Medium-term protocols for in vivo evaluation of chemical modifiers of carcinogenesis

Cancer development is a long-term multistep process which allows interventional measure before the clincial disease emerges. the detection of natural substances which can block the process of carcinogenesis is a important as the identification of anti-tumoral drugs since they might be used in chemoprevention of cancer in high-risk groups. In vivo rodent models of chemical caecinogenesis have been used to study plant-derived inhibitors of carcinofenesis such as indols, coumarins, isothiocyanates, flavones, phenols and allyl-sulfides. Since the standard in vivo rodent bioassay is prolonged and expensive, shorter reliable protocols are needed. Two in vivo medium-term protocols for evaluation of modifiers of carcinogenesis are presented, one related to liver and the other to bladder cancer. Both protocols use rats, last 8 and 36 weeks and are based on the two-step concept of carcinogenesis: initiation and promotion. The protocols use respectively the development of altered foci of hepatocytes expressing immunochistochemically the placental form of gluthation S-transferase and the appearence of pre-neoplastic urothelium and papillomas as the end-points. the use of these protocols for detection of plantpderived inhibitors of carcinogenesis appear warranted.

Wound healing and colon carcinogenesis. Enhancing effects of skin wounding on development of colon tumors induced by 1,2 Dimethylhydrazine in the rat

Este estudo foi desenvolvido com a finalidade de investigar a possível influência do processo de cicatrização sobre o desenvolvimento neoplásico à distância, em um modelo experimental de carcinogênese do colon induzido pela 1,2 dimetil-hidrazina ( DMH). Ratos Wistar machos receberam injeções semanais de DMH ( 20mg/Kg, via subcutânea) ou solução salina, durante oito semanas. Na nona semana, um grupo tratado com DMH e um controle, foram submetidos à intervenção cirúrgica para retirada de um retalho cutâneo de 4cm no flanco direito, que cicatrizou por segunda intenção. Na 12ª semana, logo após o fechamento da ferida cutânea, os animais foram submetidos à eutanásia. O colon foi dividido em segmentos proximal e distal e examinado a nível macroscópico e histológico. Foram analisadas a incidência, distribuição e morfologia das lesões. O número total de tumores na mucosa do colon e o número de tumores por animal foi significantimente maior no grupo submetido à ferida cutânea do que no grupo tratado somente com DMH. O número de carcinomas pouco diferenciados foi significantimente maior no grupo com ferida cutânea do que em seu respectivo controle. Estes resultados sugerem que o processo de reparação de uma ferida cutânea favorece o desenvolvimento neoplásico em um órgão à distância, tal como o colon e que este efeito parece estar relacionado ao tipo histológico da neoplasia.

Absence of promoting potential of bracken fern (Pteridium aquilinum) in rat urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)-nitrosamine and uracil

Conventional studies on bracken fern (Pteridium aquilinum; PA) carcinogenicity have used high dietary concentrations (around 30%) and long-term exposure (up to 52-70 weeks) without consideration of the multistep character of the chemical carcinogenesis process. The present study evaluated specifically the promoting potential of 3-5% dietary crude PA in the rat urinary bladder mucosa in a 32-week-long initiation-promotion assay for chemical carcinogenesis. Initiation of urothelial carcinogenesis was accomplished with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Uracil (U) was provided through the diet in order to expand the population of initiated cells. Seven groups (C) of male Wistar rats were submitted to the following treatments: G1 = BBN (n = 8); G2 = U (n = 10); G3 = BBN-U (n = 9); G4 = BBN-PA-U-PA (n = 16); G5 = PA (n = 8); G6 = BBN-PA (n = 10); G7 = PA-U-PA (n = 12). At the end of the experiment rats presenting epithelial papillary or nodular hyperplasia (PNH), papillomas (PAP), or simultaneous PNH plus PAP numbered, respectively G1: 2-0-1; G2: 0-0-0; G3: 3-0-2; G4: 4-3-2; G5: 1-0-1; G6: 8-0-0; and G7: 0-0-0, with no significant differences in the incidence of lesions among the groups. More frequent and more severe lesions occurred in BBN-initiated animals, predominantly in those also exposed to uracil (G3 and G4). Low-dose crude bracken fern in the diet does not promote rat urinary bladder carcinogenesis after a 32-week period of exposure, even when the initiated urothelial cell population has been expanded through a mechanical stimulus. (C) 1995 Wiley-Liss, Inc.

