Aflatoxin cell culture

The toxification of benzo[a]pyrene and most other polycyclic aromatic hydrocarbons to mutagenic intermediates by continuous cell lines has been reported dozens of times, whereas toxification of aflatoxin Bj to mutagenic intemediates in some cell lines does not occur.225 These data can be explained by the fact that the forms of P-450 necessary for polycyclic-hydrocarbon toxification remain in cultured primary and continuous cell lines, whereas the forms of P-450 responsible for the 2,3-oxide formation of aflatoxin Bj disappear rapidly in culture, for unknown reasons. Studies involving cultured human tissues may have this same major liability. The choice of cell culture for any particular compound therefore can be important. 

The toxic effects of aflatoxin in animals includes gross liver damage, development of a necrosis in the periportal zone [246], and haemorrhage in the intestinal tract and peritoneal cavity. Aflatoxins also affect several cell culture systems in vitro [247—249], and cause lesions in chick embryos [249—251 ]. The finding of multiple liver tumours and lung metastases [252] was the first indication of the hepatotoxicity of this group of mycotoxins. This observation has been several times confirmed [253-257] and only 0.005 p.p.m. failed to induce liver tumour in rats [258]. Mice are more resistant [259—261], whereas rainbow trouts are considerably more sensitive to the action of aflatoxins [262—266]. 

Initial attempts to purify enzymes from A, parasiticus mycelial extracts that catalyze aflatoxin synthesis were unsuccessful because these enzymes are present in relatively low concentrations and are extremely short-lived (23). Subsequently, several techiuques were examined for disruption of large quantities of mycelia to obtain active and stable cell-fiee preparations (23, 24) pertinent enzymes were recovered from cell-free extracts after grinding mycelia under liquid nitrogen (24). The optimum age of mycelial cultures for recovery of aflatoxin pathway enzymes was determined to be between 72 and 84 h (24a, 25). 

A number of chemicals with demonstrable suppression of immune function produce this action via indirect effects. By and large, the approach that has been most frequently used to support an indirect mechanism of action is to show immune suppression after in vivo exposure but no immune suppression after in vitro exposure to relevant concentrations. One of the most often cited mechanisms for an indirect action is centered around the limited metabolic capabilities of immunocompetent cells and tissues. A number of chemicals have caused immune suppression when administered to animals but were essentially devoid of any potency when added directly to suspensions of lymphocytes and macrophages. Many of these chemicals are capable of being metabolized to reactive metabolites, including dime-thylnitrosamine, aflatoxin Bi, and carbon tetrachloride. Interestingly, a similar profile of activity (i.e., suppression after in vivo exposure but no activity after in vitro exposure) has been demonstrated with the potent immunosuppressive drug cyclophosphamide. With the exception of the PAHs, few chemicals have been demonstrated to be metabolized when added directly to immunocompetent cells in culture. A primary role for a reactive intermediate in the immune suppression by dimethylnitrosamine, aflatoxin Bi, carbon tetrachloride, and cyclophosphamide has been confirmed in studies in which these xenobiotics were incubated with suspensions of immunocompetent cells in the presence of metabolic activation systems (MASs). Examples of MASs include primary hepatocytes, liver microsomes, and liver homogenates. In most cases, confirmation of a primary role for a reactive metabolite has been provided by in vivo studies in which the metabolic capability was either enhanced or suppressed by the administration of an enzyme inducer or a metabolic inhibitor, respectively. 

Compared with some of the other mycotoxins such as aflatoxin, the trichothecenes do not appear to require metabolic activation to exert their biological activity.50 After direct dermal application or oral ingestion, the trichothecene mycotoxins can cause rapid irritation to the skin or intestinal mucosa. In cell-free systems or single cells in culture, these mycotoxins cause a rapid inhibition of protein synthesis and polyribosomal disaggregation.35 47 50 Thus, we can postulate that the trichothecene mycotoxins have molecular capability of direct reaction with cellular components. Despite this direct effect, it is possible to measure the toxicokinetics and the metabolism of the trichothecene mycotoxins. 

In kidney, diltiazem and the atrial natriuretic peptide act together with Mn(II) in controlling haemodynamics [621,622]. In liver, ethanol alters the levels of a number of trace metals, including Mn(II) [623], while Mn(II) itself reduces the effects of aflatoxin and enhances the cytochrome P-450 mediated metabolism of hexabarbitol-type drugs [624,625]. In the nervous system, the effects of ischaemia are modulated by Ca-channel blockers such as Mn(II) [626]. Mn(II) ions have also been reported to modulate or inhibit the transient outward current in cultured sensory neurons or in taste nerve responses [627,628]. In pancreas beta-cells, interactions between the fluxes of Mn(II) and Ca(II) have also been observed [629]. 

More recently, freshly isolated diploid Syrian hamster embryo cells were used in a quantitative focus assay. Transformed foci, observed within 3 weeks on a monolayer of normal cells, were induced in secondary cultures of cells after treatment of mass cultures for 6 days with A -acetoxy-2-acetylamino-fluorene, aflatoxin Bi, benzo [a]pyrene, jS-propriolactone, dibenz [a,/i] anthracene, ethyl methanesulfonate, 3-methylcholanthrene, methyl methanesulfo-nate, or A -methyl-A -nitro-A -nitrosoguanidine. 

M. S. Legator and A. Withrow, Aflatoxin Effect on mitotic division in cultured embryonic lung cells, J. Ass. Offic. Agr. Chemists 47, 1007-1009 (1964). 

Therefore, to increase the spectrum of compounds that can be studied in the cell-mediated mutagenesis system, Langenbach et al. have used primary cultures of rat liver cells to activate two known classes of liver carcinogens (nitrosamines and aflatoxins), and V79 cells to detect their mutagenic metabolites. A schematic diagram summarising the procedure and technique is shown in Fig. 2. Conversion from ouabain susceptibility to ouabain resistance, a mutation associated with the surface member Na /K ATPase, was used as the genetic marker. Typical data obtained with several nitrosamines and aflatoxins are summarised in Table 5. 

The second method employed in biosynthetic studies involves the characterization of fermentation products closely related to the secondary metabolite under investigation. This method has been extensively used and has led to the implication of averufin (8), the versicolorins, and sterigmato-cystin (32) in aflatoxin biogenetic pathway. Some of the intermediates were isotopically labeled and their role in aflatoxin biosynthesis verified, e.g., sterigmatocystin by whole cell (Hsieh et al., 1973) and cell-free cultures of A. parasiticus (Singh and Hsieh, 1976). 

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