Sequential histologic study of rat liver during peroxisome proliferator [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]-acetic acid (Wy-14,643)-induced carcinogenesis

The livers of male inbred F344 rats fed Wy-14,643 [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (CAS: 50892-23-4) in the diet at a concentration of 0.1% (wt/wt) were examined sequentially at 5, 10, 17, 21, 26, 30, 35, 40, 52, 60, and 70 weeks. At 5 weeks the livers were markedly enlarged and histologically showed markedly enlarged hepatocytes with prominent nucleoli. At 21 weeks altered acidophilic areas were seen in 2 of 3 animals that were killed. Between 26 and 52 weeks neoplastic nodules were noted of 1-8 mm containing cells with morphologic features similar to those observed in altered areas. Hepatocellular carcinomas (HCC) were observed in 1 animal killed at 30 weeks and in all the animals sacrificed at 60 weeks and later. [3H]thymidine nuclear labeling studies showed marked proliferative activity of cells in altered areas, neoplastic nodules, and HCC. Altered areas, neoplastic nodules, and HCC were consistently negative for gamma-glutamyltransferase and showed decreased ATPase activity. Glucose-6-phosphatase (Glc-6-Pase) activity was decreased in altered areas and neoplastic nodules. However, some of the HCC showed a strong positive reaction for Glc-6-Pase.     

Relationship of hepatic peroxisome proliferation and replicative DNA synthesis to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalate and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) in rats

The mechanism of hepatocarcinogenesis caused by peroxisome proliferators (PP) is poorly understood, making it difficult to predict the carcinogenicity of PP to rodents or other species. It has been suggested that the carcinogenic potential of individual PP in rodents is correlated with the degree of PP-induced hepatic peroxisome proliferation. To evaluate this possible correlation, di(2-ethylhexyl)phthalate (DEHP) at 1.2% and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) at 0.1% were fed to male F-344 rats for up to 365 days and hepatocytic peroxisome proliferation and DNA replication were measured. All rats fed Wy-14,643 for 365 days had numerous grossly visible nodules in comparison to none in the livers of DEHP-fed or control rats. Despite this difference in the induction of tumors, both DEHP and Wy-14,643 increased the peroxisomal volume density 4- to 6-fold from 8 to 365 days of treatment. Peroxisomal beta-oxidation enzyme activities were increased 8-fold by both DEHP and Wy-14,643 after 18 days. At later time points (77 to 365 days), these enzyme activities were about 25% higher in livers of Wy-14,643- than DEHP-fed rats. DEHP or Wy-14,643 increased absolute liver weights 50 to 75% above controls after 18 to 365 days of feeding. Labeling of hepatocyte nuclei with a single injection of tritiated thymidine revealed a rapid burst in replicative DNA synthesis in both DEHP and Wy-14,643-fed rats, with a return to control levels by 4 days. Additional rats were implanted with 7-day osmotic pumps containing tritiated thymidine. With this more extended method of labeling a 5- to 10-fold increase in replicative DNA synthesis was observed in rats receiving Wy-14,643 for 39 to 365 days as compared to DEHP-fed rats or controls. In conclusion, when performed under conditions similar to the tumorigenicity studies, the degree of peroxisome proliferation correlated poorly with the relative hepatocarcinogenicity of DEHP and Wy-14,643. However, a strong correlation was observed between the relative hepatocarcinogenicity of DEHP and Wy-14,643 and the ability to induce a persistent increase in replicative DNA synthesis. These data emphasize the possible importance of cell replication in the mechanism of PP-induced hepatocarcinogenesis.     

Suppression of EGF binding in rat liver by the hypolipidemic peroxisome proliferators, 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio-(N-{beta}-hydroxyethyl)acetamide and di(2-ethylhexyl)phthalate

Dietary administration of 4-chloro-6-(2,3-xylidino)-2- pyrimidinylthio(N-ß-hydroxyethyl)acetamlde(BR931) and di(2-ethylhexyl)phthalate (DEHP), hypolipidemicagents, to rats for 3–35 days induced a marked reductionin the hepatocyte surface and intracellular binding of [¹²⁵I]EGFwithout affecting its binding affinity. The reduction was apparentafter 3 days' feeding of BR931 and the magnitude of the reductionwas consistently higher in hepatocytes of BR931-treated ratsthan those of DEHP-treated rats. The liver extracts and thesera from rats fed BR931 or DEHP for 4 weeks showed no inhibitoryeffects on the EGF binding of hepatocytes from rats fed a basaldiet. Possible significance of the changes in relation to theirhepatocarcinogenic action was discussed.     

