João Lauro Viana de Camargo

Strain Differences in Susceptibility to 2-Acetylaminofluorene and Phenobarbital Promotion of Rat Hepatocarcinogenesis in a Medium-term Assay System: Quantitation of Glutathione S-Transferase P-positive Foci Development

Strain differences in susceptibility to promotion by the liver carcinogens 2‐acetylaminofluorene (2‐AAF) and phenobarbital (PB) were examined in the medium‐term bioassay system initially developed in our laboratory using male F344 rats as the test animal and glutathione S‐transferase placental form (GST‐P)‐positive foci as the lesion end‐point. Numbers and areas per cm2 of induced GST‐P‐positive hepatocellular foci were compared in LEW, F344, NAR, SD, WBN, SHR, Wistar and ODS rats initiated with diethylnitrosamine (DEN) and subjected to partial hepatectomy during subsequent administration of 2‐AAF or PB. LEW, SD, WBN, and F344 rats were most susceptible to hepatopromotion by both compounds, with a hundred fold increase in lesion area being observed for 2‐AAF in the LEW case. NAR and SHR strains demonstrated an intermediate response, while Wistar and, in particular, the related ODS rats demonstrated very low susceptibilities. The obvious strain differences could be expressed in terms of comparative indices of promoting effects of 2‐AAF and PB as well as DEN itself regarding each of the 8 strains tested. The use of F344 rats for the bioassay model was validated by the relatively high sensitivity to both DEN and 2‐AAF initiation as well as second‐stage promotion stimulus exhibited.

A NEW MEDIUM‐TERM BIOASSAY SYSTEM FOR DETECTION OF ENVIRONMENTAL CARCINOGENS USING DIETHYLNITROSAMINE‐INITIATED RAT LIVER FOLLOWED BY D‐GALACTOSAMINE TREATMENT AND PARTIAL HEPATECTOMY

The effects of D‐galactosamine on induction of preneoplastic glutathione S‐transferase placental form positive liver foci were investigated in F344 rats pretreated with diethylnitrosamine (DEN) in an attempt to improve the predictive value of the medium‐term bioassay system developed in our laboratory. Two weeks after the initial single ip dose (200 mg/kg) of DEN, administration of test compounds was commenced simultaneously with an ip injection of D‐galactosamine at a dose of 300 mg/kg body wt. All rats were then subjected to two‐thirds partial hepatectomy (PH) at week 5 and sacrificed for assessment of lesion yield at week 8. Measurement and comparison of the numbers and areas of glutathione S‐transferase placental form positive (GST‐P+) foci per cm2 revealed a positive response to more carcinogens, including non‐hepatocarcinogens, than did the same bioassay system without injection of D‐galactosamine. Thus the results suggest that inclusion of this extra proliferative stimulus may improve the medium‐term detection of carcinogens and modifiers.

Natural Killer Activity in a Medium‐term Multi‐organ Bioassay for Carcinogenesis

Natural killer (NK) cell activity was evaluated after the initiation and promotion steps in a medium‐term multi‐organ bioassay for carcinogenesis. NK cell activity was assessed in vitro by Cr51 release assay at the 4th and 30th weeks of the experiment. Male Wistar rats were sequentially initiated with N‐diethylnitrosamine (DEN i.p.), N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN drinking water), N‐methyl‐N‐nitrosourea (MNU i.p.), dihydroxy‐di‐N‐propylnitrosamine (DHPN drinking water) and N, N′‐dimethylhydrazine (DMH s.c.) at subcarcinogenic doses for 4 weeks (DMBDD initiation). One group was evaluated at the 4th week and the other was maintained without any further treatment until the 30th week. Two initiated groups were exposed through the diet to 2‐acetylaminofluorene (2‐AAF) or phenobarbital (PB), from the 6th until the 30th week. Five additional groups were studied to evaluate the effects of each initiator on NK activity. All groups submitted to initiation only, initiation plus promotion, or promotion only, developed significantly more preneoplastic lesions than the untreated control group. The main target organs for tumor development in the initiated animals were the liver and the colon, irrespective of treatment with 2‐AAF or PB. NK cell activity was not affected by exposure to genotoxic carcinogens after initiation, at the 4th week. Treatments only with PB or 2‐AAF did not change NK cell activity. However, decreased NK cell activity was registered in the group only initiated with DMBDD and in the group given DMBDD+2‐AAF. This late depression of NK cell activity at the 30th week could be related to the production of suppressing molecules by the tumor cells.

