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Background: Obesity increases risk for postmenopausal breast cancer, and is associated with elevated blood levels of inflammation-related marker C-reactive protein (CRP), insulin, glucose, and the angiogenesis-related biomarker vascular endothelial growth factor (VEGF). The long-term effect of weight loss on these breast cancer risk biomarkers is unknown. This study aim was to test the 30-month effect of weight loss on breast cancer biomarkers in postmenopausal overweight/obese women, who had completed a randomized control trial test of behavioral weight loss and/or exercise compared to control. Methods: From 1/2005-7/2008, women (N=438) were randomized to 1 of 4 arms: reduced calorie weight loss diet (D; N=118); moderate intensity aerobic exercise (E; 225 minutes/week) (N=117); both interventions (D+E; N=116); or control (C, no intervention; N=87). The D intervention goals were: 1200-2000 calories/day, dietary fat < 30% of calories, and > 10% loss of initial weight. The E goal was > 45 minutes of moderate-intensity exercise 5 days/week. The D+E interventions were identical to those for D and E. A total of 157 women returned for a 30-month blood draw (18 months after end-of-study). Analysis: We compared the average changes in outcomes from baseline to 30 months in weight loss (combining D and D+E groups) vs. no weight loss (combining C and E groups), according to the intention-to-treat principle, using generalized estimating equations (GEE). As a secondary, pre-planned analysis, we combined data for all women regardless of randomization group, and divided them into four categories based on change from baseline to 30 months: 1) gained weight or remained weight stable; 2) lost < 5% of baseline weight; 3) lost 5% - 10% of baseline weight. We then compared 30-month analyte changes by these 30-month weight loss groups, with category 1 as referent. Results: Mean 30-month weight changes from baseline were: D+E, - 7.7%; D, - 6.3%; E, -1.9%; and C, -3%. CRP was 22.5% lower than baseline for women randomized to a diet group, compared with a 4% reduction from baseline in women in a non-diet group (p=0.07). Insulin was 33% lower in women randomized to diet, while it was 20% lower in women not randomized to a diet group (p=0.10). Women who lost 5% - 10% had a 56% lower CRP level (p 10% (p 10% lower than baseline, respectively. Glucose levels increased in women whose weight loss was < 5%, but remained stable in those whose 30-month weight was > 5% lower than baseline (each p=0.04). Conclusions: Long-term weight loss of at least 5% may have biological effects relevant to breast cancer prevention in overweight or obese postmenopausal women. Citation Format: McTiernan A, Duggan C, Tapsoba JdD, Mason C, Wang C-Y. Long-term effects of weight loss on breast cancer biomarkers in postmenopausal women [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-13-02.

Background: Random periareolar fine-needle aspiration (RPFNA) is a research-based procedure used to obtain breast epithelial cells and fluid. Data suggest that atypia in epithelial cells from RPFNA specimens is associated with an increased risk of breast cancer, and changes in the degree of atypia may be a viable biomarker for response to chemopreventive interventions. Methods: We performed RPFNA on 37 overweight or obese (body mass index (BMI≥ 25.0 kg/m2) postmenopausal (50-75 y) women enrolled in a randomized clinical trial of Vitamin D vs placebo in combination with a lifestyle (diet + exercise)-based weight loss program. Eligibility criteria included serum vitamin D concentrations 10–32 ng/mL. The procedure was performed by two trained clinicians prior to randomization to assess baseline cell counts and degree of atypia. Two areas in the breast (10:00 and 2:00 positions) were infiltrated with lidocaine, and 8 to 10 aspirations of cellular material were performed using 1.5 inch, 21-gauge needles. The aspirated fluid was placed in 9 cc of Cytolyt Thinprep (Hologic Inc.) with 1 cc of 1% neutral buffered formalin and slides were prepared according to the University of Washington Department of Pathology non-gyn protocol. Results: Thirty-seven women were included in this analysis. The mean age and BMI of study participants was 59.3 years and 32.5 kg/m2, respectively. The mean 5-year Gail risk score for study participants was 1.96%; 52% of women had a 5-year Gail risk >1.66%. Eight participants (21.6%) had >50 cells present on cytology examination. One additional participant had 10–50 cells, and one had Conclusions: RPFNA in our sample of overweight/obese postmenopausal women did not yield epithelial cells in a majority of participants. Younger age was associated with a greater cell count. We are planning further studies to determine whether other markers in RPFNA specimens can be used to assess breast tissue changes in response to chemopreventive interventions. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-11-06.

