Avaliação do potencial citotóxico e genotóxico de Plantago major L. em sistemas teste in vivo / Evaluation of the cytotoxic and genotoxic potential of Plantago major L. in test systems in vivo

A infusão das folhas de Plantago major (Plantaginaceae), conhecida como tansagem ou transagem, é usada como antibiótica, antiinflamatória, anti-séptica, anti-térmica, na prevenção de tumores e no tratamento de neoplasias. Este efeito é atribuído aos flavonóides encontrados em diversas espécies do gênero Plantago. O presente estudo objetivou avaliar os potenciais efeitos, tóxico e mutagênico, do extrato bruto hidroalcoólico de folhas de P. major, por meio dos testes in vivo de Allium cepa e do micronúcleo. Para o ensaio biológico vegetal, meristemas de raízes de A. cepa foram usados para o preparo de lâminas através da técnica de esmagamento. No ensaio do micronúcleo foram analisadas lâminas de células de medula óssea de roedores. As análises estatísticas seguiram o teste de Tukey (p<0,05) para o ensaio de Allium cepa e teste de Scott-Knott (p<0,05) para o ensaio do micronúcleo. Os resultados do teste de Allium cepa demonstram que houve redução significativa no índice de germinação em todas as concentrações testadas. P. major provoca alteração no ciclo celular pela inibição da divisão das células, como indica o índice mitótico. Os índices de efeitos clastogênico e aneugênico demonstram que, além de não determinar aumento de aberrações cromossômicas, o que indica ausência de ação genotóxica, P. major possui atividade anti-genotóxica. Os resultados do teste do micronúcleo reforçam a sugestão de que o extrato de P.major não possui atividade mutagênica, entretanto provoca alterações na divisão celular.

The infusion of leaves of Plantago major (Plantaginaceae), known as "tansagem" or "transagem", is used as antibiotic, anti-inflammatory, anti-septic, anti-thermal in the prevention of tumors and in the treatment of neoplasms. This effect is attributed to the flavonoids found in diverse species of the genus Plantago. The present study aimed to evaluate the potential toxic and mutagenic effects of the crude hydroalcoholic extract from P. major leaves by means of in vivo tests with Allium cepa and micronucleus. For the plant biological assay, meristems of A. cepa roots were used for the preparation of slides by adopting the crushing technique. In the micronucleus assay, slides of bone marrow cells from rodents were analyzed. Statistical analyses were carried out according to Tukey's test (ρ<0.05) for the Allium cepa assay and Scott-Knott test (ρ<0.05) for the micronucleus assay. Results of the A. cepa test demonstrate that there was a significant reduction in the germination index at all tested concentrations. P. major causes alteration in the cell cycle by inhibiting the division of cells, as indicated by the mitotic index. The indexes of clastogenic and aneugenic effects show that, in addition to not determining the increase in chromosomal aberrations, which indicates the absence of genotoxic action, P. major has anti-genotoxic activity. Results of the micronucleus test reinforce the suggestion that P. major extract does not have mutagenic activity but causes alterations in the cell division.

Diagnosis of gonococcal infection in high risk women using a rapid test.

OBJECTIVE: To assess the performance and acceptability for patients and health care workers of the NGThermo Biostar (GC OIA) to diagnose gonococcal infection compared with culture using modified Thayer Martin medium. METHODS: This study involved 326 high-risk women presenting with vaginal discharge or referral by sexual partner with urethral discharge at a sexually transmitted infections (STI) clinic in Manaus, Brazil. Endocervical swabs collected from the women were tested with both the NG Biostar and modified Thayer Martin culture as the reference standard test. Clinic staff were trained to perform the NG Biostar on site and the culture was performed in the laboratory of the clinic. RESULTS: The prevalence of gonococcal infection as measured by the reference standard was 15% (50/326) overall. Among asymptomatic participants, the prevalence of infection was 17.7% (25/141) and among symptomatic women it was 13.5% (25/185) (p = 0.3). Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the NG Biostar test, with 95% confidence intervals (CI), were 60% (46.4% to 73.6%), 89.9% (86.2% to 93.6%), 55.6% (42.4% to 68.8%), and 92.6% (89.5% to 95.7%), respectively; 98.8% of study participants were willing to wait approximately 1 hour in the clinic for test results. CONCLUSION: Syndromic management protocols for treatment of STI in developing countries require refinement because, as currently described, they lead to over-treatment of cervical infection. A rapid test done during patients' initial presentation and leading to immediate treatment if positive would help improve the accuracy of diagnosis and could also be used to screen asymptomatic women. Even though the NG Biostar had a low sensitivity and PPV, which is less than ideal, it could still improve the rates of treatment over the gold standard test that requires return visits for patients to receive results and to benefit from treatment. Cost-effectiveness studies using rapid point-of-care tests for Neisseria gonorrhoeae infection compared to the syndromic approach should be carried out to assess their value in STI diagnosis and treatment in developing nations.