Suppression of choline-deficient diet-induced hepatocyte membrane lipid peroxidation in rats by the peroxisome proliferators 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-beta-hydroxyethyl)- acetamide and di(2-ethylhexyl)phthalate

Many hypolipidemic peroxisome proliferators have been shown to induce liver tumors in rats after long-term feeding. In short-term assays, however, some of them prevent the development of gamma-glutamyl transpeptidase-positive foci, the putative preneoplastic lesions, in the liver of carcinogen-initiated rats and inhibit the promoting effect of a choline-deficient (CD) diet on these lesions. The CD diet-induced lipid peroxidation in the liver has been implicated as one of the underlying mechanisms of the promoting effect. In the present study, the effects of 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-beta-hydroxyethyl)acetamid e (BR931) and di(2-ethylhexyl)phthalate (DEHP) on CD diet-induced liver membrane lipid peroxidation were investigated by determining the extents of conjugated diene formation. No evidence of lipid peroxidation was detected in the microsomal lipids of the liver after administration of BR931 or DEHP at concentrations of 0.16% and 1%, respectively, for 1, 2, and 4 wk. When added to a CD diet, both BR931 and DEHP effectively protected against the diet-induced lipid peroxidation. There was an increase in cellular glutathione levels after 4 wk and an increase in catalase activity after 2 wk in the liver of rats fed BR931 or DEHP. The levels of activity of the glutathione peroxidases and glutathione-s-transferase were significantly reduced. The results suggest that, in the acute stage, hypolipidemic peroxisome proliferator-induced effects of excess production of H2O2 and potential lipid peroxidation are balanced by stimulation of some cellular detoxifying systems. The inhibition of lipid peroxidation by hypolipidemic peroxisome proliferators may account for their inhibitory effects on the CD diet-induced promotion of preneoplastic foci.     

Instability of ({+/-})-7{beta}, 8{alpha}-dihydroxy-9{beta}, 10{beta}-epoxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene (syn-BaPDE)- DNA adducts formed in benzo[a]pyrene-treated Wistar rat embryo cell cultures

One of the peaks present in HPLC profiles of [³H]benzo[a]-pyrene(BaP)-deoxyribonucleosides prepared by enzymatic degradationof [³H)BaP-DNA isolated from Wistar rat embryo cell culturesexposed to [G-³H)BaP was found to be r-7, c-9, c-10 t-8-tetrahydroxy-7,8, 9, 10-tetrahydroBaP, a BaP-DNA adduct decomposition product(Pruess-Schwartz, D. and Baird, W.M., Cancer Res., 46, 545–552,1986). To investigate the stability of the hydrocarbon-deoxyribo-nucleosidelinkages in intact BaP-modified DNA, DNA was isolated from Wistarrat embryo cells that had been exposed to [G-³H]BaP- and incubatedin darkness at 37°C at a range of pH values from 5 to 11for 72 h or for 1– 150 h at pH 7. The rate of breakdownof (³H)BaP-DNA adducts (0.25%/h) was linear over 150 h. Theamounts of the two major BaP-DNA adduct decomposition products,I and II (present in a ratio of 1: 3), increased with lengthof time of incubation. Formation of I was not affected by pH.whereas, formation of II was highest at acidic and neutral pH.Analysis of the decomposition products by immobilized boronatechromatography and reverse-phase HPLC demonstrated that bothI and II contained cis-vicinal hydroxyl groups and decompositionproduct II cochromatographed with r-7, c-9, c-10, t-8-tetrahydroxy-7, 8, 9, 10-tetrahydroBaP, a (±)- 7ß,8-dihydroxy-9ß, 10ß-epoxy-7, 8, 9, 10-tetrahydroBaP(syn-BaPDE)-derivedtetraol. At neutral pH [³H](±)-syn-BaPDE-modified calfthymus DNA formed a decomposition product identical to II. Analysisof the BaP-DNA adducts that remained covalently bound to theDNA after the above incubations demonstrated that the amountsof both major syn-BaPDE-deoxyguanosine adducts decreased withlength of time of incubation. Thus, syn-BaPDE-deoxyribonucleosideadducts formed in the DNA of [³H)BaP-treated Wistar rat embryocells are unstable and breakdown spontaneously in the absenceof light to yield syn-BaPDE-tetraol decomposition products.     