VACO-330, a nontransformed cell line established from a human colon adenoma, undergoes spontaneous apoptosis and shedding of cells into the culture medium. Shed cells were shown to be apoptotic, both by nuclear morphology and by generation of a typical "laddered" pattern of degraded DNA. Quantitation of DNA released into the medium, compared with the amount retained on the plate, demonstrated that 6.2 +/- 1.1% of the total cell mass underwent apoptotic death daily. The addition of transforming growth factor beta (20 ng/ml) accelerated this spontaneous apoptotic rate 3.2-fold. Moreover, apoptosis could be rapidly induced in up to 45% of the VACO-330 cells by using brief exposure to a calcium chelating medium to release the cells from the substratum. We suggest that transforming growth factor beta is a likely physiological regulator of apoptosis during maturation of the colonic epithelial cells. We additionally suggest the existence of an alternate pathway, which at the time of shedding from the crypt induces apoptosis in colonic epithelial cells that have escaped earlier apoptotic signals.

Biomarkers of low-grade systemic inflammation are used to study the associations of inflammation with chronic diseases, including cancers. However, little is known about the reliability (i.e., intraindividual variability) of most of these measures. Fasting (12-hour) serum samples, which were collected at the end of ≥3 week washout periods in a 4-diet crossover feeding trial, were used to measure the inflammatory markers hsCRP, IL-6, TNF-α, IL-8 and sTNFRI and II. Participants included 32 men and 31 women (5 men and 2 women were missing from analyses of IL-8, TNF-α, and sTNFR I and II), aged 20-40, who were free of factors known to influence inflammation, e.g., chronic disease, medication use, heavy alcohol use, smoking and obesity (body mass index >30 kg/m2). Intraclass correlations (ICC) were estimated using random effects analysis of variance, across all 4 time points (∼5 weeks apart). For TNF-α and sTNFRI & II, the ICCs were very high (ICC= 0.92, 95% CI: 0.89 - 0.96; 0.92, 95% CI: 0.88 - 0.95; and 0.90, 95% CI: 0.85 - 0.94, respectively). The ICCs for IL-8 and hsCRP were 0.73 (95% CI: 0.63-0.83 and 0.68 (95% CI: 0.58-0.78), respectively. The ICC for IL-6 was considerably lower, (ICC = 0.48, 95% CI: 0.36-0.62). With the exception of IL-6, reliability of all inflammatory markers in our panel over 4 time points was high, suggesting that a single measure accurately captures the variability within an individual and is a good measure for use in epidemiologic studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5486. doi:1538-7445.AM2012-5486

