Protective effect of acetaminophen against colon cancer initiation effects of 3,2'-dimethyl-4-aminobiphenyl in rats.

A previous investigation demonstrated the anticarcinogenicity of acetaminophen (APAP) against colon carcinogenesis in rats induced by 3,2'-dimethyl-4-aminobiphenyl (DMAB). DMAB was selected as a structurally related surrogate for heterocyclic amines, formed during cooking of protein, which are believed to be involved in human colon cancer. The objective of the present study was to ascertain whether the early initiating effects of this colon carcinogen are inhibited by APAP. Six groups of male F344 rats were treated over a 6-week period as follows: (1) vehicle (corn oil) for 6 weeks; (2) APAP in the diet at 1000 ppm daily for 6 weeks; (3) 50 mg/kg DMAB by gavage once a week for the last 4 weeks; (4) 5 mg/kg DMAB as for (3); (5) 1000 ppm APAP for 6 weeks and 50 mg/kg DMAB for the last 4 weeks; and (6) 1000 ppm APAP and 5 mg/kg DMAB as for (5). Colonic tissue was within normal limits in the control and APAP groups. In the APAP only group, apical enterocytic hypertrophy and hyperaemia over the entire surface epithelium was present. In the high-dose DMAB group, in the lower third of the crypts, foci of enlarged glands with hypertrophic cells exhibiting karyomegaly and anisokaryosis (FHE) of 3+ degree of severity were evident in 100% of the animals. Also, there were increases in periglandular fibrocytes, matrix and mononuclear cells (PF). In the low-dose DMAB group both FHE and PF changes with the same degree of severity were reduced. In rats given the low dose of DMAB plus APAP, FHE and PF with the same degree of severity (3+) was absent. Both DMAB exposures increased significantly the replicating fraction of colonic enterocytes in an exposure-related fashion and the replicating fractions were significantly reduced by APAP. In 32P-postlabelling of colon, liver and urinary bladder DNA, high-dose DMAB produced 2-6 distinct dose-related spots reflecting DNA adducts. These spots were reduced or were no longer detectable in all three tissues when APAP was given 2 weeks before and during DMAB exposure. Using immunohistochemical detection of DMAB adducts in the colon, a dose-related colour intensity was present for both doses of DMAB. APAP reduced this by 94-fold. Thus, APAP produced a marked protective effect in colonic enterocytes against several parameters of neoplastic development by the carcinogen.     

Dose dependence of N-hydroxy-3,2'-dimethyl-4-aminobiphenyl-induced rat prostate carcinogenesis.

Groups of F344 rats were administered biweekly intraperitoneal injections of N-hydroxy-3,2'-dimethyl-4-aminobiphenyl (N-OH-DMAB) at a dose of 5, 10 or 20 mg/kg body weight or DMAB, the parent compound, at a dose of 25 mg/kg body weight, for a total of 10 times. Prostate carcinomas in the ventral lobe developed in a N-OH-DMAB dose-dependent manner (0, 17.6 and 66.7%, respectively) with limited tumor yields in other organs. Although intraperitoneal administration of DMAB was similarly found to induce prostate tumors, it also caused severe chemical peritonitis, which resulted in a high mortality. The present data confirmed that intraperitoneal administration of N-OH-DMAB provides a relatively specific induction method for models of prostate carcinogenesis.     

Preparation and characterization of agonistic monoclonal antibodies against Toll-like receptor 4-MD-2 complex

Ligands for toll-like receptors (TLR) are known to induce a variety of immune responses. Selective induction of desirable responses would be important for the treatment of individual diseases with various pathogenesis. For this purpose, we established six MAbs against the TLR4/MD-2 complex (UT MAbs) from TLR4(-/-) mice or MD-2(-/-) mice. Three MAbs (UT12, 18, and 22) induced NF-kappaB activation and production of pro-inflammatory cytokines, but the other three (UT15, 41, and 49) did not induce such cell responses. Unlike lipopolysaccharide (LPS), agonistic UT MAbs did not require serum components for the functions. UT41 and UT49 recognized TLR4 in the absence of MD-2. On the other hand, the other four MAbs reacted to the TLR4/MD-2 complex, but not to solo TLR4. Agonistic UT MAbs shared the epitopes, but non-agonistic UT15 reacted to distinct epitope on the complex. UT MAbs appear to be useful analyzing the molecular mechanism of TLR signaling and will contribute to the development of novel immunotherapies.     

