Ameliorative effects of Lactobacillus against Aflatoxin B1 / Efeitos benéficos do Lactobacillus contra a Aflatoxin B1

Abstract Lactobacilli are probiotics with Aflatoxin (AF) detoxification ability, found in fermented products, GIT of animals and environment. Purpose of this study was to investigate the ability of broiler isolates of Lactobacillus against Aflatoxin B1 (AFB1). For this purpose, 5 isolates of Lactobacillus from broiler gut were incubated with 100 ppb AFB1 in aqueous environment and effect of different parameters (cell fractions, time, temperature, pH) on detoxification was determined by HPLC. The ameliorative effect of Lactobacillus salivarius (LS) against AFB1 was studied in broiler. The results revealed that LS (CR. 4) showed the best results (in vitro) as compared to other isolates (L. salivarius (CR. 3, CR, 4), L. agilis (CE. 2.1, CE. 3.1) and L. crispatus (CE. 28). Cell debris of CR. 4 showed significantly higher detoxification (P<0.05). Maximum amount of AFB1 was detoxified at 30°C (97%), pH 4.0 (99%) and 6 h (99.97%). In vivo study showed that AFB1 decreased weight gain (1,269 ± 0.04 gm/ bird), feed consumed (2,161 ± 0.08 gm/ bird), serum total protein (2.42 ± 0.34 gm/ dl), serum albumin (0.5 ± 0.2 2 gm/dl) and antibody titer (4.2 ± 0.83). Liver function enzymes were found (alanine transaminase (ALT): 32 ± 10.7 U/L) and aspartate transaminase (AST): 314.8 ± 27 U/L) elevated in AFB1 fed broilers. Treatment with 1% LS not only decreased the toxic effects of AFB1 (group D) but also improved the overall health of broilers due to its probiotic effects (p<0.05) as compared to control negative (group A). The detoxification ability of LS was better than commercial binder (CB) (0.2% Protmyc). It was concluded that detoxification of AFB1 by Lactobacillus was strain, temperature, pH and time dependent. LS has detoxification ability against AFB1 in vivo.

Resumo Os lactobacilos são probióticos com capacidade de desintoxicação da Aflatoxina (AF), encontrados em produtos fermentados, TGI de animais e meio ambiente. O objetivo deste estudo foi investigar a capacidade de isolados de frango de corte de Lactobacillus contra a Aflatoxina B1 (AFB1). Para tanto, 5 isolados de Lactobacillus de intestino de frango foram incubados com 100 ppb AFB1 em meio aquoso, e o efeito de diferentes parâmetros (frações celulares, tempo, temperatura, pH) na desintoxicação foi determinado por CLAE. O efeito melhorador de Lactobacillus salivarius (LS) contra AFB1 foi estudado em frangos de corte. Os resultados revelaram que LS (CR. 4) apresentou os melhores resultados (in vitro) em comparação com outros isolados [L. salivarius (CR. 3, CR. 4), L. agilis (CE. 2.1, CE. 3.1) e L. crispatus (CE. 28)]. Detritos celulares de CR. 4 mostraram desintoxicação significativamente maior (P < 0.05). A quantidade máxima de AFB1 foi desintoxicada a 30 °C (97%), pH 4.0 (99%) e 6 h (99,97%). O estudo in vivo mostrou que AFB1 diminuiu o ganho de peso (1,269 ± 0.04 g / ave), alimento consumido (2,161 ± 0.08 g / ave), proteína total sérica (2.42 ± 0.34 g / dl), albumina sérica (0.5 ± 0.22 gm / dl) e título de anticorpo (4.2 ± 0.83). As enzimas da função hepática foram encontradas (alanina transaminase (ALT): 32 ± 10.7 U / L) e aspartato transaminase (AST): 314.8 ± 27 U / L) elevadas em AFB1 alimentados com frangos. O tratamento com 1% LS não só diminuiu os efeitos tóxicos de AFB1 (grupo D), mas também melhorou a saúde geral dos frangos devido aos seus efeitos probióticos (p < 0.05) em comparação com o controle negativo (grupo A). A capacidade de desintoxicação do LS foi melhor do que o aglutinante comercial (CB) (0.2% Protmyc). Concluiu-se que a desintoxicação de AFB1 por Lactobacillus foi dependente da cepa, temperatura, pH e tempo. LS tem capacidade de desintoxicação contra AFB1 in vivo.