The comparative carcinogenicities of N-hydroxy-N-acetyl-1-aminopyrene, N-acetyl-1-aminopyrene, and 1-, 2-, and 4-nitropyrene were determined following i.p. injection into weaning female CD rats (67 mumol/kg body weight in dimethyl sulfoxide; 3 times/week for 4 weeks). At sacrifice 61 weeks after the first injection the incidences of malignant mammary tumors were increased significantly to 45 and 24% in the 4-nitropyrene- and N-hydroxy-N-acetyl-2-aminofluorene-treated groups, respectively. Cellular altered foci in the liver were increased significantly in the N-acetyl-1-aminopyrene-, N-hydroxy-N-acetyl-1-aminopyrene-, and N-hydroxy-N-acetyl-2-aminofluorene- treated groups; the latter two compounds also led to significantly increased formation of hyperplastic nodules in this organ. Significant increases in leukemia induction were observed in animals treated with 2-nitropyrene or N-hydroxy-N-acetyl-2-aminofluorene. In an experiment designed to compare the influence of the route of administration on the carcinogenic potential of this agent, 1-nitropyrene was injected i.p. or s.c. into weanling female CD rats (100 mumol/kg body weight; once a week for 4 weeks). The animals were sacrificed at 87 to 90 weeks after the first treatment. The incidences of mammary gland tumors in animals receiving injections of 1-nitropyrene by either route (59%) were significantly higher than in solvent-injected controls (37%). The incidences of adenocarcinoma in the i.p. 1-nitropyrene group (28%) and fibroadenoma in the s.c. 1-nitropyrene group (52%) were significantly higher than in the control animals (7 and 27%, respectively). These data suggest that the demonstration of the weak carcinogenicity of 1-nitropyrene is probably more a function of the length of the observation period than of the routes of administration used here. A further exploration of the effect of the route of administration involved treatment of weanling female CD rats by direct injection of 1-, 2-, or 4-nitropyrene into the mammary fat pads. A total of 2.04 mumol of the nitrocompound in dimethyl sulfoxide was injected into the mammary glands under each of the 6 left nipples. The right mammary glands were treated with the solvent only. Injections of the thoracic nipple areas were carried out on day 1; inguinal areas were treated on day 2. The animals were sacrificed after 77 weeks. The number of mammary tumor-bearing animals (23 of 28), the number with fibroadenoma (15 of 28), and the number with adenocarcinoma (19 of 28) were significantly increased in the 4-nitropyrene-treated group as compared with animals treated with only dimethyl sulfoxide.(ABSTRACT TRUNCATED AT 400 WORDS)

Male Fischer rats which had been implanted with a heterotopic bladder were randomly divided into five groups and their heterotopic bladders were instilled once a week for 20 weeks with 0.5 ml phosphate-buffered saline:dimethyl sulfoxide solution (4:1) or this solution containing 1 mumol benzidine (BZ), N'-hydroxy-N-acetylbenzidine, the N'-glucuronide of N'-hydroxy-N-acetylbenzidine, or the N-glucuronide of N-hydroxy-2-aminofluorene. These bladders were then instilled once a week for an additional 30 weeks with the phosphate-buffered saline without dimethyl sulfoxide. The experiment was terminated at the end of 50 weeks. Transitional cell carcinomas were observed in 1 of 39 (control), 1 of 29 (BZ), 18 of 30 (N'-hydroxy-N-acetylbenzidine), 28 of 28 (N'-hydroxy-N-acetylbenzidine N'-glucuronide), and 24 of 29 (N-hydroxy-2-aminofluorene N-glucuronide) rats. No histological alterations were observed in their natural bladders. These results demonstrate the urothelial carcinogenicity of the N-hydroxy metabolites of BZ and suggest that N'-hydroxy-N-acetylbenzidine N'-glucuronide may play a major role in the initiation of urothelial carcinogenesis by BZ in humans.

The objective of this study was to investigate the induction of liver tumors by arylhydroxamic acids. The potential involvement of sulfate conjugation was minimized by the administration of a N-hydroxy-4-acylaminobiphenyl to female CD rats. This experimental design provided for the exposure of a target organ that has only a low capacity for activation of hydroxamic acids by sulfate conjugation, with a carcinogen that does not induce tumors in liver that possess a high sulfotransferase activity. A single dose of the N-formyl or N-acetyl derivatives of N-hydroxy-4-aminobiphenyl was given i.p. at 0.4 mmol/kg body weight to 34-day-old animals. In attempts to amplify the hepatocarcinogenic potential of the compounds, partial hepatectomy 24 hr before the chemical injection and subsequent long-term treatment with phenobarbital in the diet were carried out. For comparative purposes, other animals were subjected to three additional partial hepatectomies subsequent to the carcinogen administration instead of the phenobarbital treatment. The experiments were terminated 64 weeks after injection. Both the N-formyl and N-acetyl derivatives of N-hydroxy-4-aminobiphenyl, in conjunction with partial hepatectomy and subsequent treatment of dietary phenobarbital, induced a high incidence of neoplastic nodules and gamma-glutamyltranspeptidase-positive foci in the liver. Only one hepatocellular carcinoma was observed in each treatment group. Repeated partial hepatectomies enhanced the yield of gamma-glutamyltranspeptidase-positive foci but were ineffective in producing neoplastic nodules. In addition to the liver lesions, mammary tumors were also induced. Importantly, an inhibitory effect of the subsequent administration of phenobarbital was observed on mammary tumor formation, possibly because of alterations in hormone metabolism resulting from the induction of microsomal enzymes by phenobarbital, which resulted in a decreased promoting effect. There was no difference in the tumorigenicity of the formyl and acetyl derivatives in these experiments.