Generation and characterization of monoclonal antibodies against FABP4

Fatty acid binding protein 4 (FABP4) is a key mediator of intracellular transport and metabolism of fatty acids in adipose tissues. FABP4 binds fatty acids with high affinity and transports them to various compartments in the cell. When in complex with fatty acids, FABP4 interacts with and modulates the activity of two important regulators of metabolism: hormone-sensitive lipase and peroxisome proliferator-activated receptor gamma. Genetic studies in mice clearly indicated that deregulation of FABP4 function may lead to the development of severe diseases such as diabetes II type and atherosclerosis. In this study, we report the production and detailed characterization of monoclonal antibodies (MAbs) against FABP4. Recombinant glutathione S-transferase (GST)-FABP4 or His-FABP4 was expressed in bacteria, affinity purified, and used for immunization of mice, enzyme-linked immunosorbent assay (ELISA) screening, and characterization of selected clones. We have isolated two hybridoma clones that produced antibodies specific for recombinant and native FABP4, as shown by Western blotting and immunoprecipitation. The specificity of generated antibodies was further tested in a cell-based model of adipogenesis. In this analysis, the accumulation of FABP4 during NIH 3T3-L1 differentiation into adipocytes was detected by generated antibodies, which correlates well with previously published data. Taken together, we produced MAbs that will be useful for the scientific community working on fatty acid-binding proteins and lipid metabolism.     

Lobe specific effects of testosterone and estrogen on 3,2'-dimethyl-4-aminobiphenyl-induced rat prostate carcinogenesis

We have previously shown that chronic administration of a pharmacological dose of testosterone propionate (TP) after treatment with the carcinogen, 3,2'-dimethyl-4-aminobiphenyl (DMAB), results in development of invasive and metastatic adenocarcinomas arising from the dorso-lateral and anterior prostate, as well as the seminal vesicles. Co-administration of ethinyl estradiol (EE) with TP increased the yield of carcinomas in the lateral and anterior lobes. In the present experiment, male F344 rats were treated with DMAB for 20 weeks and then co-administered a pharmacological dose of TP together with various doses of EE for 40 weeks. Without hormone(s) administration, carcinomas were confined to the ventral prostate and all were of intra-acinar type. TP administration suppressed development of the ventral prostate carcinomas but caused invasive carcinomas of the lateral and anterior lobes and of seminal vesicles and intra-acinar carcinomas in the dorsal prostate. The appearance of carcinomas in the lateral and anterior prostate was increased by co-administration of EE in a dose-related fashion but carcinomas of the seminal vesicles were inversely reduced. The suppressive influence of TP on ventral carcinoma development was overcome by only the highest dose of EE. It is concluded that estrogen can modify the enhancing effects of TP on induction of rat prostate and seminal vesicle carcinomas in a dose-related fashion with lobe specificity.     

Preparation and preliminary characterization of rabbit monoclonal antibodies against human midkine

We prepared rabbit monoclonal antibodies that target human midkine (MK). The MK gene was amplified by PCR from the plasmid pEGFP-MK and subcloned into the prokaryotic expression vector pGEX-1λT to generate an N-terminally glutathione S-transferase (GST)-tagged fusion protein construct. Expression of the GST-MK fusion protein was achieved by IPTG induction in Escherichia coli cells. The expressed protein was purified using the GST system. After verifying purification, the fusion protein was used to immunize rabbits to prepare monoclonal antibodies against human MK by the rabbit hybridoma technique. The hybridomas generated were screened by an enzyme-link immunoassay (ELISA) for specificity, which was further characterized by Western blotting and ELISA. SDS-PAGE analysis showed that the purified protein corresponds to the calculated molecular weight. The GST-MK fusion protein was prepared. At least one hybridoma cell line secreting anti-MK MAb was obtained. Western blotting analysis confirmed the identity of the MAb. The titer of the MAbs measured by an indirect ELISA was 1:64,000. The affinity constant, which was measured by a non-competitive ELISA, was found to be 3.0?×?10(9) M(-1). Western blotting and immunohistochemistry analysis showed that the produced MAbs bind to the MK protein in cancerous tissues. The GST-MK fusion protein was successfully expressed and purified. The MAbs against MK were subsequently prepared, which should further aid research and the application of MK MAbs in clinical settings.     

