expression of this isoformwas not found in benign naevi. TGF-ß3 expression,which was only barely detectable in benign melanocytic lesions,was enhanced in some skin metastases. Interestingly, the epidermisoverlaying melanomas revealed lower levels of TGF-ß3mRNA expression than epidermis of healthy skin or epidermisadjacent to benign naevi, thereby suggesting that paracrinemechanisms between tumour cells and keratinocytes may influencemelanoma development. In primary melanomas TGF-ß typeII receptor mRNA signals were much more heterogeneously distributedwhen compared to benign melanocytic naevi, suggesting variabledegrees of TGF-ß resistance among melanoma cells withinindividual lesions. However, melanoma progression appeared notto be correlated with a complete loss of TGF-ß typeII receptor gene expression, since all skin metastases revealedclearly detectable although heterogeneous levels of TGF-ßtype II receptor mRNA expression.     

Attenuation of proteasome-induced proteolysis in skeletal muscle by {beta}-hydroxy-{beta}-methylbutyrate in cancer-induced muscle loss

Loss of skeletal muscle is an important determinant of survival in patients with cancer-induced weight loss. The effect of the leucine metabolite beta-hydroxy-beta-methylbutyrate (HMB) on the reduction of body weight loss and protein degradation in the MAC16 model of cancer-induced weight loss has been compared with that of eicosapentaenoic acid (EPA), a recognized inhibitor of protein degradation. HMB was found to attenuate the development of weight loss at a dose greater than 0.125 g/kg accompanied by a small reduction in tumor growth rate. When EPA was used at a suboptimal dose level (0.6 g/kg) the combination with HMB seemed to enhance the anticachectic effect. Both treatments caused an increase in the wet weight of soleus muscle and a reduction in protein degradation, although there did not seem to be a synergistic effect of the combination. Proteasome activity, determined by the "chymotrypsin-like" enzyme activity, was attenuated by both HMB and EPA. Protein expression of the 20S alpha or beta subunits was reduced by at least 50%, as were the ATPase subunits MSS1 and p42 of the 19S proteasome regulatory subunit. This was accompanied by a reduction in the expression of E2(14k) ubiquitin-conjugating enzyme. The combination of EPA and HMB was at least as effective or more effective than either treatment alone. Attenuation of proteasome expression was reflected as a reduction in protein degradation in gastrocnemius muscle of cachectic mice treated with HMB. In addition, HMB produced a significant stimulation of protein synthesis in skeletal muscle. These results suggest that HMB preserves lean body mass and attenuates protein degradation through down-regulation of the increased expression of key regulatory components of the ubiquitin-proteasome proteolytic pathway, together with stimulation of protein synthesis.