1-Nitropyrene, a direct-acting mutagenic component of extracts of diesel exhaust and ambient air particulates, is one of the major nitroarenes found in products of incomplete combustion. Injection of this compound into newborn Sprague-Dawley-derived CD rats at 100 mumol/kg body weight s.c. once a week for 8 weeks induced 32 and 28% sarcomas, primarily malignant fibrous histiocytomas, at the site of injection in male and female animals, respectively. Mammary tumors, most of which were adenocarcinomas, developed distant from the site of injection in 47% of the females. Similar but lesser responses were observed in animals given one-half this dose. This carcinogenic response is consistent with the systemic effects of structurally related aromatic amines. 4-Nitrobiphenyl, given at equimolar doses, was not tumorigenic in this study.

Unscheduled DNA synthesis (UDS)-inducing activity was used as a parameter to estimate the abilities of rat mammary epithelial cells and urothelial cells from various species to activate carcinogenic aromatic amine derivatives. The N-hydroxy, N-hydroxy-N-acetyl, N-hydroxy-N-glucuronosyl derivatives of 2-aminofluorene (2-AF) and 4-aminobiphenyl (4-ABP) induced UDS in primary cultures of rat mammary epithelial cells, but 2-AF, the O-glucuronide of N-hydroxy-N-acetyl-2-AF (N-OH-AAF) and 4-ABP did not. Neither the activity of N-OH-AAF, N-hydroxy-N-formyl-2-AF, nor N-acetoxy-N-acetyl-2-AF was significantly altered by paraoxon, an inhibitor of microsomal N-deacetylase. Although N-hydroxy-3,2'-dimethyl-4-aminobiphenyl (N-OH-DMABP) also induced UDS, its N-acetyl derivative, which can not be activated by intramolecular, N,O-acetyltransfer, did not. Similarly, rat urothelial cells were responsive to the UDS-inducing activity of this hydroxylamine, but not the hydroxamic acid. In contrast, dog urothelial cells were responsive to both compounds. The UDS-inducing activity of N-OH-AAF was inhibited by paraoxon in the dog, but not in rat urothelial cells. N-Hydroxy-N,N'-diacetylbenzidine induced UDS in the urothelial cells of dog, rat, and rabbit, and a human urothelial cell line, HCV-29, whereas benzidine, N-acetylbenzidine, and N,N'-diacetylbenzidine did not. Co-treatment with 12-O-tetradecanoylphorbol-13-acetate did not enable benzidine to induce UDS in dog urothelial cells. Rat mammary epithelial cells activated N-OH-DMABP by acetyl coenzyme A-dependent O-acetylation and N-OH-AAF by N,O-acetyltransfer. They could not N-deacetylate N-OH-AAF. These results suggest that rat mammary and bladder epithelial cells are capable of activating N-arylhydroxylamine metabolites of these carcinogens, probably by N,O-acetyltransfer and O-acetylation, whereas dog urothelial cells are more likely to activate these metabolites by N-deacetylation and a reaction that has yet to be identified.

The induction of unscheduled DNA synthesis in primary cultures of urothelial cells from rats, mice, and dogs by nitrofurans and arylamines and their derivatives was examined. Electron microscopy of the culture from rat cells showed the characteristics of epithelial cells. The cultured cells were exposed to the test chemicals in a serum-free medium containing 10 mM hydroxyurea and [3H]thymidine for 24 hr, and unscheduled DNA synthesis was determined by autoradiography. N-Hydroxy-2-aminofluorene, N-hydroxy-2-acetylaminofluorene, N-hydroxy-4-amino-biphenyl, and N-hydroxy-4-acetylaminobiphenyl were active in inducing unscheduled DNA synthesis in the urothelial cells of rats, mice, and dogs. 2-Aminofluorene, N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide, and 2-amino-4-(5-nitro-2-furyl)thiazole were active only in dog cells. 2-Acetylaminofluorene and 4-aminobiphenyl were not active. These results demonstrate that urothelial cells contain enzymes that can selectively activate bladder carcinogens and their metabolites.