Tumor model-specific proviral insertional mutagenesis of the Fos/Jdp2/Batf locus.

Retroviral activation of the AP-1/ATF super family member Jdp2 was recently reported to be a common event in M-MLV-induced T cell lymphoma in p27-null C57x129 mice as compared to wild type-inoculated mice but has not been found important in other models. On the basis of retroviral tag retrieval from 1190 individual Akv- and SL3-3-induced lymphomas, we here report that insertional mutagenesis into the 250-kb Fos/Jdp2/Batf locus is associated with SL3-3 MLV-induced T but not Akv-induced B cell lymphomas of NMRI and SWR mice. Integration pattern and clonality analyses suggest that Jdp2 participates in SL3-3-induced tumorigenesis distinctly as compared to the M-MLV setting. Northern blot analysis showed Jdp2 to be alternatively spliced in various normal tissues as well as MLV-induced lymphomas. Interestingly, in some tumors, proviral insertion seems to activate different mRNA sub-species. Whereas elevated mRNA levels of the Fos gene could not be correlated with provirus presence, in one case, Northern blot analysis as well as quantitative real-time PCR indicated proviral activation of the AP-1 super family member Batf, a gene not previously reported to be a target of insertional mutagenesis. A novel integration cluster between Jdp2 and Batf apparently did not influence the expression level of either gene, underscoring the importance of addressing expression effects to identify target genes of insertion. Altogether, such distinct insertion patterns point to different mechanism of activation of specific proto-oncogenes and are consequently of importance for the understanding of proviral activation mechanisms as well as the specific role of individual oncogenes in tumor development.

Identification of a MMTV insertion mutation within the coding region of the Fgf-3 protooncogene.

The Fgf-3 protooncogene (previously called int-2) is a target of proviral insertion mutations in mammary tumors induced by the mouse mammary tumor virus (MMTV). These insertion mutations result in the transcriptional activation of Fgf-3, which is not normally expressed in the adult mammary gland. Previous mapping studies of numerous Fgf-3 insertion mutations have failed to reveal any provirus integrations within the gene coding region. This finding is consistent with the hypothesis that oncogenesis occurs in this system as a consequence of up-regulation of Fgf-3 transcription, rather than from alterations of the gene product. During an analysis of a new cohort of tumors from the WXG-2 mouse strain, a breast tumor was identified which had a MMTV provirus integrated 24 bp upstream of the Fgf-3 stop codon. This insertion mutation generated a fusion transcript which was readily detectable in tumor RNA by RT-PCR. The predicted protein product of this fusion transcript is missing 8 aa of native sequence and contains an additional 8 aa of cryptic MMTV-encoded sequence. These data document the first exception to the generalization that the Fgf-3 coding region is not disrupted by MMTV insertion mutation.

Patients with Meniére's disease possess IgE reacting with herpes family viruses.

OBJECTIVE: To determine if patients with Meniere's disease possess serum IgE specific for herpes simplex virus (HSV) type 1, HSV type 2, Epstein-Barr virus, and/or cytomegalovirus. DESIGN: A modified radioallergosorbent test method was employed wherein each serum sample was processed with recombinant protein A to remove competing non-IgE antibodies, and HSV-1, HSV-2, cytomegalovirus, and Epstein-Barr viral proteins were used as potential antigens. PATIENTS: Ten patients with long-standing active Meniere's disease were tested. Ten age- and gender-matched patients with allergic rhinitis but without Meniere's disease served as control subjects. RESULTS: IgE specific for HSV-1, HSV-2, Epstein-Barr virus, and/or cytomegalovirus was found in the serum sample of nine of 10 patients with Meniere's disease but only in four of 10 control serum samples. Of the positive subjects tested, seven patients with Meniere's disease were positive for IgE for at least three viruses compared with only two control subjects. CONCLUSIONS: (1) Most patients with Meniere's disease possess virus-specific IgE in their serum samples; (2) four viruses of the herpes family are capable of inducing such IgE-mediated sensitization; and (3) latent virus-specific, IgE-mediated inflammation may be an important factor in the initiation and/or sustenance of Meniere's disease.