Andrographia paniculata a Miracle Herbs for cancer treatment: In vivo and in vitro studies against Aflatoxin B1 Toxicity

The history of natural products used in ancient times and in folk medicine these days, around the world, is the basis for the use of many therapeutic drugs in modern day medicine. Andrographia paniculata belongs to the family Acanthaceae or Kalmegh and is commonly known as 'king of bitters'. It is extensively used as home remedy for various diseases in Indian traditional system as well as in tribal system in India for multiple clinical applications. In our present work, extracts of these ayurvedic plants were tested for their anticlastogenic, and anticarcinogenic properties against Aflatoxin Bl induced toxicity. We used the in vitro method i.e. human lymphocytes culture and in vivo method in bone marrow cells of albino mice, while the parameters studied included chromosomal aberrations [CA], sister chromatid exchanges [SCEs] and cell growth kinetics [RI] both in the presence as well as in the absence of exogenous metabolic activation system for in vitro studies, whereas total aberrant cells and the frequencies of aberrations were used for in vivo methods A. paniculata extracts significantly reduced chromosomal aberrations from 35.0%, 62.0% and 69.0% level [at 24, 48, and 72 h due to Aflatoxin B1] to 21.72%, 44.0% and 52.0%, similarly sister chromatid exchanges were reduced from 14.60 per cell to 7.50 per cell at 48 h of treatments and replication index was enhanced in vitro for each concentration and duration of treatment. In conclusion A. paniculata extracts significantly reduced the number of aberrant cells and frequencies of aberration per cell at each concentration and duration of exposure in vivo; similarly it reduced chromosomal aberrations and sister chromatid exchanges and replication index was enhanced in vitro that was statistically significant at < 0.05 level

Protective effect of pretreatment with thymoquinone against aflatoxin B1 induced liver toxicity in mice

Thymoquinone [TQ] is one of the active components of Nigella sativa. The plant has been used in herbal medicine for treatment of many diseases including liver complications. The present study aimed to investigate protective effects of TQ on Aflatoxin B1 [AFB1] induced liver toxicity in mice. Animals were divided into six groups and treated intraperitoneally. Group 1 [blank] served as vehicle, group 2 [positive control] received AFB1, Group 3 was treated with 9 mg/kg of TQ, Groups 4, 5 and 6 were treated with 4.5, 9 and 18 mg/kg of TQ, respectively. After three consecutive days, except for groups 1 and 3, animals were administered with a single dose of AFB1 [2 mg/kg]. All the animals were killed 24 hrs following the AFB1 administration under ether anesthesia. Biochemical parameters including AST, ALT and ALP in serum samples and glutathione [GSH] and malondialdehyde [MDA] contents in liver homogenates were determined. Liver sections were collected for histopathological examination. Findings of this study showed that AST, ALT, ALP and MDA levels were significantly lower in the TQ treated animals as compared to AFB1 group [group 2]. Furthermore, TQ was able to recover glutathione content [GSH] of liver tissue. The best response, however, was observed with the dose of 9 mg/kg. Liver sections of AFB1 intoxicated mice showed inflammation, necrosis, hyperplasia of kupffer and infiltration of mononuclear cells, dilation of sinusoids and disruption of hepatocytes, while treatment with TQ helped to normalize liver architecture in accordance to biochemical findings. Taken collectively, TQ has a protective role with optimum dose of 9 mg/kg in AFB1 hepatotoxicity