Promoting activities of sodium phenobarbital (PB) and sodium saccharin (SS), incorporated in a semisynthetic diet (AIN-76A), on 2-stage carcinogenesis initiated with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) or N,N-dibutylnitrosamine (DBN) in male F344 rats were investigated. For the first 4 weeks of the experiment, weanling male Fischer rats were fed Wayne diet containing 0.2% FANFT or drinking water containing 0.005% DBN. The control rats were given the basal diet and normal drinking water. Beginning at the fifth week, the rats were given the AIN-76A diet or this diet containing 0.05 or 0.15% PB or 5% SS. The experiment was terminated at the end of 100 weeks. PB significantly increased the incidence of transitional cell carcinoma of the bladder of the rats that had been treated with FANFT (P = 0.027). PB also increased the incidence of bladder carcinoma of the rats that had been treated with DBN, but the increase was not significant (P = 0.081). SS in the AIN-76A diet increased the incidence of bladder carcinoma in the rats which had been treated with FANFT or DBN, but the increase was not significant (P = 0.059 and 0.327, respectively). Both high and low doses of PB, but not SS, significantly increased the incidence of hepatocellular carcinoma in the rats that had been treated with DBN. None of the control rats that had been fed the basal diet or the basal diet containing low or high PB or 5% SS developed either bladder or liver carcinoma. These results demonstrate that PB promotes urinary bladder carcinogenesis of rats initiated with FANFT but not with DBN. In contrast to incorporation in commercial rat chows, SS incorporated in the AIN-76A diet is very weak in promoting bladder carcinogenesis. On the other hand, PB, but not SS, promotes hepatocarcinogenesis initiated with DBN. Neither PB nor SS promoted DBN-induced carcinogenesis of esophagus or forestomach.

Thirty-two heterocyclic compounds, including 24 nitroheterocycles, 7 aminoheterocycles and derivatives, and 1 thiophene lacking a nitro group, were tested for mutagenic activity in Salmonella typhimurium TA 98 and TA 100. All the nitroheterocycles (11 new), including nitrofurans, nitrothiophenes, nitropyrroles, and 1 nitroimidazole, were mutagenic in TA 100; 13 were also mutagenic in TA 98. 5-Nitro-2-furoic acid, a noncarcinogen, was mutagenic in TA 100. Seven carcinogenic nitroheterocycles were mutagenic in both strains. Seven aminoheterocycles (4 new), aminothiophenes and aminothiazole derivatives, and 1 thiophene without a nitro group were not mutagenic. Both TA 98 and TA 100 were uvrB and lacked the ability of excision repair of DNA. Among the 24 mutagenic nitroheterocycles, only 13 compounds exhibited bacterial killing effects, suggesting that more than 1 mechanism may be involved in the interaction of nitroheterocycles with bacterial DNA.

A murine monoclonal antibody (anti-BL7) was raised by immunization of BALB/c mice with a precursor B-cell line (Josh-7) which detects a heat-stable, nonimmunoprecipitable antigen. The expression of BL7 was investigated in peripheral blood and/or bone marrow leukemic cell suspensions stained by indirect immunofluorescence and analyzed by flow cytometry. Lymphoblasts from 43 of 43 cases of "null" acute lymphoblastic leukemia were BL7-. Five cases of T-acute lymphoblastic leukemia and 5 cases of terminal deoxynucleotidyl transferase-positive blastic chronic myelogenous leukemia were also BL7-. All 63 cases of B-cell chronic lymphocytic leukemia were BL7+. Neoplastic cells in 22 of 28 cases of B-cell non-Hodgkin's lymphomas in leukemic phase were also BL7+. Expression of BL7 showed some correlation with Rappaport's histological classification. Four cases of multiple myeloma and plasma cell leukemia were BL7-. Twenty-three cases of acute nonlymphocytic leukemias were also analyzed. Of these, only the acute promyelocytic (M3,4 cases) and acute myelomonocytic (M4, one case) varieties expressed BL7 on a small proportion (approximately 15%) of the leukemic cells. All other subgroups were BL7-. The reactivity of anti-BL7 was compared to other B-cell antibodies on selected samples and was shown to be different from B1, B2, and the BA antibodies. Anti-BL7 is a unique monoclonal antibody useful in the study of B-cell cancers.