Lack of modification by naturally occurring antioxidants of 3,2'-dimethyl-4-aminobiphenyl-initiated rat prostate carcinogenesis

The modifying effects of 6 naturally occurring antioxidants on 3,2'-dimethyl-4-aminobiphenyl (DMAB)-initiated rat prostate carcinogenesis were investigated in male F344 rats. Animals were pretreated with DMAB in a 20-week initiation protocol and then administered basal diet containing 0.8% catechol, 0.8% resorcinol, 0.8% hydroquinone, 2 ppm selenium, 2% gamma-orysanol or 1% alpha-tocopherol for 40 weeks. The experiment was terminated at week 60 for histopathological assessment of lesion development. Atypical hyperplasias and carcinomas of the prostate were observed in the ventral lobe in all groups treated with DMAB. However, the incidences of these lesions were not significantly different between carcinogen control and antioxidant-treated groups. There were also no significant increases or decreases in the incidences of tumors in any other organs.     

Formation of DNA adducts in vivo in rat liver and intestinal epithelium after administration of the carcinogen 3,2'-dimethyl-4-aminobiphenyl and its hydroxamic acid

Administration of the ³H-labeled colon carcinogen, 3,2'-dimethyl-4-aminobiphenyl(DMABP) and its hydroxamic acid derivative, N-hydroxy-N-acetyl-DMABP,to male F344 rats resulted in high levels of covalent bindingto hepatic and intestinal DNA, RNA and protein. For both compounds,binding to hepatic macromolecules was 2–4 times higherthan in the intestine. High pressure liquid chromatographicanalysis of the enzymatically hydrolyzed DNA from liver andintestinal epithelium indicated the presence of two cardnogen-DNAadducts: 5-(deoxyguanosin-N²-yl)-DMABP (15%), N-(deoxyguanosin-8-yl)-DMABP(50%), and a decomposition product of the latter (15%). N-acetylatedadducts were not detected. When measured after 7 days, all adductsin the intestinal DNA had decreased by 70%, while only a 29%decrease had occurred in the hepatic DNA. To determine if theloss of DMABP products was a consequence of cell turnover orrepair, rats were treated wtih [³H]thymidine and DMABP, andthe specific activity of hepatic liver and intestinal DNA wasmeasured. Between 1 and 7 days only a slight decrease in [³H]thymidinecontent occurred in hepatic DNA as compared with a 95% reductionin intestinal DNA. Thus, the higher rate of DNA synthesis inthe intestine versus that in the liver may serve to promotefixation of the initiating lesion and account for the preferentialinduction of intestinal cancer by DMABP. Furthermore, comparisonof these data with metabolic activation pathways reported earlierstrongly suggest that N-hydroxy-DMABP is the proximate carcinogenicmetabolite of both DMABP and N-hydroxy-N-acetyl-DMABP.     

Low tumorigenic response to 3,2'-dimethyl-4-aminobiphenyl administration in the prostate of rats castrated at birth

Six-week-old rats which had been orchiectomized at birth were given 3,2'-dimethyl-4-aminobiphenyl (DMAB) at various doses combined with a stimulus to prostate epithelial cell proliferation in the form of oral administration of methyltestosterone (MT) for 4 weeks. Thereafter MT treatment was continued or the animals received subcutaneous implants of testosterone propionate (TP) and were maintained until sacrifice at week 60. Although prostatitis and prostatic enlargement were frequently observed, especially in the TP group, numbers of atypical hyperplastic lesions were low and only one prostatic carcinoma in situ developed. Thus, despite the presence of proliferation, castration brought about a significant reduction in susceptibility to DMAB.     

Dietary supplementation with silymarin inhibits 3,2'-dimethyl-4-aminobiphenyl-induced prostate carcinogenesis in male F344 rats.