EXPRESSION OF C-ERB B-2 PROTEIN AND DNA-PLOIDY IN BREAST CARCINOGENESIS

Objective.-to examine the c-erb B-2 expression and nuclear DNA content in samples of breast lesions to ascertain any relationship between c-erb B-2 expression and aneuploidy in the different types of proliferative breast lesions and in intraductal and invasive carcinomas.Design and Setting.-lmmunohistochemical analysis of c-erb B-2 expression and cytometric nuclear DNA assessment were performed in a series of 39 cases of intraductal hyperplasia without atypia, 7 cases of intraductal hyperplasia with atypia, 64 cases of intraductal carcinoma, and 85 cases of invasive breast carcinoma (30 of which had extensive intraductal component).Results.-Overexpression of c-erb B-2 was seen only in cases of carcinoma: 28 (43.7%) intraductal carcinomas and 15 (17.6%) invasive carcinomas. Aneuploidy was demonstrated in 3 (43.0%) cases of intraductal hyperplasia with atypia, in 54 (84.4%) cases of intraductal carcinoma, and in 63 (74.2%) cases of invasive carcinoma. All cases of intraductal hyperplasia without atypia were euploid and none expressed c-erb B-2. Among the carcinomas (intraductal and invasive) there was a strong relationship between aneuploidy and c-erb B-2 expression. In most instances, the intraductal and invasive components of the 30 invasive carcinomas with extensive intraductal component displayed similar DNA content and c-erb B-2 immunoreactivity; whenever there was a difference, the intraductal component tended to be aneuploid (five out of six cases) and c-erb B-2 positive (one case), in contrast to the respective invasive component.Conclusions.-The higher frequency of aneuploidy and c-erb B-2 expression in intraductal carcinomas in comparison with invasive carcinomas suggests there is not a linear relationship between DNA content abnormalities and neoplastic progression and that some invasive breast carcinomas evolve without an identifiable intraductal phase or are unrelated to disturbances at the c-erb B-2 locus.

Dose- and sex-related carcinogenesis by N-bis(2-hydroxypropyl)nitrosamine in Wistar rats

An initiation-promotion medium-term bioassay for detection of chemical carcinogens, developed in the male F344 rat, uses 0.1% N-bis(2-hydroxypropyl)nitrosamine (DHPN) among five genotoxic chemicals for the initiation of carcinogenesis in multiple organs. To establish this bioassay in the Wistar strain, the effects of two dose levels of DHPN were evaluated on the main DHPN rat target organs: lung, thyroid gland, kidneys and liver. Four groups of male and female animals were studied: Control--untreated group; Multi-organ initiated group (also referred to as DMBDD, based on the initials of the five initiators)-treated sequentially with N-diethylnitrosamine (DEN, i.p.), N-methyl-N-nitrosourea (MNU, i.p.), N-butyl-N-(4-hydroxy butyl)nitrosamine (BBN, drinking water), N, N'-dimethylhydrazine (DMH, s.c.) and DHPN (drinking water) for 4 weeks; a third group treated with 0.1% DHPN in drinking water for 2 weeks and the last group treated with 0.2% DHPN in drinking water for 4 weeks. The animals were sacrificed after 30 weeks. DHPN at 0.2% induced preneoplasia in the liver and kidneys of rats of both sexes, the number and area of the putative preneoplastic liver glutathione S-transferase-positive hepatocyte foci being significantly increased in these animals. It also induced benign and malignant tumors in female and in male rats. However, there was no relationship between the increased incidence of preneoplastic lesions and tumor development in the 0.2% DHPN-exposed groups of both sexes. DHPN at 0.1% induced only a few preneoplastic lesions in the liver and kidney and no tumors in both male and female rats. A clear dose and sex-related carcinogenic activity of DHPN was registered, although Wistar rats of both sexes showed a relative resistance to the carcinogenic activity of this compound.

Placental glutathione S-transferase correlates with cellular proliferation during rat tonguie carcinogenesis induced by 4-nitroquinoline 1-oxide

Taking into consideration that glutatione S-transferase (GST) and cellular proliferation play a crucial role during carcinogenesis, the goal of this study was to investigate the expression of placental GST, called GST-P, and proliferating cellular nuclear antigen (PCNA) by means of immunohistochemistry during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO). This is a useful model for studying oral squamous cell carcinoma phase by phase. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution by drinking water for 4, 12 or 20 weeks. Ten animals were used as negative control. GST-P positive foci were detected in non-neoplastic oral cells at 4 weeks of 4NQO administration. In the same way, GST-P positive cells were detected in pre-neoplastic lesions and squamous cell carcinomas induced after 12 and 20 weeks-treatment, respectively. None of the control animals expressed GST-P positive cells. Regarding cellular proliferation, PCNA positive nuclei were higher at 12 and 20 weeks following 4NQO exposure (p < 0.05) when compared to negative control. These results suggest that the expression of GST-P is correlated with cellular proliferation, in which GST-P is associated with risk and progression of oral cancer, whereas PCNA is closely involved during neoplastic conversion. (c) 2007 Published by Elsevier GmbH.