To investigate the binding of a nitrofuran to tissue macromolecules in vivo, the urinary bladder carcinogen N-[4-(5-nitro-2-furyl)- 2-[35S]thiazolyl]formamide [[35S]FANFT; 94 mCi/mmol] was given p.o. to conventional [n = 4; 115 +/- 8 g (S.D.)] and germfree [n = 4; 105 + 5 g] female CD rats [1.23 mCi/rat]. After 18 hr, organs were removed, and macromolecules were then isolated from individual livers and kidneys and from pooled urinary bladders. A hydroxylapatite isolation procedure was followed (Beland et al., J. Chromatogr., 174: 177-186, 1979), and the nucleic acids obtained were further purified by digestion with appropriate nucleases and/or centrifugation (105,000 X g). The results are as follows and are given in pairs (conventional/germ-free) expressed as pmol FANFT bound per mg macromolecule. Protein binding levels were: liver, 165 +/- 40/307 +/- 31; kidney, 72 +/- 19/88 +/- 24; bladder, 272/322. RNA levels were: liver, 217 +/- 184/413 +/- 196; kidney, 219 +/- 60/617 +/- 196; bladder, 448/1373. DNA levels were: liver, 9.8 +/- 7.5/17.0 +/- 7.5; kidney, 0.69 +/- 0.38/4.5 +/- 1.0. The quantity of bladder DNA was insufficient for accurate measurement. Diethylaminoethyl cellulose chromatography of liver RNA from a germfree rat, either before or after RNase digestion, showed that the majority of the radioactivity was associated with a polynucleotide fraction that appeared to be RNase resistant and accounted for only a small portion of the total RNA but that also permitted the intercalation of ethidium bromide. The deformylated FANFT metabolite, 2-amino-4-(5-nitro-2-fury)thiazole, reacted with transfer RNA upon reduction with sodium dithionite in vitro to give adduct(s) that also appeared to be RNase resistant. Thus, these results show that the urinary bladder carcinogen FANFT or its metabolites react in vivo with protein and nucleic acid of both target and nontarget organs and that binding levels are elevated in germfree rats.

A monoclonal antibody (anti-BL4) recognizing a previously characterized Mr 54,000 glycoprotein (gp54) was developed by immunizing BALB/c mice with cells from a precursor B-cell line (Josh-7). In normal individuals, this antigenic molecule was present on tonsillar B-cells (60-80%) and on a fraction of peripheral blood B-cells (5-25%). BL4 (gp54) expression was investigated in 186 patients with a variety of hematological malignancies using indirect immunofluorescence and flow cytometric analysis. Twenty-six of 37 cases of B-cell chronic lymphocytic leukemia (CLL) and 18 of 33 cases of B-cell non-Hodgkin's lymphoma were BL4 positive. Surface expression of BL4 on reactive cases of CLL and non-Hodgkin's lymphoma was brighter than those of B1, B2, and B4, BL4 positive CLL cases expressed a higher proportion of mouse rosette forming cells and Leu-1 positive cells than the BL4 negative subgroup and were not associated with elevated serum immunoglobulin levels. Four of 7 BL4 negative CLL cases were associated with increased serum levels of immunoglobulin M. Lymphoblasts from 14 of 14 cases of non-T acute lymphoblastic leukemia and 3 of 3 pre-B lymphoid blast crisis of chronic myeloid leukemia were BL4 negative. Neoplastic cells from 2 of 3 cases of Waldenstrom's macroglobulinemia and 4 of 7 cases of hairy cell leukemia were BL4 reactive. None of 7 cases of multiple myeloma and plasma cell leukemia were BL4 positive. All 11 T acute lymphoblastic leukemia cases, 6 other T-cell malignancies, 5 cases of Hodgkin's disease, 51 cases of acute nonlymphocytic leukemia, and 9 cases of chronic myeloid leukemia in chronic phase thus far studied were BL4 negative. An in vitro induction experiment using phorbol ester on a case of B-CLL demonstrated disappearance of BL4 accompanied with further B-cell differentiation. Our study further substantiates the previous finding that gp54 is a differentiation antigen restricted to the B-cell lineage and expressed during the intermediate stage of B-cell ontogeny.