PURPOSE: Silymarin has been shown to be a potent anticarcinogenic agent. Here, we investigated the modifying effects of dietary feeding with a naturally occurring polyphenolic antioxidant flavonoid silymarin on 3,2'-dimethyl-4-aminobiphenyl (DMAB)-induced prostatic carcinogenesis in male F344 rats. EXPERIMENTAL DESIGN: Male F344 rats were given s.c. injections of DMAB (25 mg/kg body weight) every other week for 20 weeks. They also received the experimental diet containing 100 or 500 ppm silymarin for 40 weeks, starting 1 week after the last dosing of DMAB. All of the rats were sacrificed 60 weeks after the start of the experiment. Histopathology and immunohistochemistry for proliferative cell nuclear antigen, cyclin D1, and apoptotic indices were done in the prostatic lesions, including invasive adenocarcinomas, intraepithelial neoplasms, and nonlesional glands. RESULTS: Dietary feeding with 500 ppm silymarin significantly inhibited the incidence of prostatic adenocarcinoma when compared with the DMAB-alone group (17.6% versus 50.0%, P < 0.05). The proliferative cell nuclear antigen- and cyclin D1-positive indices in adenocarcinomas, prostatic intraepithelial neoplasm, and nonlesional glands in rats treated with DMAB and silymarin were slightly lower than that of the DMAB-alone group. Also, dietary administration of silymarin increased apoptotic index in prostatic adenocarcinoma by measuring immunohistochemically positive nuclei for ssDNA. CONCLUSIONS: Our results indicate that silymarin exerts chemopreventive ability against chemically induced prostatic carcinogenesis through apoptosis induction and modification of cell proliferation.     

Preparation and characterization of monoclonal antibodies against mouse Jab1/CSN5 protein

Jab1, also known as the fifth component of the COP9 signalosome complex (CSN5), directly interacts with and regulates the activity and stability of multiple intracellular regulatory molecules, such as c-Jun, p27, p53, Cullin, Smad4, and HIF1alpha. In addition, a high level of Jab1 is observed in a variety of human cancers and is sometimes correlated with a poor prognosis, suggesting that Jab1 contributes to cancer cell proliferation and survival and could be a novel target of cancer therapy. In this report, we generated five mouse monoclonal antibodies to a bacterially produced recombinant mouse Jab1 protein and examined their capabilities and limitations in commonly used assays, including enzyme linked immunosorbent assay (ELISA), Western blotting with denatured and native polyacrylamide gel electrophoresis (PAGE), immunoprecipitation, and immunofluorescence microscopy, finding the most suitable antibody for each application. Because these antibodies proved useful for immunohistochemical staining for Jab1 in fixed sections of human cancer samples, they should be useful in determining the expression and subcellular distribution of Jab1 in human tumors.     

Preparation and characterization of monoclonal antibodies against placental alkaline phosphatase and other human trophoblast-associated determinants

We have prepared monoclonal antibodies by immunizing BALB/c mice with purified human term placental plasma membranes. The antibodies were selected to show predominant specificity for trophoblast and trophoblast derivatives. Four of these antibodies have been found to recognize the placenta-specific isozyme of alkaline phosphatase (EC 3.1.3.1), and to cross-react with the closely related testis form of this enzyme. One antibody recognized transferrin, a serum protein with an abundant placental receptor. The specificities of the other antibodies, whose target antigens are unknown, are described. Their reactivity with some human tumour-derived epithelial cell lines suggests that they may provide useful markers of human trophectoderm differentiation, as well as for properties selected for during tumour progression.     

