Modulation of aflatoxin B1 cytotoxicity and aflatoxin M1 synthesis by natural antioxidants in a bovine mammary epithelial cell line.

Aflatoxin (AF) B1, a widespread food and feed contaminant, is bioactivated by drug metabolizing enzymes (DME) to cytotoxic and carcinogenic metabolites like AFB1-epoxide and AFM1, a dairy milk contaminant. A number of natural antioxidants have been reported to afford a certain degree of protection against AFB1 (cyto)toxicity. As the mammary gland potentially participates in the generation of AFB1 metabolites, we evaluated the role of selected natural antioxidants (i.e. curcumin, quercetin and resveratrol) in the modulation of AFB1 toxicity and metabolism using a bovine mammary epithelial cell line (BME-UV1). Quercetin and, to a lesser extent, resveratrol and curcumin from Curcuma longa (all at 5 µM) significantly counteracted the AFB1-mediated impairment of cell viability (concentration range: 96-750 nM). Moreover, quercetin was able to significantly reduce the synthesis of AFM1. The quantitative PCR analysis on genes encoding for DME (phase I and II) and antioxidant enzymes showed that AFB1 caused an overall downregulation of the detoxifying systems, and mainly of GSTA1, which mediates the GSH conjugation of the AFB1-epoxide. The negative modulation of GSTA1 was efficiently reversed in the presence of quercetin, which significantly increased GSH levels as well. It is suggested that quercetin exerts its beneficial effects by depressing the bio-transformation of AFB1 and counterbalancing its pro-oxidant effects.

Constitutive expression of the AHR signaling pathway in a bovine mammary epithelial cell line and modulation by dioxin-like PCB and other AHR ligands.

Environmental pollutants, such as dioxin-like (DL) PCBs, benzo(a) pyrene (B[a]P), and flavonoids are aryl hydrocarbon receptor (AHR) ligands and may be excreted in dairy milk. The expression of AHR-target genes, particularly those involved in xenobiotic biotransformation, and their modulation by two DL-PCBs, B[a]P, and ß-naphthoflavone was investigated in a bovine mammary epithelial cell line (BME-UV). As assessed by quantitative PCR, BME-UV cells expressed a functional AHR signaling pathway. All the AHR ligands induced a concentration-related increase in the transcription of cytochrome P450 1A1 and 1B1, known to be implicated in the bioactivation of several xenobiotics. Conversely, genes encoding for antioxidant and detoxifying enzymes, like quinone oxidoreductase or glutathione S-transferase A2, were not affected or even depressed. This study demonstrates the occurrence and the modulation by different AHR-ligands of genes involved in xenobiotic metabolism in BME-UV cells, with the potential generation of (re) active metabolites that may damage mammary tissue and/or affect animal or human health via the contaminated milk.

Modulation of aryl hydrocarbon receptor target genes in circulating lymphocytes from dairy cows bred in a dioxin-like PCB contaminated area.

Animal productions (i.e. fish, eggs, milk and dairy products) represent the major source of exposure to dioxins, furans, and dioxin-like (DL) polychlorobiphenyls for humans. The negative effects of these highly toxic and persistent pollutants are mediated by the activation of the aryl hydrocarbon receptor (AHR) that elicits the transcriptional induction of several genes, including those involved in xenobiotic metabolism. Previously we demonstrated the presence and functioning of the AHR signaling pathway in primary cultures of bovine blood lymphocytes. The aim of the present study was to investigate by real time PCR the expression and the inducibility of selected target genes (i.e. AHR, AHR nuclear translocator (ARNT), AHR repressor, CYP1A1 and CYP1B1) in uncultured cells from dairy cows naturally exposed to DL-compounds. The study was carried out on two groups of animals bred in a highly polluted area and characterized by a different degree of contamination, as assessed by bulk milk TEQ values, and a control group reared in an industry free area. Bovine lymphocytes expressed only AHR, ARNT and CYP1B1 genes to a detectable level; moreover, only CYP1B1 expression appeared to be correlated to TEQ values, being higher in the most contaminated group, and decreasing along with animal decontamination. Finally, lymphocytes from exposed cows displayed a lower inducibility of both CYP1A1 and CYP1B1 after the in vitro treatment with a specific AHR ligand. In conclusion, our results indicate that DL-compound contaminated cows may display significant changes in AHR-target gene expression of circulating lymphocytes.

Gene expression and inducibility of the aryl hydrocarbon receptor-dependent pathway in cultured bovine blood lymphocytes.

The exposure to dioxin-like (DL) compounds, an important class of persistent environmental pollutants, results in the altered expression of target genes. This occurs through the binding to the aryl hydrocarbon receptor (AhR), the subsequent dimerization with the AhR nuclear translocator (ARNT), and the binding of the complex to DNA responsive elements. A number of genes are up-regulated, including, among others, the AhR repressor (AHRR) and several biotransformation enzymes, such as the members of CYP1 family and NAD(P)H-quinone oxidoreductase (NOQ1). The expression and the inducibility of the above genes were investigated in mitogen-stimulated cultured blood lymphocytes from cattle, which represent a notable source of DL-compound human exposure through dairy products and meat. As assessed by real-time PCR, all the examined genes except CYP1A2 and NQO1 were detected under basal conditions. Cell exposure to the DL-compounds PCB126 or PCB77 in the 10(-6)-10(-9)M concentration range resulted in a 2-4-fold induction of CYPIA1 and CYP1B1, which was antagonized by α-naphthoflavone or PCB153. This study demonstrates for the first time the presence and inducibility of the AhR pathway in easily accessible cells like bovine peripheral lymphocytes and prompts further investigations to verify whether similar changes could occur under in vivo conditions.