Genotoxicity and mutagenicity of Rosmarinus officinalis (Labiatae) essential oil in mammalian cells in vivo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Assessment of the potential genotoxic risk of medicinal Tamarindus indica fruit pulp extract using in vivo assays

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

Genotoxicity assessment of the antimalarial compound artesunate in somatic cells of mice

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Diretrizes para diagnóstico morfológico em síndromes mielodisplásicas

As síndromes mielodisplásicas são reconhecidas como doenças que se originam nas células-tronco da medula óssea e que requerem avaliação sistemática e criteriosa de sangue periférico e medula óssea para seu correto diagnóstico. O objetivo deste relato é estabelecer os critérios morfológicos (cito-histológicos) como parâmetros para o diagnóstico de SMD em amostras de sangue periférico e medula óssea, com especial direcionamento aos hematologistas e patologistas clínicos que exercem a hematologia laboratorial na sua rotina de trabalho. Os principais achados morfológicos são listados no final deste relato, na forma de check-list, objetivando a sistematização sobre estes achados.

Determinação da expressão da molécula de adesão CD56 em plasmócitos no mieloma múltiplo através de estudo imuno-histoquímico

O papel das moléculas de adesão celular, na fisiopatologia do mieloma múltiplo (MM), tem sido alvo de vários estudos nos últimos anos. A expressão de CD56 pelos plasmócitos tumorais está associada a comportamento clínico menos agressivo da doença, e sua perda tem sido descrita na fase de leucemização plasmocitária. A determinação da expressão da molécula CD56 pelos plasmócitos tumorais pode ser obtida através de citometria em fluxo, revelando positividade em 55% a 78% dos casos. No presente estudo, objetivamos verificar a expressão da molécula CD56 por plasmócitos tumorais na medula óssea de portadores de MM, utilizando o estudo imuno-histoquímico das amostras histológicas obtidas ao diagnóstico. Analisamos as amostras de medula óssea de vinte portadores de MM, e realizamos o estudo imuno-histoquímico para determinação da expressão das cadeias leves kappa e lambda e da molécula CD56 pelos plasmócitos tumorais. A expressão da molécula CD56 foi importante em três casos, moderada em seis, discreta em quatro e negativa em sete. O estudo imuno-histoquímico mostrou-se válido para determinação da expressão de CD56 por plasmócitos tumorais em portadores de MM, fornecendo resultados semelhantes aos descritos para os obtidos por citometria em fluxo. Através do estudo imuno-histoquímico, foi possível observar variações da expressão da molécula CD56.

Linfoma não-Hodgkin de órbita: relato de caso

O objetivo é relatar manifestação incomum de linfoma não-Hodgkin de órbita. Paciente masculino, de 75 anos, se apresentou com queixa de lacrimejamento crônico bilateral. Havia feito dacriocistorrinostomia endonasal à direita e à esquerda por duas vezes, sem sucesso. Ao exame, massas de consistência fibroelástica, em topografia das bolsas de gordura das pálpebras inferiores e proptose axial. O paciente negava outros sintomas ou sinais sistêmicos. Hemograma sem alteração, hormônios tireoidianos normais. A tomografia computadorizada mostrava infiltrado difuso na órbita e proptose axial. Biópsia de gordura orbitária e de medula óssea diagnosticaram linfoma não-Hodgkin. O paciente foi tratado com quimioterapia, sendo em seguida submetido à cirurgia da via lacrimal bilateral, com resolução do quadro. A doença sistêmica que exigia diagnóstico e tratamento adequados para que se tivesse bom prognóstico estava mascarada pelo quadro de epífora bilateral.