Plasma haptoglobin and immunoglobulins as diagnostic indicators of deoxynivalenol intoxication

This study aimed to discover potential biomarkers for dioxynivalenol (DON) intoxication. B6C3F1 male mice were rally exposed to 0.83, 2.5 and 7.5 mg/kg body weight (bw) DON for 8 days and the differential protein expressions in their blood plasma were determined by SELDI - Time-of-Flight/Mass Spectrometry (TOF/MS) and the immunoglobulins (Igs) G, A, M and E in the serum were investigated. 11.7 kDa protein was significantly highly expressed according to DON administration and this protein was purified by employing a methyl ceramic HyperD F column with using optimization buffer for adsorption and desorption. The purified protein was identified as a haptoglobin precursor by peptide mapping with using LC/Q-TOF/MS and MALDI-TOF/MS and this was confirmed by western blotting and ELISA. IgG and IgM in serum were decreased in a dose-dependent manner and IgA was decreased at 7.5 mg/kg bw DON administration, but the IgE level was not changed. To compare the expressions of haptoglobin and the Igs patterns between aflatoxin B1 (AFB1), zearalenone (ZEA) and DON intoxications, rats were orally administered with AFB1 1.0, ZEA 240 and DON 7.5 mg/kg bw for 8 days. Haptoglobin was increased only at DON 7.5 mg/kg bw, while it was slightly decreased at ZEA 240 mg/kg bw and it was not detected at all at AFB1 1.0 mg/kg bw. IgG and IgA were decreased by DON, but IgG, IgA, IgM and IgE were all increased by AFB1. No changes were observed by ZEA administration. These results show that plasma haptoglobin could be a diagnostic biomarker for DON intoxication when this is combined with examining the serum Igs.

Modulatory effect of Gynandropsis gynandra L. on glucose metabolizing enzymes in aflatoxin B1-induced hepatocellular carcinoma in rats.

The modulation of glucose-metabolizing enzymes activities play a vital role in the depletion of energy metabolism and leads to inhibition of cancer growth. In the present study, the effect of Gynandropsis gynandra L. extract on aflatoxin B1 (AFB1)-induced hepatocellular carcinoma (HCC) was studied on glucose-metabolizing enzymes in rats. A significant increase (p < 0.001) in the activities of the key glycolytic enzymes viz., hexokinase and phosphoglucoisomerase, with a significant decrease (p < 0.001) in the gluconeogenic enzymes glucose-6-phosphatase and fructose-1,6-bisphosphatase were observed in HCC-bearing rats, when compared with the control. Administration of G. gynandra extract caused a significant decrease in the activities of glycolytic enzymes and an increase in the gluconeogenic enzymes activities to near normal values. Thus, findings suggest the G. gynandra extract has a definite modulating role on the key enzymes of glucose metabolism in HCC. The modulatory effect may be due to the phytoactive constituents present in the extract of G. gynandra.

Preventive role of Gynandropsis gynandra L., against aflatoxin B1 induced lipid peroxidation and antioxidant defense mechanism in rat.

G. gynandra extract was found to potentially diminish the rate of lipid peroxidation, with a significant increase in the levels of enzymatic (superoxide dismutase, catalase and glutathione peroxidase) and non-enzymatic (reduced glutathione vitamins E and C, and uric acid) antioxidants, which were found, altered during aflatoxin B1 (AFB1) injection. The result confirmed that G. gynandra extract exerts its chemopreventive efficacy by preventing the rate of lipid peroxidation and influenced the enzymatic and non-enzymatic antioxidants in AFB1 induced male albino rats.