Myenteric Denervation Downregulates Galectin-1 and-3 Expression in Gastric Carcinogenesis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Viral infections in workers in hospital and research laboratory settings: a comparative review of infection modes and respective biosafety aspects

Objectives: To compare modes and sources of infection and clinical and biosafety aspects of accidental viral infections in hospital workers and research laboratory staff reported in scientific articles. Methods: PubMed, Google Scholar, ISI Web of Knowledge, Scirus, and Scielo were searched (to December 2008) for reports of accidental viral infections, written in English, Portuguese, Spanish, or German; the authors' personal file of scientific articles and references from the articles retrieved in the initial search were also used. Systematic review was carried out with inclusion criteria of presence of accidental viral infection's cases information, and exclusion criteria of absence of information about the viral etiology, and at least probable mode of infection.Results: One hundred and forty-one scientific articles were obtained, 66 of which were included in the analysis. For arboviruses, 84% of the laboratory infections had aerosol as the source; for alphaviruses alone, aerosol exposure accounted for 94% of accidental infections. of laboratory arboviral infections, 15.7% were acquired percutaneously, whereas 41.6% of hospital infections were percutaneous. For airborne viruses, 81% of the infections occurred in laboratories, with hantavirus the leading causative agent. Aerosol inhalation was implicated in 96% of lymphocytic choriomeningitis virus infections, 99% of hantavirus infections, and 50% of coxsackievirus infections, but infective droplet inhalation was the leading mode of infection for severe acute respiratory syndrome coronavirus and the mucocutaneous mode of infection was involved in the case of infection with influenza B. For blood-borne viruses, 92% of infections occurred in hospitals and 93% of these had percutaneous mode of infection, while among laboratory infections 77% were due to infective aerosol inhalation. Among blood-borne virus infections there were six cases of particular note: three cases of acute hepatitis following hepatitis C virus infection with a short period of incubation, one laboratory case of human immunodeficiency virus infection through aerosol inhalation, one case of hepatitis following hepatitis G virus infection, and one case of fulminant hepatitis with hepatitis B virus infection following exposure of the worker's conjunctiva to hepatitis B virus e antigen-negative patient saliva. of the 12 infections with viruses with preferential mucocutaneous transmission, seven occurred percutaneously, aerosol was implicated as a possible source of infection in two cases, and one atypical infection with Macacine herpesvirus 1 with fatal encephalitis as the outcome occurred through a louse bite. One outbreak of norovirus infection among hospital staff had as its probable mode of infection the ingestion of inocula spread in the environment by fomites.Conclusions: The currently accepted and practiced risk analysis of accidental viral infections based on the conventional dynamics of infection of the etiological agents is insufficient to cope with accidental viral infections in laboratories and to a lesser extent in hospitals, where unconventional modes of infection are less frequently present but still have relevant clinical and potential epidemiological consequences. Unconventional modes of infection, atypical clinical development, or extremely severe cases are frequently present together with high viral loads and high virulence of the agents manipulated in laboratories. In hospitals by contrast, the only possible association of atypical cases is with the individual resistance of the worker. Current standard precaution practices are insufficient to prevent most of the unconventional infections in hospitals analyzed in this study; it is recommended that special attention be given to flaviviruses in these settings. (C) 2011 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Simple mucin-type carbohydrate antigens (T, sialosyl T, Tn and sialosyl-Tn) in breast carcinogenesis