Serum immunoglobulins specific for intracellular proteins of squamous cell carcinoma.

OBJECTIVE: To determine an autologous humoral immune response to squamous cell carcinoma (SCC) intracellular proteins in patients with SCC. DESIGN: Intracellular proteins were isolated from 25 different cultured SCC lines. The proteins were used as a source of antigens to measure IgA, IgE, and IgG responses in the serum samples of patients and controls. Antibody response was assessed in both unfractionated and fractionated intracellular proteins. PATIENTS: The serum samples of 65 patients with SCC and of 65 age- and gender-matched controls were tested. RESULTS: Antibodies to SCC intracellular proteins were detected in the serum samples of 40 (62%) of the 65 patients with SCC and in the serum samples of 46 (71%) of 65 controls. Thirty (46%) of the patients with SCC and 40 (62%) of the controls had IgE responses, 18 (28%) of the patients and one (2%) of the controls had IgA responses, and 17 (26%) of the patients and 14 (22%) of the controls had IgG responses. An inverse relation was noted between detectable IgE responses and IgA or IgG responses in the patients and the controls. The analysis of antibody response indicated that 28 molecules were recognized predominantly by the serum samples of patients with SCC, but not by the serum samples of controls. CONCLUSIONS: A substantial proportion of patients with SCC and of controls exhibited an autologous humoral immune response to SCC intracellular proteins. The IgE responses to SCC intracellular proteins were inversely related to IgA or to IgG responses. Different antibody isotypes normally cause markedly different immune functions, and may suggest different roles for the existent immune responses to SCC antigens. We identified many tumor-associated antigens that were selectively recognized by the serum samples of patients with SCC. These antigens could be used to define molecular studies of immune surveillance and selection, and may represent appropriate targets for immunotherapy.

Inhibition of succinate dehydrogenase and beta-hydroxybutyrate dehydrogenase activities by methylmalonate in brain and liver of developing rats.

The effects of methylmalonate (MMA) on succinate dehydrogenase (SDH) and beta-hydroxybutyrate dehydrogenase (HBDH) activities in brain and liver of 15-day-old rats were studied. The apparent Km of SDH for succinate was 0.45 mmol/L in brain and 0.34 mmol/L in liver. MMA inhibited the enzyme activity in both tissues with Ki values of 4.5 mmol/L and 2.3 mmol/L in brain and liver, respectively, and the inhibition was of the reversible competitive type. The calculated Km for HBDH with beta-hydroxybutyrate as substrate was 1.26 mmol/L in brain and 0.36 mmol/L in liver. MMA inhibited the enzyme with a Ki value of 0.015 mmol/L in brain and 0.275 mmol/L in liver. These results are probably relevant to our understanding of cerebral metabolism in methylmalonic acidaemic children, especially during ketoacidotic and hypoglycaemic crises, and may be related to the pathogenesis of cerebral dysfunction of methylmalonic acidaemia.

Effect of methylmalonate on in vitro lactate release and carbon dioxide production by brain of suckling rats.

Methylmalonate (MMA) accumulates in the tissues of patients with methylmalonic acidaemia, who present severe neurological signs soon after birth and later mental retardation. Attempting to understand the pathophysiology of the disorder, we investigated the effects of MMA on brain glucose uptake, lactate release and CO2 production. Glucose uptake and lactate release were studied by incubating 40 microns wide brain prisms from 15-day-old rats in Krebs-Ringer bicarbonate buffer, pH 7.0, containing 5.0 mmol/L glucose and one of three concentrations of MMA (1.0, 2.5 and 5.0 mmol/L). Controls did not contain MMA in the incubation medium. MMA induced a significant increase of lactate production in a dose-dependent pattern that was proportional to glucose uptake by the brain prisms. We also studied the influence of MMA on brain CO2 production from [2-14C]glucose and [U-14C]acetate by incubating brain prisms in the same buffer in the presence of the substrates with (experimental groups) or without (controls) 5.0 mmol/L MMA. MMA significantly reduced CO2 formation from both substrates.

Effects of methylmalonate and propionate on uptake of glucose and ketone bodies in vitro by brain of developing rats.