Antitumor Activity and Pharmacokinetics of Estra-1,3,5 (10)-Triene-3,17{beta}-Diol, 3-Benzoate, 17-((4-(4-(Bis (2-Chioroethyl)Amino)Phenyl)-1-Oxobutoxy) Acetate) (Bestrabucil) in Human Tumor Xenografts Serially Transplanted into Nude Mice

Bestrabucil (KM2210), the benzoate of an estradiol-chlorambucilconjugate, was used experimental cancer chemotherapy against13 human tumor xenografts serially transplanted into nude mice,and its pharmacokinetics was studied. The tumors were one esophageal,two gastric, six colon, one cholecystic and three breast carcinomas.Two tumor tissue fragments approximately 3x3x3 mm were inoculatedinto BALB/cA nude mice, which were then treated with KM2210at doses of 100, 200 and 300 mg/Kg/day orally starting 24 hrafter the transplantation or when the tumor reached a weightof 100–300 mg. The concentration of KM2210 and its derivativesin the tumor xenografts, normal muscular tissue and blood wereassayed by high performance liquid chroma-tography. Six out of 13 xenografts were found to be sensitive to KM2210.The concentrations of KM2210 and its derivatives in the tumortissues of the sensitive xenografts were five to 10 times higherthan those in blood and muscular tissue, and the antitumor activitycorrelated well with the area under the curve of active metabolitesof KM2210 in the tumor.     

In vivo formation and persistence of DNA adducts in mouse and rat skin exposed to ({+/-})-trans-7, 8-dihydroxy-7, 8-dihydrobenzo[a]-pyrene and ({+/-})-7{beta}, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7, 8, 9, 10-tetrahydro-benzo(a)pyrene

The in vivo DNA adduct formation of (±)-trans-7, 8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BPD) and (±)-7ß, 8-dihydroxy-9,10-epoxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene (anti-BPDE) werecompared and the persistence and disappearance of the adductsin both mouse and rat epidermis determined. BPD (100 nmol/mousein 150 µl acetone and 200 nmol/rat in 300µl acetone)and anti-BPDE (77 nmol/mouse in 150 µJ tetrahydrofuran)and 154 nmol/rat in 300 µ tetra-hydrofuran) were topicallyapplied to 50-day-old male Swiss mice and 35-day-old Wistarrats. To improve the identification of the DNA adducts formed,an acid hydrolysis technique was used to convert the BPD- andanti-BPDE- de-oxyribonucleoside adducts formed in mouse andrat skin to BP tetrols. The modified deoxyribonucleosides andBP tetrols obtained by hydrolysis of adducts were isolated byreverse-phase h.p.l.c. At approximately similar doses per unitarea of treated skin, the initial total binding of these compoundsto epidermal DNA and the level of modified deox-yribonucleosideswas 6-fold lower in rat skin epidermis than in mouse skin epidermis.Similar ratios of (±)-anti-BPDE-deoxyguanosine (dGuo)to (±)-syn-BPDE-dGuo adducts (5.7 and 6.1, determinedby h.p.l.c. analysis of BP tetrols obtained by hydrolysis ofmodified dGuo) were found in both mouse and rat epidermis ashort time (6 h)after topical application of (±)-trans-BPD.Three hours after topical application of (±)-anti-BPDE,the ratios of BP-7, 10/8, 9-tetrol to 7/8, 9, 10-tetrol were9: 1 in mouse epidermal DNA and 6: 1 in rat epidermal DNA. Oneand three weeks after application of these two compounds, only(+)-anti-BPDE-dGuo was detected in mouse epidermis; 2 and 0.2%of the initial (+)-anti-BPDE-dGuo level was found to persistin the epidermal DNA from BPD- and anti-BPDE-treated mice respectively.No DNA adducts were detected in rat epidermis 3 weeks afterBPD and anti-BPDE treatment. Thus, 3 weeks after topical applicationof BPD and anti-BPDE to mouse and rat skin, the DNA adductscompletely disappeared form rat epidermis while they persistedin mouse epidermis. The results suggest that: (i) the persistenceof (+)-anti-BPDE-dGuo may be related to carcinogenesis in mouseepidermis by BPD and anti-BPDE; (ii) the complete disappearanceof the anti-BPDE-dGuo adduct may also account in part for therelative resistance of tissue from this species to the carcinogenicaction of benzo(a) pyrene.     

Immunohistochemical detection of a substituted 1,N(2)-ethenodeoxyguanosine adduct by omega-6 polyunsaturated fatty acid hydroperoxides in the liver of rats fed a choline-deficient, L-amino acid-defined diet

