Effects of aflatoxin B1 on murine lymphocytic functions

We recently reported on the immunotoxic effects of Aflatoxin B1 (AFB1), a secondary fungal metabolite of Aspergillus flavus in mice. The present paper describes the effect of AFB1 on cellular functions in lymphocytes after in vivo or in vitro exposures. Male BALB/c mice received 0, 0.03, 0.145 or 0.7 mg/kg body weight of AFB1 for 4 weeks. A dose-related decrease in DNA synthesis in lymphocyte cultures with or without the mitogens (lipopolysaccharide, phytohemagglutinin, pokeweed mitogen and concanavalin A) was observed. No effects on protein or ribonucleic acid synthesis were observed. There were dose-dependent decreases in peripheral leucocyte counts and natural killer cell function. Splenic lymphocytes from normal mice cultured with 10(-6) to 10(-4) M AFB1 had decreased DNA synthesis at greater than 1 X 10(-5) M, a decreased protein synthesis at 10(-4) M and decreased RNA synthesis at greater than 2.5 X 10(-5) M. In vitro addition of AFB1 (greater than 50 microM) reduced generation of concanavalin A-induced suppressor cells. The results suggest that AFB1 had a direct and complex effect on lymphocytes and there is a differential sensitivity of various subpopulations.     

In vitro inhibitory effect of aflatoxin B1 on acetylcholinesterase activity in mouse brain

Growing concern on the problem of mycotoxins in the alimentary chain underlines the need to investigate the mechanisms explaining the cholinergic effects of aflatoxin B(1) (AFB(1)). We examined the effect of AFB(1), a mycotoxin produced by Aspergillus flavus, on mouse brain acetylcholinesterase (AChE) and specifically on its molecular isoforms (G(1) and G(4)) after in vitro exposure. AFB(1) (from 10(-9) to 10(-4)M), inhibited mouse brain AChE activity (IC(50) = 31.6 x 10(-6)M) and its G(1) and G(4) molecular isoforms in a dose-dependent manner. Michaelis-Menten parameters indicate that the K(m) value increased from 55.2 to 232.2% whereas V(max) decreased by 46.2-75.1%. The direct, the Lineweaver-Burk and the secondary plots indicated a non-competitive-mixed type antagonism, induced when the inhibitor binds to the free enzyme and to the enzyme-substrate complex. AFB(1)-inhibited AChE was partially reactivated by pyridine 2-aldoxime (2-PAM) (10(-4)M) but the AChE-inhibiting time courses of AFB(1) (10(-4)M) and diisopropylfluorophosphate (DFP) (2 x 10(-7)M) differed. Overall these data suggest that AFB(1) non-competitively inhibits mouse brain AChE by blocking access of the substrate to the active site or by inducing a defective conformational change in the enzyme through non-covalent binding interacting with the AChE peripheral binding site, or through both mechanisms.     

Immunotoxic effect of beta-chlorolactic acid on murine splenocyte and peritoneal macrophage function in vitro

β-Chlorolactic acid is a major intermediate of 3-monochloro-1,2-propanediol (MCPD) in mammalian species, which a well-known by-product of acid-hydrolyzed soy sauce during its manufacturing process. β-Chlorolactic acid has not been studied on immunotoxicity. To evaluate the immunomodulatory effect of β-chlorolactic acid on murine splenocyte and macrophage in vitro, we investigated splenocyte blastogenesis by concanavalin A (Con A), anti-CD3 and lipopolysaccharide (LPS), the production of cytokines from splenocyte, and the activity of mouse peritoneal macrophages. β-Chlorolactic acid suppressed significantly splenic blastogenesis to Con A or anti-CD3 from 8.5 to 54.7% at doses comprised between 200 and 800 μM. β-Chlorolactic acid also suppressed significantly splenic blastogeneis to LPS from 8.5 to 71.5% at doses comprised between 200 and 800 μM. The production level of interferon (IFN)-g on splenocyte culture with Con A was significantly reduced from 21.5 to 51.4% at the higher concentration than 100 μM of β-chlorolactic acid. The levels of interleukin (IL)-2 and IL-4 were also decreased 22.6–58.4 and 10.2–36.6%, respectively, at high concentrations of β-chlorolactic acid. There was a significant decrease from 6.1 to 40.8% in the production of nitric oxide (NO) by peritoneal macrophages treated with 400–1000 μM β-chlorolactic acid. These results indicate that β-chlorolactic acid might be able to induce immunotoxic effect on immune response of lymphocytes and peritoneal macrophages in vitro.     

