Gonzalo J. Diaz

Cytochrome P450 enzymes involved in the metabolism of aflatoxin B1 in chickens and quail

A study was conducted to identify the cytochrome P450 (CYP, CYP450) enzyme orthologs involved in the bioactivation of aflatoxin B(1) (AFB(1)) into the highly toxic metabolite known as aflatoxin-8,9-epoxide (AFBO) in quail and chicken hepatic microsomes. The strategies used included the use of specific CYP450 inhibitors and the correlation of prototype substrate activities with AFBO production. Additionally, the presence of the enzymes was qualitatively determined using an immunoblotting technique. The results showed that both quail and chicken microsomes have CYP1A1, CYP1A2, CYP2A6, and CYP3A4 enzymatic activity. A strong relationship between CYP1A1 and CYP2A6 activities and AFB(1) bioactivation was found in both species. Inhibition studies provided more evidence for the role of CYP2A6 in the bioactivation of AFB(1). The immunoblot results showed clear bands for the CYP2A6 and CYP3A4 orthologs in both species. The results of the present study indicate that CYP2A6 and, to a lesser extent, CYP1A1 are responsible for the bioactivation of AFB(1) into AFBO in both quail and chicken hepatic microsomes.

Aflatoxicosis in Chickens (Gallus gallus): An Example of Hormesis?

Poultry has commonly been considered highly susceptible to aflatoxins. However, among domestic fowl there is wide variability in specific species sensitivity to these mycotoxins. Comparative toxicological studies in avian species have shown that ducklings and turkey poults are the most sensitive species to aflatoxins, quails show intermediate sensitivity, whereas chickens are the most resistant. Hormesis is a dose-response phenomenon characterized by low-dose stimulation and high-dose inhibition. The low-dose stimulation is typically maximal at only about 30 to 60% greater than controls. Hormesis has been noted in regards to changes in body weight in numerous studies, including those performed for the US National Toxicology Program, with over 50 chemicals. The present paper assesses how relatively low levels of aflatoxin consumption in feed may affect the growth rate of chickens. In general, multiple independent investigations have shown that such aflatoxin consumption affects growth in a hormetic-like biphasic manner with a low dose stimulation and a high dose inhibition. Such observations were then generalized to other toxic agents and animal models, suggesting that low doses of stressor agents induce adaptive responses as reflected in accelerated growth rates. The implications of such hormetic dose responses are briefly discussed.

Individual and combined effects of t‐2 toxin and das in laying hens

1. The individual and combined effects of T-2 toxin and 4,15-diacetoxyscirpenol (DAS) on laying hens were investigated in an experiment consisting of a 2 x 2 completely randomised factorial design with dietary concentrations of 0 and 2 mg/kg T-2 toxin and 0 and 2 mg/kg DAS. 2. Individually, T-2 toxin and DAS induced oral lesions in half of the hens and decreased significantly egg production and food intake. 3. The effects of T-2 toxin and DAS were additive for reduced food consumption and incidence of oral lesions. However, a synergism for reduced egg production was observed during the last experimental period. 4. No effects on body weight were observed during this study. Mild changes in selected plasma enzymes activities and no change in liver malondialdehyde content were detected. 5. The combination of T-2 toxin and DAS was more toxic than the single mycotoxins, for some parameters, and therefore, may pose a greater economic threat to the poultry industry than either of the toxins individually.

Occurrence of aflatoxin M1 in retail milk samples from Bogotá, Colombia

A study was conducted to establish the occurrence and levels of contamination of aflatoxin M1 (AFM1) in retail milk from Bogotá, Colombia. A total of 241 samples were analysed during 2004 and 2005. Samples were cleaned up by an immunoaffinity column and AFM1 was quantified by liquid chromatography with fluorescence detection. A total of 69.2 and 79.4% of the samples analysed during 2004 and 2005, respectively, were found to contain levels of AFM1 above 10 ng l−1. Levels of contamination ranged from 10.7 to 213.0 ng l−1 in 2004, and from 10.6 to 288.9 ng l−1 in 2005. Despite the high incidence of AFM1 found in the milk samples analysed, all samples complied with current local regulations, which allow AFM1 content in milk up to 400 ng l−1. However, due to the high incidence of AFM1 in milk found in the present study, it is recommended that a permanent surveillance programme be established for milk consumed in Bogotá in order to prevent milk lots containing levels above the regulatory level entering the food chain.

Microsomal and cytosolic biotransformation of aflatoxin B1 in four poultry species

1. This research evaluated differences in hepatic in vitro metabolism of aflatoxin B1 (AFB1) on selected avian species. 2. Microsomal and cytosolic liver fractions were obtained from chickens, ducks, quails and turkeys; eight males and eight females of each. 3. All microsomes studied produced AFB1-8,9-exo-epoxide (AFBO), a metabolite regarded as the active product of AFB1. Turkey microsomes produced 1·8 and 3·5 times more AFBO than quails and chickens microsomes, respectively. 4. Males from evaluated birds produced more AFBO than females, but statistically-significant differences between genders were observed only in ducks and turkeys. 5. The cytosolic fraction from all four species produced aflatoxicol (AFL). Turkey and duck hepatic cytosol produced more AFL than from quail and chickens. 6. It is known that turkeys are very sensitive to AFB1, quails are intermediate and chickens are particularly resistant; the differences in AFBO production shown in our study may help to explain the different in vivo responses among turkeys, quail and chickens. 7. Moreover, AFL may be related to AFB1 toxicity; it was produced in larger amounts by hepatic cytosol from the more susceptible species. 8. Because AFBO production by microsomes in ducks was relatively low, it is possible that other toxicity mechanisms are involved in this highly susceptible species.