Endogenous lipid peroxidation products react with DNA and form exocyclic DNA adducts. The purpose of this study was to investigate the in vivo formation of 7-(2-oxo-heptyl)-substituted 1,N(2)-etheno-2'-deoxyguanosine adduct (Oxo-heptyl-varepsilondG), one of the major products from the reaction of 13-hydroperoxyoctadecadienoic acid (13-HPODE) with dG. The monoclonal antibody specific to Oxo-heptyl-varepsilondG was prepared using a chemically synthesized conjugate of Oxo-heptyl-varepsilondG and carrier protein as immunogen. The characterization showed that the obtained antibody (mAb6A3) is specific to the Oxo-heptyl-varepsilondG moiety. Using the novel antibody, the presence of the Oxo-heptyl-varepsilondG adduct in vivo was immunohistochemically revealed in the liver of rats fed a choline-deficient, L-amino acid-defined diet, an endogenous carcinogenesis model, for 3 days. In addition, the Oxo-heptyl-varepsilondG formation was confirmed in DNAs treated with peroxidized linoleic acid, arachidonic acid and gamma-linolenic acid, respectively, suggesting that the hydroperoxides of omega-6 polyunsaturated fatty acids could be the potential sources of Oxo-heptyl-varepsilondG formation in vivo. Collectively, the results in this study suggest the first evidence that the formation of Oxo-heptyl-varepsilondG, the omega-6 lipid hydroperoxide-mediated DNA adduct, may be a potential biomarker for the early phase of carcinogenesis.     

5-Fluorouracil, interferon-{alpha}-2b and cisplatin (FAP) for advanced urothelial cancer. A phase II study

Purpose: To evaluate the efficacy and toxicity of the FAP combinationchemotherapy as first-line treatment in advanced urothelial cancer.Patients and methods: Thirty-four patients with histologicallyconfirmed advanced urothelial cancer, with measurable disease and withoutprevious chemotherapy entered the study; all 34 are evaluable. The 28 malesand 6 females had a median age of 65 (19–75) and a median ECOGperformance status of 1 (0–2). Twenty-eight patients had bladder cancer,four had renal pelvic cancer and two ureteral cancer. Thirty patients hadtransitional cell carcinoma and four mixed, mostly of grade 3. Sites ofdisease included lymph nodes (18), bladder (9), liver (9), pelvic mass (9),lung (7), etc. The treatment plan was as follows: 5-fluorouracil 500mg/m² continuous infusion D1–D5 and D22–D26;interferon--2b 5 million I.U./m² D1–D5 followed by3×/week and then D22–D26; cisplatin 25 mg/m² D1,D8, D15, D22. Cycles were repeated every 36 days.Results: The median number of cycles administered was 3 (1–6).The relative dose intensities for 5-fluorouracil, interferon and cisplatinwere 76%, 71% and 75%, respectively. Twenty-two of 34patients (65%, 95% confidence interval [95% CI],46% to 80%) had objective responses, including six completeclinical responses (CR) (18%, 95% CI, 7% to 35%)and 16 partial responses (PR) (47%, 95% CI, 30% to65%). Three patients had stable disease and seven progressed. Twopatients discontinued treatment after the first cycle because of toxicity. Themedian survival is 15.30 months (1.40–37.60), the median time toprogression 11.60 months (4.13–37.60), and the median survival ofcomplete responders 20.75+ months (8+ to 38+). The only significanthematologic toxicity was the grade 3–4 neutropenia in 44%.Non-hematologic toxic effects were unremarkable.Conclusion: The FAP combination as first-line chemotherapy is highlyactive in the treatment of advanced urothelial cancer, and has limitedtoxicity. Further phase III studies are in progress to compare FAP and M-VAC.     

Presence of cytidine 5'-monophospho-N-acetylneuraminic acid:Gal beta 1-3GalNAc-R alpha(2-3)-sialyltransferase in normal human leukocytes and increased activity of this enzyme in granulocytes from chronic myelogenous leukemia patients

We have examined granulocytes from patients with chronic myelogenous leukemia (CML) and from normal subjects to determine whether activity of a specific sialyltransferase might account for the aberrant sialylation of O-linked membrane oligosaccharides in CML cells. Total membrane preparations of morphologically mature CML and normal granulocytes were tested for sialyltransferase activity using the substrates galactosyl-beta 1-3-N-acetyl-D-galactosamine-alpha-O-nitrophenyl and N-acetyl-D-galactosamine-alpha-phenyl. N-Acetyl-D-galactosamine-alpha-phenyl was not an acceptor with either CML or normal cells. With galactosyl-beta 1-3-N-acetyl-D-galactosamine-alpha-O-nitrophenyl, sialyltransferase activity was 2.8 times higher in CML cells compared to normal cells. Product identification by high performance liquid chromatography showed that enzyme from both normal and CML granulocytes linked sialic acid to galactosyl-beta 1-3-N-acetyl-D-galactosamine-R by the alpha(2-3) and not the alpha(2-6) linkage. The enzyme CMP-N-acetylneuraminic acid: galactosyl-beta 1-3-N-acetyl-D-galactosamine-R alpha(2-3)-sialyltransferase has not previously been described in human granulocytes. The marked increase in activity of this enzyme in CML and the resulting increase in sialylation may contribute to the pathophysiological behavior of CML granulocytes.