Evaluation of Carcinogenic Potential of Diuron in a Rat Mammary Two-Stage Carcinogenesis Model

This study aimed to evaluate the carcinogenic potential of the herbicide Diuron in a two-stage rat medium-term mammary carcinogenesis model initiated by 7,12-dimethylbenz(a)anthracene (DMBA). Female seven-week-old Sprague-Dawley (SD) rats were allocated to six groups: groups G1 to G4 received intragastrically (i.g.) a single 50 mg/kg dose of DMBA; groups G5 and G6 received single administration of canola oil (vehicle of DMBA). Groups G1 and G5 received a basal diet, and groups G2, G3, G4, and G6 were fed the basal diet with the addition of Diuron at 250, 1250, 2500, and 2500 ppm, respectively. After twenty-five weeks, the animals were euthanized and mammary tumors were histologically confirmed and quantified. Tumor samples were also processed for immunohistochemical evaluation of the expressions of proliferating cell nuclear antigen (PCNA), cleaved caspase-3, estrogen receptor-α (ER-α), p63, bcl-2, and bak. Diuron treatment did not increase the incidence or multiplicity of mammary tumors (groups G2 to G4 versus Group G1). Also, exposure to Diuron did not alter tumor growth (cell proliferation and apoptosis indexes) or immunoreactivity to ER-α, p63 (myoephitelial marker), or bcl-2 and bak (apoptosis regulatory proteins). These findings indicate that Diuron does not have a promoting potential on mammary carcinogenesis in female SD rats initiated with DMBA.

Diuron lacks promoting potential in a rat liver bioassay.

The promoting activity of the herbicide Diuron was evaluated in a medium-term rat liver carcinogenesis bioassay that uses as endpoint immunohistochemically identified glutathione S-transferase positive (GST-P+) foci. Male Wistar rats were allocated to the following groups: G1 to G6 were initiated for liver carcinogenesis by a single dose of diethylnitrosamine (DEN, 200 mg/kg) while groups G7 and G8 received only 0.9% NaCl (DEN vehicle). From the 2nd week animals were fed a basal diet (G1 and G7) or a diet added with Diuron at 125, 500, 1250, 2500 and 2500 ppm (G2 to G5 and G8, respectively) or 200 ppm Hexaclorobenzene (HCB; G6). The animals were submitted to 70% partial hepatectomy at the 3rd week and sacrificed at the 8th week. The herbicide did not alter ALT or creatinine serum levels. No conspicuous GST-P+ foci development was registered in non-initiated rats fed Diuron at 2500 ppm. While DEN-initiated animals fed Diuron at 1250 or 2500 ppm developed mild centrilobular hypertrophy, DEN-initiated HCB-fed animals showed severe liver centrilobular hypertrophy and significant GST-P+ foci development. These findings indicate that the medium-term assay adopted in this study does not reveal any liver carcinogenesis initiating or promoting potential of Diuron in the rat.