Methylmalonate (MMA) and propionate effects on glucose and ketone body uptake in vitro by brain of fed and 30-hour-fasted 15-day-old rats were studied. In some experiments cerebrum prisms were incubated in the presence of glucose and either MMA or propionate in Krebs-Ringer bicarbonate buffer, pH 7.0. In others, the incubation medium contained beta-hydroxybutyrate (HBA) or acetoacetate (AcAc) instead of glucose. We verified that MMA increased glucose uptake by brain of fasting animals, whereas propionate had no effect. In addition, MMA diminished HBA but not AcAc incorporation into brain prisms, whereas propionate provoked a diminished utilization of both ketone bodies by brain. The in vitro effect of MMA and propionate on brain and liver beta-hydroxybutyrate dehydrogenase activity was also investigated. It was shown that MMA but not propionate significantly inhibited this activity. Rats were also injected subcutaneously three times with a MMA buffered solution, and the in vivo effects of MMA on the above-mentioned parameters assessed. Results from these experiments confirmed the previously found in vitro MMA effects. Methylmalonic acidemic patients accumulate primarily methylmalonate and secondarily propionate and other metabolites in their tissues at levels comparable to those we used in our assays. Most patients who survive early stages of the disease show a variable degree of neuromotor delay. Since glucose and sometimes ketones are the vital substrates for brain metabolism, it is possible that our findings may contribute to a certain extent to an understanding of the biochemical basis of mental retardation in these patients.

Effect of postnatal methylmalonate administration on adult rat behavior.

1. Methylmalonate (MMA) levels (2.0-2.5 mM) comparable to those of human methylmalonic acidemia were achieved in blood of young rats from the 5th to the 25th day of life by injecting the drug subcutaneously twice a day with an interval of 8 h. MMA doses ranged from 0.76 to 1.69 mumol/g body weight as a function of animal age. MMA-treated rats had normal body and brain weights. 2. Behavioral studies using aversive and nonaversive tasks were performed at 60 days of life. Motor activity was similar in MMA-treated and saline-treated controls. No differences in performance between these groups were identified in the shuttle-avoidance responses and in the inhibitory avoidance tasks. However, MMA-injected rats escaped footshock faster than the controls (1.22 +/- 0.11 vs 1.76 +/- 0.14 (mean +/- SEM) for 24 rats in each group (P less than 0.01)) suggesting that they may be hyperreactive to this stimulus. 3. In the open field, a nonaversive behavior task, MMA-injected rats, in contrast to control rats, presented no habituation. 4. Our results suggest that MMA by itself may impair central nervous system function, causing minor disabilities which result in specific learning deficiencies.

Effect of postnatal methylmalonate administration on adult rat behavior

Methylmalonate (MMA) levels (2.0-2.5 mM) comparable to those of human methylmalonic acidemia were achieved in blood of young rats from the 5th to the 25th day of life by of life by injecting the drug subcutaneously twice a day with an interval of 8h. MMA doses ranged from 0.76 to 1.69 *mol/g body weight as a function of animal age. MMA-treated rats had normal body and brain weights. Behavioral studies using aversive and nonaversive tasks were performaed at 60 days of life. Motor activity was similar in MMA-treated and saline-treated controls. No differences in performance between these groups were identified in the shuttle-avoidance responses and in the inhibitory avoidance tasks. However, MMA-injected rats escaped footshock faster than the controls (1.22 ñ 0.11 vs 1.76 ñ 0.14 (mean ñ SEM) for 24 rats in each group (P<0.01)) suggesting that they may be hyperreactive to this stimulus. In the open field, a nonaversive behavior task, MMA-injected rats, in contrast to control rats, presented no habituation. Our results suggest that MMA by itself may impair central nervous system function, causing minor disabilities which result in specific learning deficiencies

Inborn errors of metabolism. Sensitivity of screening tests in high risk patients.