Cobalt-induced polycythaemia causing right ventricular hypertrophy and ascites in meat-type chickens.

Cobalt increases the red cell mass in both man and animals by increasing the production of erythropoietin. Since meat-type chickens can develop pulmonary hypertension from increased erythropoiesis and polycythaemia, two trials were conducted to investigate the role of cobalt on broiler chicken erythropoiesis and pulmonary hypertension. The results showed that feeding cobaltous chloride at 500 parts/10(6) to meat-type chickens from 1-day-old for 42 days significantly increased haemoglobin content and, to a lesser extent red blood cell count, and haematocrit. No effect was observed on mean corpuscular volume. Increased haemoglobin content was linearly correlated with pulmonary hypertension as measured by the right ventricle weight to total ventricle weight ratio (RV:TV). Levels of malondialdehyde in cardiac tissue were also correlated with the RV:TV ratio, suggesting that peroxidative damage may be related to ventricular hypertrophy. Chickens fed cobalt showed a significantly higher incidence of right ventricular hypertrophy and right ventricular failure and 18.3% developed ascites.

Occurrence of aflatoxins in selected colombian foods.

A survey of aflatoxin contamination in selected Colombian foods was conducted over a 12-month period on a total of 248 samples. Samples were collected in supermarkets, retail stores and stock centres and were grouped into five categories: (1) corn and corn products, (2) cereal grains, (3) rice and rice products, (4) legume seeds; and (5) snacks and breakfast cereals. Aflatoxins were identified and quantitated using a liquid chromatographic technique with a limit of detection of 1 ng/g for each aflatoxin. Aflatoxins were detected in 14 of 109 samples of corn and corn products, 4 of 40 samples of rice and rice products, 2 of 30 samples of legume seeds, and 2 of 11 samples of snacks and breakfast cereals. None of the cereal grains samples analysed contained detectable levels of aflatoxins. Twelve of the total of 22 positive samples exceeded the maximum tolerable level of aflatoxin B1 adopted in most countries (5 ng/g); 10 of these 12 samples corresponded to corn and corn products. The results of the present study indicate that aflatoxin B1 contamination in certain foods in Colombia is a major public health concern. Continuous monitoring of aflatoxin B1 levels in Colombian foods is advised.

Bioactivation of aflatoxin B1 by turkey liver microsomes: responsible cytochrome P450 enzymes.

1. A study was conducted to determine the cytochrome P450 enzymes responsible for the bioactivation of aflatoxin B1 into its epoxide form (AFBO) in turkey liver microsomes. 2. The strategies used included the measurement of prototype substrate activity for specific human P450s, use of selective inhibitors, determination of correlation between aflatoxin bioactivation and enzymatic activity of prototype substrates and the determination of immunoreactive proteins using antibodies against human P450s. 3. Enzymatic activity and immunoreactive proteins corresponding to the turkey orthologs CYP1A1, CYP1A2, CYP2A6 and CYP3A4 were detected, but not for the CYP2D6 ortholog. 4. The results of the inhibition and correlation studies strongly suggest that the turkey CYP2A6 ortholog and, to a lesser extent, the CYP1A1 ortholog, are involved in the bioactivation of aflatoxin B1 in turkey liver microsomes. 5. This is the first study reporting the role of CYP2A6 in the bioactivation of AFB1 in an avian species and the role of CYP1A1 in any species.

Biotransformation of Aflatoxin B1 and Its Relationship with the Differential Toxicological Response to Aflatoxin in Commercial Poultry Species

Aflatoxin B1 (AFB1) is a highly toxic compound (LD50 = 1-50 mg/kg) for most animal species, although it is extremely toxic (LD50 turkey poults > goslings > pheasant chicks > quail chicks > chicks (Leeson et al., 1995). Even though there is still no clear explanation for this differential sensitivity, differences in susceptibility could be due to differences in AFB1 biotransformation pathways among species. The aim of the present chapter is to review the current knowledge on AFB1 biotransformation, with emphasis on

Stability of cyclopiazonic acid in solution

Cyclopiazonic acid (CPA) is an important mycotoxin given its toxicity and prevalence in foods and feeds. There is tremendous interest in developing analytical methods that include CPA as part of a multi-residue mycotoxin routine, but there appears to be considerable difficulty in analysing it using liquid chromatography with electrospray ionisation tandem mass spectrometry (LC-MS/MS). During the development of a multi-residue method for mycotoxins including CPA, a number of issues were discovered under routine and common analytical conditions that have an impact on the determination of CPA, including: (1) at the ng/ml level CPA reacts with ambient oxygen from the headspace of the vial, an effect that decreases its concentration linearly; (2) CPA readily adsorbs to plastic in a reversible fashion; (3) CPA is acid hydrolysed with formic acid; (4) CPA reacts with the column stationary phase affecting chromatographic parameters; and (5) CPA presents significant carry-over issues. In an effort to find solution...