Evaluation of Diuron (3-[3,4-dichlorophenyl]-1,1-dimethyl urea) in a Two-stage Mouse Skin Carcinogenesis Assay

Diuron (3-[3,4-dichlorophenyl]-1,1-dimethyl urea) is an herbicide with carcinogenic activity in rats and mice, which have developed respectively urothelial and mammary gland tumors in long-term studies. Accordingly, diuron has been categorized as a “likely human carcinogen” by the U.S. Environmental Protection Agency. Although the carcinogenesis-initiating activity of diuron has been reported in an early initiation-promotion mouse skin study, its genotoxic potential has been disputed. It is necessary to clarify the mode of action through which it has caused rodent neoplasia and verify its relevance to humans. Herein, two experiments were developed to verify the initiating and promoting potentials of diuron in a twenty-three- and a twenty-one-week–long mouse skin carcinogenesis protocol. In one, dimethylsulfoxide (DMSO) was the solvent for the herbicide; in the other, acetone was the alternative solvent in order to verify whether DMSO had inhibitory influence on a potential cutaneous carcinogenic activity. The adopted schedule for the tumor-promoting agent 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in skin ulcers, which demonstrates the need for careful selection of TPA dose levels and frequency of application in this model. In both studies, diuron did not exert any influence on the skin carcinogenesis process, in contrast with results already reported in the literature.

Summation effects of uracil and other promoters on epithelial lesion development in the F344 rat urinary bladder initiated by N-butyl-N(4-hydroxybutyl)nitrosamine

Five non‐genotoxic chemicals previously demonstrated to be bladder cancer promoters in 36‐week in vivo assays for carcinogenesis were reevaluated in a 20‐week experiment in order to assess the summation influence of dietary nracil, a component of RNA, on the development of (pre)neoplastic lesions. The test chemicals, sodium bicarbonate, sodium L‐ascorbate, sodium citrate, butylated hydroxytoluene and ethoxyquin, were mixed into the diet at concentrations of 3%, 5%, 5%, 1% and 0.8%, respectively, and administered to male F344 rats after initiation with 0,05% N‐butyl‐N‐(4‐hydroxybutyDnitrosamine (BBN) in their drinking water for 4 weeks. The test chemicals were given from the 4th to the 8th and the llth to 20th experimental weeks, uracil being administered at the level of 3% in the diet during the intervening period. Rats in the control group received only BBN and uracil. All animals were killed at week 20 and the bladders were evaluated for the occurrence of putative preneoplastic papillary or nodular (PN) hyperplasia and tumors. Significant increase in the occurrence of FN hyperplasia was observed in all groups initiated with BBN and fed uracil and test chemicals. Quantitative values for papillomas were also significantly increased except in the ethoxyquin‐treated group. The results confirm that uracil given in the middle of the post‐initiation stage enhances the promoting activity of chemicals and suggest that the use of this chemical might be useful to reduce the duration of current bioassays for bladder chemical carcinogens.

Infiltrating CD8+ T Lymphocytes, Natural Killer Cells, and Expression of IL-10 and TGF-β1 in Chemically Induced Neoplasms in Male Wistar Rats

The present study aimed to estimate the number of CD8+ T and natural killer (NK) infiltrating cells and the expression of interleukin-10 (IL-10) and transforming growth factor beta 1 (TGF-β1) in chemically induced neoplasms in an initiation-promotion bioassay for carcinogenesis. Male Wistar rats were treated withN-nitrosodiethylamine, N-methyl-N-nitrosourea, N-butyl-N-(4-hydroxybutyl)nitrosamine, dihydroxy-di-N-propylnitrosamine, and 1,2-dimethylhydrazine for 4 weeks. Two groups were subsequently exposed through diet to phenobarbital (0.05%) or 2-acetylaminofluorene (0.01%) for 25 weeks. An untreated group was used as a control. Immune cells and cytokines were immunohistochemically evaluated in neoplasms and in surrounding normal tissues at the liver, kidneys, lung, and small and large intestines. When compared to the respective normal tissues, an increased number of NK cells was verified infiltrating the colon, lung, and kidney neoplasms, while the number of CD8+ T cells decreased in the intestine and lung neoplasms. Expression of IL-10 was found mainly in kidney tumors. TGF-β1 was expressed mainly in the liver and kidneys tumors. The results indicate that the differential occurrence of immune cells between neoplastic and normal tissues could be dependent upon tumor microenvironment.

Alternative Multiorgan Initiation-Promotion Assay for Chemical Carcinogenesis in the Wistar Rat.