Inactivación de aflatoxina B1 y aflatoxicol por nixtamalización tradicional del maíz y su regeneración por acidificación de la masa / Inactivation of aflatoxin B1 and aflatoxicol through traditional

OBJETIVO: Confirmar el efecto de la nixtamalización tradicional sobre la aflatoxina, identificar el compuesto remanente en masa, evaluar su toxicidad y su regeneración por tratamiento ácido. MATERIAL Y MÉTODOS: Se utilizó maíz, sin y con aflatoxina, y se nixtamalizó. La toxicidad se evaluó en pollos de ocho días de edad. Se aplicó el tratamiento ácido a la masa. La cuantificación de aflatoxinas se realizó por cromatografía líquida de alta resolución (HPLC). RESULTADOS: La nixtamalización destruyó la aflatoxina (96 por ciento) y el aflatoxicol (70 por ciento); el remanente en masa fue aflatoxina B1. El tratamiento ácido in vitro no eleva las concentraciones de ninguna de las dos micotoxinas. Los pollos murieron al ingerir 260 mg de AFB1, y la masa con aflatoxina remanente no fue tóxica. CONCLUSIONES: Los resultados ilustran el beneficio de la nixtamalización tradicional en la inactivación de las aflatoxinas presentes en maíz y en su no reconstitución por efecto del tratamiento ácido.

Hepatocellular carcinoma--a model of human cancer: clinico-pathological features, etiology and pathogenesis.

Hepatocellular carcinoma (HCC), a unique human neoplasm, has interested many in several fields of biological and health sciences. This cancer is credited as the first that can be largely eliminated by a safe anti-viral vaccine and other transmission control measures. It is also the first cancer for which a reliable diagnostic tumour marker was identified and studies on this tumor in humans and animals have provided a large body of information on causation and step-wise evolution of cancers. Being a common and rapidly fatal tumour, mainly affecting males in the more populous developing countries, HCC may well be the commonest cancer of the human male. Clinical features are not specific for HCC but manifestations represent varying combinations of those due to cirrhosis which is a very frequently associated and pre-existent disease, those due to tumour and those due to malignancy. This tumour commonly takes a massive form with satellite nodules or a scattered multinodular form. A fibrolamellar variant is biologically and clinically quite different from the usual HCC and the small capsulated variant is seen in some geographic areas. Microscopically the trabecular variety is common and differentiation from metastatic cancers and benign lesions may need use of newly described immunohistochemical markers in addition to clinical evidence of cirrhosis. Hepatitis B and C viruses, dietary aflatoxin B1 and cirrhosis from any cause are common risk factors in that order of importance. Several lines of evidence including molecular biology and animal studies support these etiological linkages. Studies in experimental animals using chemical carcinogens, different hepatotropic viruses and aflatoxin have greatly helped our understanding of carcinogenesis in general and hepatocarcinogenesis in particular. Individual HCC risk factors may contribute to summation of mutations subsequent to repeated and continued liver cell injury act as carcinogen/co-carcinogen or be involved in both capacities.

Promotion of aflatoxin B1-induced hepatocarcinogenesis by dichlorodiphenyl trichloroethane (DDT).

A study of the effect in rats of dichlorodiphenyl trichloroethane (DDT) on hepatocarcinogenesis that is initated by aflatoxin B1 (AFB1). In the first experiment, Buffalo rats were given a single oral dose of AFB1 (5 mg/kg) followed by dietary DDT (100 ppm) for 20 weeks. Neoplastic nodules were observed in 1 of the 14 AFB1-exposed rats, compared with 3 of the 19 rats in the AFB1/DDT group. In the second experiment, Wistar rats were given dietary aflatoxin B, (4 ppm) for 6 weeks followed by a 6-week exposure to DDT (500 ppm) in a plain semisynthetic diet. Five altered hepatic foci were displayed by seven rats in the AFB1 group, compared with 6 foci and one neoplastic focus in five of the AFB1/DDT rats at 32 weeks. Subsequently, the AFB1 group produced 8 (27.5%) tumor-bearing rats while 10 of the 28 (35.7%) AFB1/DDT-exposed rats were tumor-bearing by 60 weeks. The results suggest that DDT slightly potentiates hepatocarcinogenesis induced by either a single dose of AFB1 or short term-dietary AFB1.