Extração de DNA de materiais de arquivo e fontes escassas para utilização em reação de polimerização em cadeia (PCR)

Este trabalho visou a comparação de cinco métodos diferentes de extração de DNA de materiais de arquivo (tecidos incluídos em parafina, esfregaços de sangue periférico - corados e não corados com Leishman, lâminas com mielogramas, gotas de sangue em Guthrie Card) e de fontes escassas (células bucais, um e três bulbos capilares e 2 mL de urina), para que fossem avaliadas a facilidade de aplicação e a facilidade de amplificação deste DNA pela técnica da reação de polimerização em cadeia (PCR). Os métodos incluíram digestão por proteinase K, seguida ou não por purificação com fenol/clorofórmio; Chelex 100® (BioRad); Insta Gene® (BioRad) e fervura em água estéril. O DNA obtido foi testado para amplificação de três fragmentos gênicos: Brain-derived neutrophic factor (764 pb), Factor V Leiden (220 pb) e Abelson (106 pb). de acordo com o comprimento do fragmento gênico estudado, da fonte potencial de DNA e do método de extração utilizado, os resultados caracterizaram o melhor caminho para padronização de procedimentos técnicos a serem incluídos no manual de Procedimentos Operacionais Padrão do Laboratório de Biologia Molecular do Hemocentro - HC - Unesp - Botucatu.

HISTORICAL PERSPECTIVE and HUMAN CONSEQUENCES of AFRICANIZED BEE STINGS IN THE AMERICAS

In 1956, Africanized bees began to spread in the American continent from southern Brazil, where original African bees mated with European bees. A few years later, in 1990, these Africanized bees reached the United States and were found in Texas. Currently, these hybrid bees are found in several North American states and will probably reach the Canadian border in the future. Although the presence of Africanized bees had produced positive effects on Brazilian economy, including improvement in crop pollination and in honey production, turning Brazil into a major exporter, the negative impacts-such as swarming, aggressive behavior, and the ability to mass attack-resulted in serious and fatal envenomation with humans and animals. Victims of bee attacks usually develop a severe envenomation syndrome characterized by the release of a large amount of cytokines [interleukins (IL) IL-1, IL-6, IL-8], and tumor necrosis factor (TNF). Subsequently, such cytokines produce an acute inflammatory response that triggers adverse effects on skeletal muscles; bone marrow; hepatic and renal functions; and cardiovascular, central nervous, and immune systems. Finally, the aim of the present review is to study historical characteristics and current status of Africanized bees' spread, the composition of their venom, the impact of the bees on the Brazilian economy and ecology, and clinical aspects of their stings including immune response, and to suggest a protocol for bee sting management since there is no safe and effective antivenom available.

Thymic lymphomas in Wistar rats exposed to N-methyl-N-nitrosourea (MNU)

The Brazilian Agency for the Environment (IBAMA) recently adopted an alternative medium-term multiple-organ assay system with the Wistar rat strain for detection of the carcinogenic potential of pesticides. Originally, this initiation-promotion protocol was established in Japan with the isogenic Fischer 344 male rat. Among the initiating agents used in that assay, N-methyl-N-nitrosourea (MNU) rapidly induces malignant lymphoma and leukemia and early mortality of rats from different strains. This study was developed to evaluate whether the outbred Wistar rats are also similarly susceptible to MNU. Particularly, it aimed to evaluate the dose-response relationship and to register the MNUinduced pre-neoplasia and neoplasia that may develop in the lympho-hematopoietic system (LHS) of the Wistar rat within a medium-term period. Four groups of male Wistar rats were treated during 2 weeks with vehicle or with MNU (80, 160 or 240 mg/kg body weight, i.p.). After sacrifice at the 12th and 20th weeks, the thymus, spleen, bone marrow, cervical and mesenteric lymph nodes and liver were collected for analysis. At the 20th week, LHS malignant tumors and benign vascular tumors occurred only in the high- and intermediate-dose MNU-treated animals. Four animals treated with 240 mg/kg developed diffuse thymic lymphomas; two others, treated respectively with 240 mg/kg and 160 mg/kg, developed spleen hemangiomas. The present observations indicate that the Wistar strain is as susceptible as other strains to the early development of MNU-induced LHS (pre)neoplasia. Therefore, this strain seems suitable to be used as test system in bioassay protocols that adopt MNU as an initiating agent for carcinogenesis.