The human promyelocytic leukemic cell line HL-60 can be induced to mature monocytes and macrophages in vitro by lymphocyte-conditioned medium. We are reporting sequential changes in surface antigenic expressions, which are sensitive markers of the characteristic events in the process of cell differentiation. The promyelocyte membrane antigen, detected by a monoclonal antibody produced using HL-60 cells as an immunogen, was shown to be associated with immature myeloid cells and was used to determine HL-60 cell development. The expression of this membrane antigen, determined to have a molecular weight of 85,000 was lost early in the differentiation period. In the following stage, in which the promyelocytes developed into monocytic cells, a steady increase of cells bearing the OKM1 normal monocyte antigen was observed. When macrophages became predominant in the final culture period, the expression of the OKM1 antigen decreased. The usefulness of these differentiation antigens in studying cellular development is discussed.

Urinary N-hydroxy metabolites of the bladder carcinogens, 2-aminofluorene and 4-aminobiphenyl, were examined for the induction of unscheduled DNA synthesis (UDS) in urothelial cells of several susceptible species. N-Hydroxy-2-aminofluorene, N-hydroxy-2-acetylaminofluorene (N-OH-AAF), N-hydroxy-4-aminobiphenyl, N-hydroxy-4-acetylaminobiphenyl, and the N-glucuronides of these two hydroxylamines induced UDS in the urothelial cells of dogs, rats, and rabbits. N-Hydroxy-2-aminonaphthalene, N-hydroxy-2-acetylaminonaphthalene, and the N-glucuronide of the hydroxylamine were not active. The induction of UDS in dog cells by N-OH-AAF or N-acetoxy-2-acetylaminofluorene, but not by N-hydroxy-2-aminofluorene, was inhibited by paraoxon. The microsomal fraction of dog urothelial cells catalyzed the binding of N-OH-AAF to transfer ribonucleic acid; the enzyme activity was completely inhibited by paraoxon, suggesting that N-deacetylase, but not N-,O-acetyltransferase, was responsible for the binding. The O-glucuronide of N-OH-AAF did not induce UDS in the urothelial cells of dogs, rats, or rabbits, nor did it bind to tRNA in the presence of dog urothelial enzymes, which suggest that N-OH-AAF is detoxified by O-glucuronidation. These results are consistent with the hypothesis that nonacetylated, N-hydroxylated metabolites play a major role in arylamine-induced bladder carcinogenesis. The importance of arylacethydroxamic acid metabolites in bladder carcinogenesis for various species may be inversely related to the rate of hepatic O-glucuronidation.

The effects of allopurinol on the induction of bladder cancer by N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), excretion of urinary tryptophan metabolites, hepatic nitroreductase activity, and the acid-soluble thiol content of liver and blood in weanling female Fischer rats were investigated. Four groups of rats were given normal diet or normal diet supplemented with 0.005% allopurinol, 0.188% FANFT, or 0.005% allopurinol-0.188% FANFT. Transitional cell carcinomas appeared in 3 of 30 rats (10%) at 15 weeks and in 7 of 44 rats (16%) at 20 weeks in the FANFT-treated group; the carcinomas appeared in 14 of 35 rats (40%) at 15 weeks and in 27 of 50 rats (54%) at 20 weeks in the FANFT-allopurinol-treated group. Growth rate was not affected by allopurinol and FANFT. Allopurinol alone caused no morphological change in the epithelial cells of the urinary bladder but decreased hepatic cytosol nitroreductase activity. FANFT alone had no effect on hepatic cytosol or microsomal nitroreductase activity but increased hepatic and blood acid-soluble thiol content. FANFT increased the urinary excretion of anthranilic acid glucuronide, kynurenine, acetylkynurenine, and 3-hydroxykynurenine and decreased indican and o-aminohippurate excretion. Allopurinol did not alter the effects of FANFT on the acid-soluble thiol content of liver and blood or the excretion of urinary tryptophan metabolites.