Effect of dietary wheat bran and dehydrated citrus fiber on 3,2'-dimethyl-4-aminobiphenyl-induced intestinal carcinogenesis in F344 rats

The effect of dietary wheat bran and dehydrated citrus fiberon 3,2'-dimethyl-4-aminobiphenyl (DMAB)-induced colon and smallintestinal carcinogenesis was studied in male F344 rats. Weanlingrats were fed semipurified diets containing 5% alphacel, 5%alphacel + 15% wheat bran or 5% alphacel + 15% citrus fiber.At 7 weeks of age, all animals, except vehicle-treated controls,received weekly s.c. injections of 50 mg DMAB/kg body weightfor 20 weeks. The DMAB- or vehicle-treated groups were autopsied20 weeks after the last injection of DMAB. The animals fed thewheat bran diet and treated with DMAB had a lower incidence(number of animals with tumors) and multiplicity (number oftumors/tumor-bearing animal) of colon and small intestinal tumorsthan did those fed the control diet and treated with DMAB. Animalsfed the diet containing citrus fiber developed fewer small intestinaltumors (incidence and multiplicity) than did the rats fed thecontrol diet; the number of adenocarcinomas was reduced in ratsfed the citrus fiber diet. This study thus indicates that dietscontaining wheat bran and citrus fiber reduce the risk for DMAB-inducedintestinal cancer and that the protection against colon cancerdepends on the type of fiber.     

Selective Induction of Prostate Carcinomas in F344 Rats Treated with Intraperitoneal Injections of N-Hydroxy-3,2'-dimethyl-4-aminobiphenyl

Groups of F344 and Wistar rats were given an intraperitoneal injection of N-hydroxy-3,2'-dimethyl-4-aminobiphenyl (N-OH-DMAB) at a dose of 5 mg/kg body weight with a 1-week dietary pretreatment with ethinyl estradiol (EE), and this regimen was repeated 10 times at one-week intervals. Additional groups were given N-OH-DMAB 10 times without the dietary EE pretreatment. The total experimental period was 60 weeks. Carcinomas and atypical hyperplasias of the prostate developed in 8 (42%) and 16 (84%) of 19 F344 rats without the dietary EE treatment and in 1 (6%) and 7 (39%) of 18 rats with the EE diet, respectively. No prostatic tumors were found in Wistar rats, although atypical hyperplasias were observed in 6 of 18 rats with and 4 of 8 rats without the EE supplementation. Tumor yields in other organs were extremely low, resulting in good survival of the animals. A comparison of the results with those obtained for DMAB suggests that intraperitoneal administration of N-OH-DMAB in F344 provides a better induction method for models of prostate carcinogenesis.     

Effect of dietary corn bran and autohydrolyzed lignin on 3,2'-dimethyl-4-aminobiphenyl-induced intestinal carcinogenesis in male F344 rats

The effect of dietary corn bran and autohydrolyzed lignin on 3,2'-dimethyl-4-aminobiphenyl (DMAB)-induced intestinal carcinogenesis was studied in male inbred F344 rats. Groups of weanling rats were fed semipurified diets containing 15% corn bran or 7.5% lignin or a semipurified diet without these fibers (control diet). At 7 weeks of age, all animals, except vehicle-treated controls, were given sc injections of 50 mg DMAB/kg body weight/week for 20 weeks. All animals were autopsied 20 weeks after the last injection of DMAB. The incidence of colon tumors (percentage of animals with tumors) and colon tumor multiplicity (tumors/animal) were increased in rats fed the corn bran diet as compared to the tumor incidence and multiplicity in rats fed the control diet. The incidence of small intestinal tumors was slightly lower in rats fed the corn bran diet as compared to the incidence in rats fed the control diet. The concentrations (mg/g dry feces) of fecal deoxycholic acid and total bile acids and the daily output of fecal deoxycholic acid, lithocholic acid, hyodeoxycholic acid, and total bile acids were increased in rats fed the corn bran diet as compared to the concentrations and daily output in rats fed the control diet. The incidence and multiplicity of small intestinal tumors as well as the number of colon adenocarcinomas per tumor-bearing animal were lower in animals fed the lignin diet than in those fed the control diet. Lignin had no effect on the concentrations of fecal bile acids, but the daily output of total bile acids was increased in animals fed the lignin diet as compared to the daily output in rats fed the control diet. This study thus indicates that the protection against colon cancer depends on the type of fiber.     