We estimated the sensitivity of a screening procedure (SP) for inborn errors of metabolism (IEM) in 566 referred, high-risk patients. The 143 (25.3% of the total sample) patients with initial abnormal results in at least one screening test (ST) were recalled for further investigations. An IEM was diagnosed in 40.6 percent of the 106 patients who came for reevaluation. In 114 of the remaining 423 patients who had normal initial ST, an IEM was still suspected on basis of clinical, radiological, and/or laboratory findings and was confirmed in 30 of such patients (5.3% of the total sample and 7.1% of the patients with normal results in the SP). The sensitivity of the SP was estimated maximally as 67.4 percent and the efficiency as 80.4 percent. Twenty-five of the 30 cases undetected with the SP were patients with sphingolipidoses. The simple inclusion of thin-layer chromatography of urinary oligosaccharides in the SP should allow the detection of at least one half of these cases, increasing its sensitivity by 14.1 percent and its efficiency by 4.6 percent. In at least 7.1 percent of patients with an initial normal ST, an IEM was detected. These would have remained undiagnosed if the limitations of the SP employed had not been fully understood.

Diminished concentrations of ganglioside N-acetylneuraminic acid (G-NeuAc) in cerebellum of young rats receiving chronic administration of methylmalonic acid.

Sustained levels of methylmalonate comparable to those of human methylmalonic acidemia were achieved in the blood of young rats from the 5th till the 25th day of life by injecting them subcutaneously with buffered methylmalonic acid (MMA) twice a day at 8-h intervals. A matched group of rats (controls) was treated with saline. The animals were weighed and killed by decapitation at 25 days of age. Cerebellum and cerebrum were weighed and their contents of protein, DNA and ganglioside N-acetylneuraminic acid (G-NeuAc), as well as the protein/DNA ratio determined. Body weight, cerebral and cerebellar weight did not differ in both groups. The concentrations of protein, DNA and the protein/DNA ratio were also similar in the experimental and control groups. The results indicate that MMA per se does not interfere with the appetite of the animals and does not affect cellular proliferation and growth in cerebrum and cerebellum. We also found that G-NeuAc concentration is significantly reduced in the cerebellum. Therefore, since a deficit of an important component of brain closely related to the dendritic surface (synaptogenesis) occurs in MMA-treated rats, it is tempting to speculate whether this alteration may be associated or even partly responsible for the mental retardation in patients affected by methylmalonic acidemia.

GM1 gangliosidosis: clinical and laboratory findings in eight families.

GM1 Gangliosidosis is an autosomal recessive genetic disorder due to deficiency of the lysosome enzyme beta-galactosidase, with consequent tissue accumulation of glycolipids, oligosaccharides, and especially GM1 ganglioside. In the present paper we report the clinical and laboratory findings obtained for eight families starting from eight index cases exhibiting the childhood form of the disease. The total number of cases in these families may be as high as 14, thus causing GM1 gangliosidosis to be the inborn metabolic error most frequently diagnosed in our service. Hypotonia, neuromotor retardation, hepatosplenomegaly, macrocephaly, and hydrocele are some of the most frequent clinical findings. The disease evolves towards convulsions and bronchopneumonia, leading to patient death generally during the first half of the second year of life. The presence of vacuolated lymphocytes, alterations of the lumbar vertebrae, and cherry spots on the retina were observed in almost all patients. When tested for inborn metabolic errors, all patients gave normal results, a fact that may have confused and delayed diagnosis. Diagnosis was made by urine oligosaccharide chromatography and confirmed by beta-galactoside measurement in peripheral blood leukocytes. This method proved to be accurate also for the detection of heterozygotes, which permitted post-mortem diagnosis in two families. The authors speculate that increased fetal loss and tendency towards macrosomy may be possible characteristics of the disease, suggest that testing for vacuolated lymphocytes be used as a screening method, and propose that urine oligosaccharide chromatography be included in the routine screening for inborn metabolic errors.

Tumor induction by Agrobacterium tumefaciens prevented in Vigna sinensis seedlings systemically infected by ribonucleic acid viruses.

Cowpea (Vigna sinensis) seedlings failed to develop tumors after being inoculated with crown gall bacteria (Agrobacterium tumefaciens) if, at times earlier than 1 day later, they were inoculated on the primary leaves with a cowpea mosaic virus that systemically infects them. Inoculation with buffer or with a virus that is restricted to a localized infection, or to which the cowpea is immune, did not interfere with the subsequent development of tumors. The virus infection did not appear to affect directly the titer of A. tumefaciens in the inoculation sites. Nor did mixing of virus particles with A. tumefaciens prevent subsequent appearance of tumors. The influence of virus infection extended across grafts (into tissue that is not susceptible to the virus) and there prevented tumor formation. The sap from infected plants, but not purified virus, decreased tumor formation on carrot disks. Systemic virus infection may induce in cowpeas a translocated substance that prevents tumor induction by A. tumefaciens.