Black bean (Phaseolus vulgaris L.) as a protective agent against DNA damage in mice

This study was designed to evaluate the toxicogenetic or protective effect of cooked and dehydrated black beans (Phaseolus vulgaris L.) in bone marrow and peripheral blood cells of exposed mice. The frequency of micronuclei detected using the bone marrow erythrocyte micronucleus test and level of DNA lesions detected by the comet assay were chosen as end-points reflecting mutagenic and genotoxic damage, respectively. Initially, Swiss male mice were fed with a 20% black bean diet in order to detect mutagenic and genotoxic activity. However, no increase in the frequency of bone marrow micronucleated polychromatic erythrocytes (MN PCEs) or DNA lesion in leukocytes was observed. In contrast, received diets containing 1, 10 or 20% of black beans, a clear, but not dose-dependent reduction in the frequency of MN PCEs were observed in animals simultaneously treated with cyclophosphamide, an indirect acting mutagen. Similar results were observed in leukocytes by the comet assay. Commercial anthocyanin was also tested in an attempt to identify the bean components responsible for this protective effect. However, instead of being protective, the flavonoid, at the highest dose administered (50 mg/kg bw), induced primary DNA lesion, as detected by the comet assay. These data indicate the importance of food components in preventing genetic damage induced by chemical mutagens, and also reinforce the role of toxicogenetic techniques in protecting human health. (C) 2003 Elsevier Ltd. All rights reserved.

Chemically induced immunotoxicity in a medium-term multiorgan bioassay for carcinogenesis with Wistar rats

A variety of chemicals can adversely affect the immune system and influence tumor development. The modifying potential of chemical carcinogens on the lymphoid organs and cytokine production of rats submitted to a medium-term initiation-promotion bioassay for carcinogenesis was investigated. Male Wistar rats were sequentially initiated with N-nitrosodiethylamine (DEN), N-methyl-N-nitrosourea (MNU), N-butyl-N-(4hydroxybutyl)nitrosamine (BBN), dihydroxy-di-n-propylnitrosamine (DHPN), and 1,2-dimethylhydrazine (DMH) during 4 weeks. Two initiated groups received phenobarbital (PB) or 2-acetyl amino fluorene (2-AAF) for 25 weeks and two noninitiated groups received only PB or 2-AAF. A nontreated group was used as control. Lymphohematopoietic organs, liver, kidneys, lung, intestines, and Zymbal's gland were removed for histological analysis. Interleukin (IL)-2, IL-12, interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), IL-10, and transforming growth factor betal (TGF-beta1) levels were determined by ELISA in spleen cell culture supernatants. At the fourth week, exposure to the initiating carcinogens resulted in cell depletion of the thymus, spleen and bone marrow, and impairment of IL-2, IL-12, and IFN-gamma production. However, at the 30th week, no important alterations were observed both in lymphoid organs and cytokine production in the different groups. The results indicate that the initiating carcinogens used in the present protocol exert toxic effects on the lymphoid organs and affect the production of cytokines at the initiation step of carcinogenesis. This early and reversible depression of the immune surveillance may contribute to the survival of initiated cells facilitating the development of future neoplasia. (C) 2003 Elsevier B.V. All rights reserved.

Antimutagenic effect of Agaricus blazei Murrill mushroom on the genotoxicity induced by cyclophosphamide

Agaricus blazei Murrill extracts have previously been shown to have anticarcinogenic and antimutagenic proper-ties. These results suggest that antimutagenic activity, besides the modulation of the immune system, might be involved in the anticarcinogenic action of A. blazei. To investigate the possible antimutagenic effect of A. blazei in vivo, we evaluated its effect on clastogenicity induced by cyclophosphamide (CP) in mice, using the micronucleus test in bone marrow (MNPCE) and in peripheral blood (MNRET). Male Swiss mice were treated with CP (25 or 50 mg/kg i.p.) or with CP plus mushroom solution at three different temperatures: 4, 21, and 60 degreesC. Aqueous solution of a mixture from various lineages of the mushroom inhibited induction of micronuclei by CP in bone marrow and in peripheral blood of mice. In contrast to the mixture of lineages, a single isolated lineage did not lead to a reduction of CP-induced MN frequencies in either bone marrow or blood cells of mice. The results suggest that under certain circumstances these mushrooms exhibit antimutagenic activities that might contribute to an anticarcinogenic effect. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Letinula edodes (Berk.) Pegler (Shiitake) modulates genotoxic and mutagenic effects induced by alkylating agents in vivo