In vitro effect of aflatoxin B1 on rat liver macrophages (Kuffer cells)

The effect of aflatoxin B1 on the uptake and incorporation of [3H]leucine and [3H]uridine and on phagocytosis of latex particles was studied using cultures of rat liver macrophages (Kuffer cells). Aflatoxin B1 inhibited the incorporation of both isotopes, but inhibition of uridine incorporation was greater than that of leucine, suggesting that RNA synthesis was a major site of inhibition. Aflatoxin B1 also inhibited phagocytosis of latex particles in a time- and dose-dependent manner.     

In vitro studies on the metabolism of aflatoxin B1 and aldrin

As Drosophila melanogaster occupies an important position within the test battery for mutagens and carcinogens, it is of interest to study the xenobiotics metabolism of this insect. Likewise, the genetic control of these important enzyme systems falls within this interest.Our attempt was to get new strains, which show changes in their xenobiotics metabolism. This was done by a mutagenization and selection procedure for the second chromosome. The 44 fertile homozygous inbred strains produced by this selection were first tested for DDT resistance. Some of them showed LT50 values which were remarkably higher than that of the original strain Berlin K.Aflatoxin B₁ metabolism in two of the new strains (H349 and H362), Berlin K, and Hikone-R was compared, whilst aldrin epoxidase activity was compared in strains H349, H362, Berlin K, vestigial, and Karsnäs-R. The metabolism studies were carried out in vitro with testes tissue of the different strains. The metabolism in testes is of specific interest because this tissue is most often used in mutagenicity testing.In the AFB₁ assays of the up to 12 observed metabolites three could be identified as AFB_2a, AFM₁, and AFR₀. Hikone-R produced mostly AFR₀ (3.43% of the initial AFB₁ concentration) and small amounts of AFM₁ (0.59% AF) and AFB_2a (0.36% AF). The strain Berlin K showed only a low production of AFB_2a (0.48% AF), while the strain H349 formed AFR₀ (6.02% AF) and AFM₁ (0.75% AF). The AFM₁ appeared in even higher amounts than with Hikone-R. On the other hand, H362 showed the lowest activity in AFB₁ metabolism. With this strain none of the determined metabolites could be detected in levels significantly higher than the control. The difference between H349 and the original strain Berlin K was highly significant. The production of AFR₀ and the binding of aflatoxin to macromolecules show a linear correlation. In both parameters measured, the strain H349 yielded the highest results. The determination of aldrin epoxidase activity gave the following results (in pmol dieldrin · mg^–1 protein · min^–1): H349: 0.74; Karsnäs-R: 0.57; vestigial: 0.57; Berlin K: 0.32; H362: 0.27. Again the difference between H349 and Berlin K was statistically significant. The measured activities match values obtained with extrahepatic tissue of mammals.It is concluded that the line H349 is a mutant in the xenobiotic metabolism. For the strains Hikone-R, Karsnäs-R, and H349 AFR₀ could be confirmed to be the main metabolite of AFB₁. The metabolism pattern was shown to differ strongly from strain to strain.Abbreviations AFB₁, AFB2_2a and AFM₁ aflatoxins b₁, B_2a, and m₁ - AFR₀ aflatoxicol - DDT 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane - EMS ethyl methanesulfonate     

Effects of aflatoxin B1 on myelopoiesis in vitro.