Immunohistochemical demonstration of carcinogen-DNA adducts in target and non-target tissues of rats given a prostate carcinogen, 3,2'-dimethyl-4-aminobiphenyl

An immunohistochemical procedure was applied which allows accurate localization of DNA lesions within organs and tissues of rats given 3,2'-dimethyl-4-aminobiphenyl (DMAB) using polyclonal antibodies against DMAB-DNA adducts. Dose-related nuclear staining was observed in organs regardless of DMAB-carcinogenic organotropism. In the male accessory sex organs, the lateral lobe of the prostate, a non-target site, demonstrated a similar staining intensity to that found for the ventral prostate and seminal vesicle, target sites. Orchiectomy and pretreatment with ethinyl estradiol resulted in a moderate to slight decrease in binding in the accessory sex organs. No observable decrease in staining intensity was evident in most organs 168 h after the administration of DMAB. These findings suggest that DNA adduct formation itself is not necessarily sufficient for tumor induction.     

Preparation and characterization of neutralizing monoclonal antibodies against FMDV serotype O with synthetic peptide antigen

A short linear peptide was designed according to the antigenic site analysis of VP1 protein of foot-and-mouth virus (FMDV) serotype O and synthesized as the peptide immunogen. The peptide, which covers the region from amino acid 133 to 160 of VP1 of FMDV, was linked to the N-terminal cysteine and conjugated with the carrier protein of keyhole limpet hemocyanin (KLH). Normal 6- to 8-week-old BALB/c mice were immunized with the 20?μg dose conjugated peptide antigen four times. The splenocytes from the immunized mice were fused with SP2/0 mouse myeloma cells, and positive hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA) and subcloned four times with limiting dilution. Five stable hybridoma cell lines, designated as 4F9, 1B11, 1E10, 1D4, and 4B8, were obtained. Isotyping of all obtained MAbs indicated that the MAbs of 4F9, 1E10, and 4B8 belonged to IgG2b; the 1B11 and 1D4 belonged to IgG1 and IgM, respectively. The micro-neutralization test indicated that the MAbs of 4F9, 4B8, and 1B11 were capable of neutralizing FMDV serotype O with neutralization indices ranging from 1.81 to 2.11. These results suggest that linear synthetic peptide conjugate can elicit antibodies against native FMDV virus and can be used as an alternative immunogen for production of MAbs with exact epitope.     

Preparation and identification of monoclonal antibodies against daintain

Daintain is a 17-kDa polypeptide originally purified from porcine intestine. This polypeptide is associated with insulin secretion and inflammatory responses. Daintain is highly similar in amino acid sequence to allograft inflammatory factor-1 (AIF-1). Here we report the preparation and identification of monoclonal antibodies (MAbs) against daintain. To enhance its immunogenicity, daintain was coupled to carrier protein bovine serum albumin (BSA) by a two-step glutaraldehyde method. Using conventional procedures, we obtained four stable hybridoma cell lines that can produce and secret anti-daintain MAbs. We further analyzed their isotypes, titer, and affinity, and found that those MAbs belong to the G1 subclass with kappa light chains. The MAbs were capable of recognizing daintain as determined by Western blotting. The produced MAbs will be a useful tool for further investigation of daintain functions in organisms.     

Selective induction of prostate carcinomas in F344 rats treated with intraperitoneal injections of N-hydroxy-3,2'-dimethyl-4-aminobiphenyl.

Groups of F344 and Wistar rats were given an intraperitoneal injection of N-hydroxy-3,2'-dimethyl-4-aminobiphenyl (N-OH-DMAB) at a dose of 5 mg/kg body weight with a 1-week dietary pretreatment with ethinyl estradiol (EE), and this regimen was repeated 10 times at one-week intervals. Additional groups were given N-OH-DMAB 10 times without the dietary EE pretreatment. The total experimental period was 60 weeks. Carcinomas and atypical hyperplasias of the prostate developed in 8 (42%) and 16 (84%) of 19 F344 rats without the dietary EE treatment and in 1 (6%) and 7 (39%) of 18 rats with the EE diet, respectively. No prostatic tumors were found in Wistar rats, although atypical hyperplasias were observed in 6 of 18 rats with and 4 of 8 rats without the EE supplementation. Tumor yields in other organs were extremely low, resulting in good survival of the animals. A comparison of the results with those obtained for DMAB suggests that intraperitoneal administration of N-OH-DMAB in F344 provides a better induction method for models of prostate carcinogenesis.