Isolation, partial characterization and growth characteristics of a bacterium resistant to aflatoxin B1.

A bacterium was isolated from the soil of dumping ground for cattle yard waste by enrichment culture containing aflatoxin B1. This bacterium was closely related to Bacillus firmus that was found to be a non-pathogenic bacterium. The minimum inhibitory concentration of aflatoxin B1 to the bacterium was found to be 80 microg ml(-1) as measured by total viable count and soluble protein content methods. The bacterium was sensitive to all the tested antibiotics. Plasmid curing by chemical agents did not show the resistance character residing in the plasmid. Protein profiles of cell extracts of aflatoxin B1 resistant bacterium grown in the presence and absence of the toxin showed 46 and 44 protein bands respectively in SDS-PAGE. It was observed that 39 bands were common in both the extracts and the remaining bands were showing differences near the high molecular weight range.

Determinación de aflatoxina M1 en leche / Detection of aflatoxin M1 in milk samples

La presencia de micotoxinas en productos lácteos se debe, por lo general, a la ingesta por parte del ganado lechero de alimentos contaminados con aflatoxina B1 (AFB1). La AFM1 es el principal metabolito hepático 4-hidroxilado de la AFB1 que se excreta por la leche. Es tan tóxica como la AFB1, aunque algunos autores han demostrado que no es tan mutagénica. Recientemente se han clasificado a las AFB1 y AFM1 como carcinógenos humanos de la clase 1 y 2B respectivamente y también se ha observado que la AFM1 tiene una alta actividad genotóxica. El objetivo de este trabajo es determinar por TLC (cromatografía en capa delgada) la presencia de AFM1 en 50 muestras de leche, recolectadas en los meses de otoño, tanto de origen comercial como provenientes de pequeños tambos, donde se ordeñan artesanalmente, para estimar los niveles de exposición a la AFM1 en la población. No se detectó la presencia de AFM1 en ninguna de las muestras estudiadas hasta este momento. Esto muestra que en esta época del año, donde las vacas lecheras se alimentan de pasto natural, no existe contaminación. Seguiremos estudiando muestras recolectadas en los meses de invierno para controlar que sucede cuando además reciben suplemento alimentario. La leche es el alimento básico consumido sobre todo por niños en etapa de crecimiento; por lo tanto, debe ser monitoreada de contaminantes, incluyendo AFM1, más aún en países como el nuestro donde los factores climáticos pueden favorecer la incidencia de la misma. En nuestro país hay muy escasos datos de incidencia de AFM1 en la leche fluida, leche en polvo y en otros derivados lácteos. La dificultad para realizar estudios toxicológicos en humanos con la ingesta de AFM1 y la dificultad para la detoxificación de micotoxinas de las dietas hacen de los programas de monitoreo la principal estrategia para disminuir el riesgo de exposición a estas micotoxinas, por eso creemos que más estudios deberían efectuarse para intensificar los conocimientos acerca de toda nuestra producción lechera

Actividad antimutagénica de extractos vegetales sobre la Aflatoxina B1 / Antimutagenic activity of vegetable extracts on aflatoxin B1

Se evaluó la capacidad de los extractos de diferentes vegetales (berro, rábano, tomate, zanohoria, lechuga, col, remolacha y pepino) para reducir la mutagenicidad de la aflatoxina B1, por medio del ensayo de Ames. Se tomaron 200 µL del jugo natural de los vegetales y se combinaron con 10(8) células de la cepa TA 98 de Salmonella typhimurium, 50 ng de agflatoxina B1 y 0,5 mL de la mezcla S9. Los resultados indican que el berro fue capaz de reduir la mutagenicidad de la toxina en el 64 por ciento, en tanto el rábano lo hizo en el 36 por ciento, el tomate en el 17 por ciento y la zanahoria en el 15 por ciento; en el caso de los restantes exctractos la reducción fue muy poca o nula