The short-term cultures of mouse myeloid progenitor cells for granulocytes and monocytes, granulocyte-monocyte colony-forming units (CFU-GM) (CFU-GM assay) and mouse long-term bone marrow culture (LTBMC) were used to investigate the hemopoietic suppression caused by aflatoxin B1 (AFB1). A dose-related suppression of granulopoiesis in short-term bone marrow cultures was seen when the cultures were treated with 10, 5, 1, 0.5 and 0.1 micrograms of AFB1/ml. Two selected doses of AFB1 (5 and 0.5 micrograms/ml) considered to be highly and slightly suppressive in CFU-GM assay exerted a strong suppression of myelopoiesis in LTBMC when applied long-term. Short-term (2 h) exposure of LTBMC to 5 micrograms of AFB1/ml caused a small damage to the myelopoiesis detected in the non-adherent layer. Short-term exposure to 0.5 micrograms AFB1/ml was without any effect on myelopoiesis in LTBMC. The production of colony-stimulating activity (CSA) by an adherent layer of LTBMC was decreased on the second and fifth day after the short-term exposure to both doses of AFB1 and comparable with non-treated culture on the seventh day after the exposure. Presented results indicate that both short-term culture of CFU-GM and LTBMC can be used in the definition and the prediction of host toxicity of AFB1 to hemopoiesis. However, comparing these two in vitro systems, the LTBMC appears to be more sensitive and discriminatory in an evaluation of hemopoietic toxicity.     

In vitro effects of aflatoxin B1 on the activities of three hydrolases

The in vitro effects of aflatoxin B1 on the activities of trypsin, amylase and lipase in the pancreatic extract of male albino Wistar rats have been studied. Activities of the 3 hydrolases were inhibited at 1 microgram/ml, 200 microgram/ml and 400 microgram/ml concentrations of aflatoxin B1, with the exception that at the 1 microgram/ml concentration the activity of lipase was stimulated. Lineweaver-Burk reciprocal plots indicate non-competitive inhibition of the 3 hydrolases.     

The immunomodulatory effects of the herbicide simazine on murine macrophage functions in vitro.

We examined the immunomodulating effects of simazine, a triazine herbicide, on murine peritoneal macrophages after in vitro pre-exposure. When thioglycollate-elicited macrophages pre-exposed to simazine were stimulated with lipopolysaccharide (LPS), the antitumor activity induced by LPS was suppressed by simazine. Simazine also inhibited poly I:C-induced antiviral activity and interferon (IFN) production in macrophages. In addition, the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) which have been known to be major effector molecules in macrophage-mediated cytotoxicity was decreased by simazine pretreatment in a dose-dependent manner. However, simazine had little effect on phagocytosis and the level of hydrogen peroxide (H(2)O(2)), interleukin-1 (IL-1) and IL-6 by LPS-stimulated macrophages. Taken together, these data indicate that simazine has a differential immunomodulating effect on macrophage secretory and cellular activities.     

In vitro time-dependent vancomycin-resistant Staphylococcus aureus-induced free radical generation and status of antioxidant enzymes in murine peritoneal macrophage

Staphylococcus aureus is most frequently isolated pathogen causing bloodstream infections, skin and soft tissue infections, and pneumonia. The immune cells use reactive oxygen species (ROS) for carrying out their normal functions, while an excess amount of ROS can attack cellular components that lead to cell damage. The aim of the present study was to evaluate the free radical generation and status of the antioxidant enzymes in murine peritoneal macrophage during in vitro vancomycin-resistant S. aureus (VRSA) treatment with different time intervals. Peritoneal macrophages were treated with 5?×?10(6) colony-forming units (CFU)/mL VRSA cell suspension in vitro for different time intervals (1, 2, 3, 6, 12, and 24 h), and superoxide anion generation, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, myeloperoxidase (MPO) activity, nitric oxide (NO) generation, antioxidant enzyme status, and components of glutathione cycle were analyzed. Superoxide anion generation, NADPH oxidase activity, MPO activity, and NO generation got peak at 3 h indicates maximum free radical generation through activation of NADPH oxidase in murine peritoneal macrophages during VRSA infection. Reduced glutathione level, glutathione peroxidase, glutathione reductase, and glutathione S-transferase activity were decreased significantly (P?相似文献   

In vitro stimulation of murine peritoneal monocytes induced by alginates

In this trial we assessed the effect of soluble alginates on murine cells. Mouse peritoneal monocytes were stimulated in vitro with a solution of alginate. The production of TNF-alpha and nitric oxide (NO), the expression of surface molecules CD80 and CD86, and the ability of monocytes to phagocyte bacteria were assessed, in order to evaluate the effect of alginate on cell functionality. We showed that mouse peritoneal monocytes stimulated with alginate produce NO and TNF-alpha. In addition, alginate is able also to increase their phagocytic activity and to a lesser extent also to increase the expression of CD80. Even with different degrees, it implies that alginates per se act directly on immune response, being able to effectively stimulate proinflammatory activity. These findings corroborate the idea that alginates can represent interesting adjuvants to use to increase the efficacy of antigenic stimulation.     