The medium-term multiorgan initiation–promotion chemical bioassay (diethylnitrosamine, methyl-nitrosourea, butyl-hydroxybutylnitrosamine, dihydroxypropylnitrosamine, dimethylhydrazine [DMBDD]) with the Fischer 344 rat was proposed as an alternative to the conventional 2-year carcinogenesis bioassay for regulatory purposes. The acronym DMBDD stands for the names of five genotoxic agents used for initiation of multiorgan carcinogenesis. The Brazilian Agency for the Environment officially recognized a variation of this assay (DMBDDb) as a valid method to assess the carcinogenic potential of agrochemicals. Different from the original protocol, this DMBDDb is 30-week long, uses Wistar rats and two positive control groups exposed to carcinogenesis promoters sodium phenobarbital (PB) or 2-acetylaminofluorene (2-AAF). This report presents the experience of an academic laboratory with the DMBDDb assay and contributes to the establishment of this alternative DMBDD bioassay in a different rat strain. Frequent lesions observed in positive groups to evaluate the promoting potential of pesticides and the immunohistochemical expressions of liver cytochrome P450 (CYP) 2B1/2B2 and CYP1A2 enzymes were assessed. Commonly affected organs were liver, kidney, intestines, urinary bladder, and thyroid. PB promoting activity was less evident than that of 2-AAF, especially in males. This study provides a repository of characteristic lesions occurring in positive control animals submitted to a modified alternative 2-stage multiorgan protocol for carcinogenesis in Wistar rat.

Nobuyuki Ito, MD, PhD

In October 2010, Dr. Nobuyuki Ito (born December 4, 1928), a long-standing friend and supporter of the Society of Toxicologic Pathology (STP), died after a prolonged illness. Dr. Ito’s contributions to the fields of toxicologic pathology, rodent pathology, and human pathology were extraordinary, with significant contributions to the science, the education, and the administration of the American and Japanese STPs. He was one of the founders of the International Federation of the Societies of Toxicologic Pathology (IFSTP) and served as its first president, and he was also one of the founding members of the International Academy of Toxicologic Pathology. He served on a variety of committees devoted to the development of standardized pathology nomenclature in experimental animals. Dr. Ito’s contributions to science and education were numerous and varied. In addition to significant contributions to the field of toxicologic pathology, he was an innovative and productive scientist in the fields of toxicology, pathology, and carcinogenesis. Educated originally at Nara Medical College (MD, 1952) and at Osaka University (PhD, 1961), he undertook a fellowship in carcinogenesis research with Emanuel Farber at the University of Pittsburg (1962–64) and was a guest scientist at Heidelberg, Germany. His contributions were innovative, many, and varied. His career was that of an outstanding and productive scientist. He had more than 600 publications, served on numerous editorial boards, and was invited to numerous major symposia internationally. He truly epitomized the global nature of science long before it became fashionable. He was a significant developer of several animal models of human diseases, including cancers of the bladder, prostate, liver, glandular stomach, forestomach, kidney, thyroid, lung, and pancreas. He was active in the development of new screening assays for chemical carcinogenesis, especially the medium-term assay (Ito assay), and also extensively investigated the chemoprevention of cancer. Dr. Ito is notably also known for his research on antioxidants. His investigations on the carcinogenicity of BHA and other antioxidants, as well as their chemopreventive properties, led one symposium organizer to suggest that antioxidant research could be dated as either being before Ito (BI) or after Ito (AI), based on the seminal nature of his work. Dr. Ito was recognized worldwide for his contributions to carcinogenesis research and to toxicologic pathology, having served on numerous panels of the U.S. National Cancer Institute, advisory boards of the U.S. Food and Drug Administration and U.S. Environmental Protection Agency, and the International Agency for Research on Cancer, beginning as early as 1974. His research was recognized with numerous awards, including the Yomiuri Tokai Medical Award in 1991, the Princess Takamatsu Cancer Research Award in 1985, the Chunichi Culture Award in 1985, and the Tomizo Yoshida Award from the Japanese Cancer Association.