Identificación fisicoquímica de algunos aluminosilicatos y de su actividad adsorbente sobre aflatoxina B1 in vitro / In vitro physico-chemical identification and adsorbent efficacy of some commercial aluminosilicates on aflatoxin B1 in Mexico

Se tomaron muestras de 6 aluminosilicatos comerciales distribuidos en México. A cada muestra se la practicaron los siguientes análisis: contenido de potasio (K), calcio (Ca), sodio (Na), aluminio (Al) y silicio (Si), por medio de espectrofotometría de absorción atómica; el contenido de K en las muestras analizadas varía de 0 a 0.89 por ciento, el Ca de 0 a 0.76 por ciento, el Na de 0.42 a 1.68 por ciento, el AI de 0.90 a 8.15 por ciento y el de Si de 13.29 a 62.71 por ciento. Resalta la muestra de Fusox en cuento a contenido de Si (62.7 por ciento). La proporción de A1/Si en las muestras analizadas fue la siguiente: Milbond, 0.416; trisil, 0.356; Quibenzil, 0.13; Rekasil, 0.276; Boldclay, 0.337; Fusox, 0.14. Se midió el tamaño de las partículas por microscopía electrónica de barrido y se encontró que Milbond, Trisil, Quibensil y Rekasil según el tamaño de sus partículas corresponden a una zeolita, a diferencia de Boldclay, con 72.62µ. Los resultados obtenidos en la confrontación in vitro, de los 6 aluminosilicatos comerciales, mostraron que sólo dos de ellos presentaron actividad adsorbente de aflatoxina B1. El porcentaje de esta actividad fue de 97.5 en Milbond y de 90 en Fusox. Después de la medición del pH se encontró que no existe relación entre la capacidad adsorbente de aflatoxina B1 y el pH del aluminosilicato

Histological study of the effect of contaminated food by mycotoxin AFBI and CPA in albino rats

Fungal flora of mixed food belonging to Aspergillus, Mucor, Penicillium and Rbizopus species were the most frequently isolated, from mixed poultry feed, especially A. flvus This study was conducted to determine whether exposure to cyclopiazonic acid [CPA] and Aflatoxin[B1] [AFB1] wouId alter the toxicity with exposure to either toxin individually. Seventy-two adult rats of both sexes were used, classified into 6 groups, 12 for each. The groups were dosed 0, 0.1 and 4 mg/kg B. W. [CPA: cyclopiazonic acid] and 0, 0.1 and 0.1 mg/kg B. W. [AFB1: Aflatoxin [B1]] daily for 3 successive doses for each group. 6 rats of each group were sacrificed alter 4 days from the initial dose, the remainder were left for a recovery period of 4 days prior to being killed. Small pieces of liver, kidney and spleen were processed histologically and paraffin sections were stained, then examined microscopically. Grossly, loss of weight of the rats receiving 0.2 mg/kg B. W. aflatoxin within 24 hours from the first dose and food consumption was 60% of controls. Rats of recovery lost 31-38 gm of their weights, the food consumption was 50%. In groups receiving CPA, food consumption was 75 of controls. Microscopically, the liver showed severe interstitial haemorrhages and lymphocytosis in the portal tracts in [lethal doses]. In animals receiving low doses, focal areas of haemorrhages were discerned with some lymphocytic infiltration, while in animals of [one dose], there were hypertrophied liver cells with many pyknotic nuclei of many cells. The kidneys of one dose had congested cortical blood vessels with pyknotic nuclei in the epithelial cell of the renal tubules. In [lethal doses], the congestion of renal tissue blood vessels was not so extensive as in the liver which was the most organ to be affected. The spleen, revealed delayed red pulp area on the expense of the white pulp in the lethal dose. In other doses, only vacuolation of many large Iympbocytes were seen in the splenic follicles