The immunomodulatory effects of 3-monochloro-1,2-propanediol on murine splenocyte and peritoneal macrophage function in vitro.

3-Monochloro-1,2-propanediol (MCPD) is a well-known by-product of acid-hydrolyzed soy sauce during its manufacturing process. MCPD has been reported genotoxic in vitro, and reproductive toxicity and carcinogenicity in rats. To evaluate the immunomodulatory effect of MCPD on murine splenocyte and macrophage in vitro, we investigated splenocyte blastogenesis by concanavalin A (Con A), anti-CD3, and lipopolyssacharide (LPS), the production of cytokines from splenocyte, and the activity of mouse peritoneal macrophages. There was a significant decrease in lymphocyte blastogenesis to Con A or anti-CD3 at subtoxic dose of MCPD. A significant decrease in splenocyte blastogenesis to LPS was also observed. The production level of interferon (IFN)-gamma on splenocyte culture with Con A was significantly reduced at the higher concentration than 1.0mM of MCPD. The levels of interleukin (IL)-4 and IL-10 were also decreased at high concentrations of MCPD. There was a significant decrease in production of nitric oxide (NO) by peritoneal macrophages treated with MCPD. MCPD also inhibits tumor necrosis factor (TNF)-alpha production of stimulated macrophages. These results indicate that MCPD might be able to reduce the functionality of lymphocytes and peritoneal macrophages in vitro.     

杂色曲霉素对体外小鼠腹腔巨噬细胞白细胞介素12表达与分泌的影响

目的　探讨杂色曲霉素(ST)对巨噬细胞免疫功能的影响。方法　分别采用半定量逆转录聚合酶链反应及酶联免疫吸附法,研究0 .12 5 ,0 .2 5 ,0 .5 ,1.0和2 .0mg·L- 1ST预处理对小鼠腹腔巨噬细胞白细胞介素(IL) 12mRNA表达及其蛋白分泌的影响。结果　在0 .12 5～1.0mg·L- 1范围内,ST处理对小鼠腹腔巨噬细胞IL 12p4 0和IL 12p35mRNA的表达均有一定的抑制作用。但在蛋白水平,对IL 12分泌的影响不明显。当ST浓度达到2 .0mg·L- 1时,可明显抑制IL 12p4 0mRNA的表达及IL 12的分泌。结论　ST可抑制巨噬细胞IL 12的表达或分泌,提示ST可能在一定程度上对机体的细胞免疫功能产生不利影响。     

In vitro toxicity of aflatoxin B1 and its photodegradation products in HepG2 cells

The effects of aflatoxin B₁ (AFB₁) were studied using the HepG2 cell line. Cytotoxicity, apoptosis and p53 expression were assessed after exposure to different concentrations of AFB₁ (0–100 μm ) and its two types of degradation products, namely the mixtures of photodegradation products in water (Pw) and the mixtures of photodegradation products in peanut oil (Po) for different time periods (0, 24, and 48 h). After exposure of the HepG2 cells to these compounds for different times and concentrations, the cytotoxicity of Pw and Po decreased approximately 40 and 100% compared with AFB₁, respectively. The expression of p53 protein decreased significantly in AFB₁‐exposed cells, decreased slightly in Pw‐treated cells and did not decrease compared to the untreated cells. The results of the in vitro cytotoxicity assay indicate that Pw is less toxic than AFB₁, and Po has almost no toxicity, which can be explained by the differences in the chemical nature of the various kinds of the test compounds. Copyright © 2011 John Wiley & Sons, Ltd.     

In vitro effect of methionine-enkephalin and thymosin on murine expression of Thy1-2 antigen

The present study investigates the in vitro effect of methionine-enkephalin (Met-Enk) on Thy1-2 antigen density and the interaction between this opioid peptide and a thymic hormone (thymosin fraction V (TFV]. In vitro, it was found that Met-Enk at various concentrations (28.10(-11), 28.10(-10), 28.10(-9) and 28.10(-8) M) had an increased positive effect on the Thy1-2 antigen density of Balb/C mouse thymocytes. This action was observed at all concentrations of Met-Enk. The addition of TFV to Met-Enk thymocyte cultures showed an additive effect of these two hormones. The physiological significance of these observations is that they indicate an interaction between immune and endocrine system functions.     