Immunohistochemical analysis of the expression of simple mucin-type carbohydrate antigens (Tn, sialyl-Tn and T) was performed in a series of 43 cases of intraductal hyperplasia without atypia, 9 cases of intraductal hyperplasia with atypia, 54 cases of ductal carcinoma in situ (DCIS) and 26 cases of invasive breast carcinoma. We also studied 36 cases of isolated breast normal epithelium, 20 cases of 'normal' breast epithelium adjacent to neoplasms and 14 cases of apocrine metaplasia. All antigens were detected in different frequencies in normal, hyperplastic, metaplastic and neoplastic breast epithelium. Tn and sialyl-Tn are expressed more frequently in malignant than in benign breast epithelium; while Tn expression increases from normal to invasive carcinomas, sialyl-Tn increases until DCIS and drops in invasive carcinomas, suggesting that either there is a failure of a proportion of DCIS to progress to invasive carcinoma or loss of expression of sialyl-Tn when some carcinomas become invasive. The high frequency of Tn and sialyl-Tn expression in breast intraductal proliferations probably reflects incomplete glycosylation in these lesions, which is a well-known tumour-associated phenomenon and supports the assumption that such lesions are putative precursors of breast cancer. T antigen was expressed in all groups studied, but its prevalence differed significantly between normal and neoplastic epithelium. The expression of these antigens in epithelium adjacent to carcinomas is similar to that found in isolated normal breast epithelium, whereas apocrine metaplasia has a pattern of simple mucin-type glycosylation that is specific and distinct from that of the normal breast epithelium, with a high frequency of marked expression of Tn and sialyl-Tn. The similarity of the pattern of expression of simple mucin-type antigens in metaplasia and malignant neoplasia reduces the usefulness of these markers from a diagnostic standpoint.

Cigarette smoking and high-risk HPV DNA as predisposing factors for high-grade cervical intraepithelial neoplasia (CIN) in young Brazilian women

Background. This cross-sectional study was designed to evaluate the role of cigarette smoking and high-risk HPV types as risk factors of CIN 2 and 3 in young, sexually active Brazilian women. Materials and method. A series of 100 consecutive women with abnormal Pap smears were recruited, subjected to colposcopy, punch biopsy, and questionnaire for their social, sexual and reproductive factors. Of these, 77 women between 20 and 35 years of age (median 26.5 years) with biopsy-confirmed CIN 1 or CIN 2 and 3, were enrolled in this study. Representative samples from the exocervix and endocervix were obtained for HPV testing with the Hybrid Capture HPV-DNA assay, including the probes for the oncogenic HPV types (16, 18, 31, 33, 35, 45, 51, 52 and 56). Results. The overall rate of CIN 2 and 3 was 23/77 (29.8%). The women with CIN 1, 2 and 3 did not differ from each other with regard to their age, race, schooling, marital status, life-time number of sexual partners, age at first intercourse, use of oral contraceptives, or parity. However, current cigarette smoking was strongly associated with CIN 2 and 3 (p < 0,001), and among smokers, the risk of high-grade CIN increased in parallel with the time of exposure (years of smoking) p = 0.07), HPV-DNA of the oncogenic types was detected in 43 (56%) women, the risk of being HPV DNA-positive was significantly higher in CIN 2 and 3 as compared with CIN 1 (p = 0.037). Importantly, the prevalence of high-risk HPV types was significantly higher in cigarette smokers than in non-smokers (p = 0.046). Conclusions. The results indicate that the severity of CIN lesions was clearly related to two fundamental risk factors: 1) high-risk HPV types, and 2) current cigarette smoking. These two risk factors were closely interrelated in that the high-risk HPV types were significantly more frequent in current smokers than in non-smokers, suggesting the possibility of a synergistic action between these two risk factors in cervical carcinogenesis.

Comparison of uroplakin expression during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in rats and mice

The expression of uroplakins, the tissue-specific and differentiation- dependent membrane proteins of the urothelium, was analyzed immunohistochemically in N butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-treated rats and mice during bladder carcinogenesis. Male Fischer 344 rats were treated with 0.05% BBN in the drinking water for 10 wk and were cuthanatized at week 20 of the experiment. BBN was administered to male B6D2F1 mice; it was either provided at a rate of 0.05% in the drinking water (for 26 wk) or 5 mg BBN was administered by intragastric gavage twice weekly for 10 wk, followed by 20 wk without treatment. In rats, BBN-induced, noninvasive, low grade, papillary, transitional cell carcinoma (TCC) showed decreased uroplakin-staining of cells lining the lumen but showed increased expression in some nonluminal cells. In mice, nonpapillary, high-grade dysplasia, carcinoma in situ, and invasive carcinoma were induced. There was a marked decrease in the number of uroplakin-positive cells lining the lumen and in nonluminal cells. This occurred in normal-appearing urothelium in BBN-treated mice and in dysplasic urothelium, in carcinoma in situ, and in invasive TCC. The percentage of uroplakin-positive nonluminal cells was higher in control mice than in rats, but it was lower in the mouse than in the rat after BBN treatment. Uroplakin expression was disorderly and focal in BBN-treated urothelium in both species. These results indicate that BBN treatment changed the expression of uroplakins during bladder carcinogenesis, with differences in rats and mice being related to degree of tumor differentiation.

Papilomavírus humano (HPV) em lesões benignas e malignas de mucosa oral pelos métodos de imuno-histoquímica e hibridização in situ

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)