We evaluated the antimutagenic effect of Letinula edodes (Berk.) Pegler (Shiitake) on the frequency of micronuclei in mice treated with N-ethyl-N-nitrosourea (ENU) or cyclophosphamide (Cl?). Mice were orally (gavage) pretreated for 15 consecutive days with solutions of Shiitake (0.6 ml per day, gavage) prepared at three different temperatures: 4, 21 (RT), and 60 degreesC. Then, the animals were intraperitoneally injected on day 15 with CP (25 or 50 mg/kg) or ENU (50 mg/kg) and killed 24 or 48 h after treatment for evaluation of micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow and micronucleated reticulocytes (MNRETs). A mixture of L. edodes lineages (LE 95/016, 96/14, 96/17, 96/22, 96/23, 97/27, and 97/28) significantly decreased the frequencies of MNPCEs and MNRETs induced by CP (25 and 50 mg/kg). When a single lineage from the mixture (LE 96/17) was tested we also found a significant reduction in the frequencies of MNPCEs and MNRETs induced by both CP or ENU (50 mg/kg). The comet assay was also performed 3 h after ENU treatment using mice pretreated with the single lineage (LE 96/17) of L. edodes. The results showed a high degree of variability with some indications of an antigenotoxic effect. Taken together, our data show that solutions from Shiitake inhibit in vivo mutagenicity of CP and ENU. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Granulocytic sarcoma of the small intestine with CBF beta/MYH11 fusion gene: report of an aleukaemic case and review of the literature

Granulocytic sarcomas (GS) are rare extramedullary tumours composed of immature myeloid cells. Inversion of chromosome 16 [inv(16)] is a cytogenetic marker for M4Eo subtype of acute myeloid leukaemia (AML). The possibility of an association between the development of granulocytic sarcoma of the small intestine (GSSI) and the M4Eo subtype of AML was suggested in nine previous case reports.Here we report an aleukaemic case of GSSI with inv(16) and its molecular equivalent, the CBFbeta/MYH11 fusion gene, detected by reverse transcriptase-polymerase chain reaction (RT-PCR), that after treatment with conventional AML chemotherapy followed by autologous bone marrow transplantation, achieved complete haematological and molecular remission on bone marrow examination. After chemotherapy, a thickened ileum wall positive for CBFbeta/MYH11 on tumour mass samples was still observed on computed tomography (CT) studies, raising the question of residual GS representing a reservoir of malignant cells. This case demonstrates the critical need of multidisciplinary diagnosis and follow-up of this entity combining immunopathologic, cytogenetic and molecular studies, reinforcing the potentiality of risk-adapted therapy strategies, as it is increasingly claimed for patients with overt AML. (C) 2003 Elsevier Ltd. All rights reserved.

Cocaine mutagenicity and hepatocarcinogenicity evaluations in rodents

The mutagenicity (clastogenicity) and the carcinogenicity (promoting potential) of cocaine were evaluated, respectively, by the mouse bone marrow micronucleus test (study I) and by the initiated rat liver bioassay (study II). In study I, two administration routes (i.p. and i.v.) and two sampling times (24 and 48 hours) after cocaine treatment were studied. Swiss male mice were treated with cocaine at doses of 0, 18, 37, and 75 mg/kg and 0, 2, 4, and 8 mg/kg by i.p. and i.v. routes, respectively. No significant differences were observed between treated and negative control groups regarding the frequencies of micronuclei and the polichromatic/normochromatic erythrocyte (PCE/NCE) ratios. In study II, the development of putative preneoplastic foci of hepatocytes expressing the enzyme glutathione S-transferase placental form (GST-P+) was utilized as the end-point marker in a 8-week rat liver bioassay. The animals were initiated for carcinogenesis by a single i.p. sub-carcinogenic dose of diethylnitrosamine (DEN). After a 6-week exposure to 5 or 10 mg/kg of cocaine i.v. twice a week there was no enhancement of GST-P+ foci development above the values of the control DEN-only treated animals. Also, cocaine did not induce any toxicity as evidenced by the absence of alterations of rat body and liver weights and of liver biochemical function and morphology. The results suggest that cocaine does not have a mutagenic effect on the mouse bone marrow cells or promoting activity on the rat hepatocarcinogenesis process. Teratogenesis Carcinog. Mutagen. 18:199-208, 1998. (C) 1998 Wiley-Liss, Inc.