Spermatotoxic effect of aflatoxin B(1) in the albino mouse.

With the background that the foodborne mycotoxin aflatoxin B(1) (AFB(1)) could be toxic to the male reproductive mechanism in man as well as wild and domestic animals, the present study was aimed at finding the effect of AFB(1) on sperm. The Swiss albino mouse was the test animal. AFB(1,) suspended in corn oil and ethanol (95:5, v/v), was administered intraperitoneally to 90-day-old mice at a daily dose of 50 microg/kg body weight for 7, 15, 35 and 45 days. The analysis consisted of fertility testing and counts, motility and abnormalities of the cauda epididymidal sperm, adopting light- as well as electron-microscopy. The fertility of the treated mice was reduced drastically. Sperm concentration in the epididymis and sperm motility decreased whereas sperm abnormalities increased. In particular, sperm abnormalities like two axonemes in a common cytoplasm, sticking together of heads/tails, etc., were noted. A higher percentage of cauda epididymidal spermatozoa than in the control mice retained the cytoplasmic droplet (CD) and such retention was dependent on the duration of the treatment. Spermatozoa retaining the CD were inhibited in motility. Sperm CD of AFB(1)-treated mice contained electron-dense spherical inclusions, which are hypothesized as lipid inclusions produced from the lamellae through the spherical vesicles of the CD. The results indicate disruption of the spermatogenic as well as androgenic compartments of the testis by AFB(1). The results also reflect an alteration of epididymal function towards the post-testicular sperm maturation process by AFB(1).     

In vitro effect of ethanol on cell-mediated cytotoxicity by murine spleen cells

The effects of ethanol on murine spleen cell-mediated lysis have been studied. Concentrations of 5.5-176 mM ethanol produced progressive inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC). Binding of spleen cells to antibody-sensitized target cells was not inhibited by comparable concentrations of ethanol. Kinetic analysis revealed decreased rates of lysis with increasing concentrations of ethanol. Changes of effector to target cell ratios revealed an inhibition of maximum lysis and decreased lytic efficiency in the presence of 88 mM ethanol. Preincubation experiments showed the inhibitory effect of ethanol to be reversible. Macrophage-depleted spleen cells appeared to be as susceptible to inhibition by ethanol as unfractionated spleen cells. Ethanol also inhibited natural killer and alloimmune cytotoxic T cell activity. The ADCC data were analysed by using a mathematical model which incorporates the kinetics of lysis, dose-response relationships, heterogeneity of the lytic effectors, reversibility of inhibition and ethanol loss during incubation. An inhibition constant (KI) of 373 mM-2 when two ethanol molecules interact with the site of inhibition was calculated. 50% inhibition of lysis is produced by 52 mM (0.24%) ethanol. The results are consistent with a model which assumes that lysis is due to a critical number of interactions which ultimately trigger the lytic event. Alcohol interferes with lysis by reacting with sites which are required for triggering the lytic event. Although the molecular details of the mechanism of inhibition are as yet undefined, we infer that ethanol inhibits ADCC at the programming for lysis or the lethal hit stages.     

Phagocytic activity of ethyl alcohol fraction of deer antler in murine peritoneal macrophage.

The mechanism of phagocytic activity of the ethyl alcohol fraction of Cervus nippon (CN-E) was investigated in vivo. The administration of CN-E (100 mg/kg, p.o.) enhanced lucigenin chemiluminescence and the engulfment of fluorescein-conjugated E. coli particles in murine peritoneal macrophages. Phagocytic activity was suppressed by the treatment of S-nitrosoglutathione (GSNO) which is an exogenous nitric oxide donor depending on the concentration of dose. CN-E suppressed the production of nitric oxide and enhanced the concentration in [Ca2+]i. The enhancement in [Ca2+]i was diminished by the treatment of EGTA. These results indicate that CN-E enhances the phagocytic activity of murine peritoneal macrophage via a suppression of nitric oxide production and an increase in [Ca2+]i.