A. flavus is the main species of fungus involved in AF production ^[1][2][3][9,11,22]. Four AF naturally present in agricultural products are described (AFB₁, AFB₂, AFG_1, AFG₂); other forms of AF are derived from the metabolic process of these primary forms within the human or animal body ^[4][28]. AF are not destroyed by boiling nor do they confer, color, aroma, or flavor to contaminated ingredients, so they usually go unnoticed by both the owner and the dog ^[5][13]. When dogs ingest CDF made with ingredients contaminated with AF, the mycotoxin are absorbed in the duodenum and bind to plasma albumin and proceed to be transported through the bloodstream ^[6][29].

In various tissues, especially in the liver and kidneys, AF are biotransformed and bioactivated by isoenzymes of the multiple function oxidase system or cytochromes (CyP₄₅₀), giving rise to highly active electrophilic forms called 8,9 endo-epoxide and 8, 9 exo-epoxide, which bind and exert an electrophilic attack on subcellular structures ^[7][30]. When AF are metabolized (hydrolysis, demethylation, or ketoreduction), they form less toxic intermediate compounds with greater solubility in water (AFM₁, AFM₂, AFQ₁, AFQ_2, AFP₁, AFP_2, and aflatoxicol); therefore, they are eliminated through feces, urine, milk, or egg ^[8][9][31,32]. The most common ways of elimination of metabolites in the urine of dogs is AFM₁, as well as traces of AFQ₁ ^[10][11][33,34]. When the epoxide binds to DNA, then an AF-DNA adduct (dihydro-8-(N⁷-guanyl)-9hydroxy-1 AF-N⁷–guanidine) is formed, which rearranges as AF-formaminopyrimidine (AF-FAPy), or it is excreted through urine as AF-N⁷guanine, which is considered a biomarker of genotoxic damage from AF ^{[8][9][10][11]}[31,32,33,34]. In general, all compounds derived from natural forms of AF, due to the activity of the enzymes that participate in the detoxification process, are considered biomarkers of exposure and damage ^[12][35].

An important detoxification mechanism in many animal species is the involvement of a group of enzymes called Glutathione S-transferases (GST) ^[13][36]. The function of GST is to bind the epoxide with the reduced glutathione tripeptide (GSH), which loses two amino acids (glycine and glutamate) to be eliminated as a cysteine residue linked to AF, called mercapturic acid or N-acetylcysteine-AF, which is eliminated through bile or urine ^[14][37]. Dogs have a reduced GST activity, which makes them especially susceptible to AF damage; in addition, a deficiency of GSH or its three precursor amino acids facilitates the occurrence of the most extensive oxidative injury ^[15][16][17][38,39,40].

2. Contamination of Feed Ingredients by Aflatoxins

Cereals are usually integrated into dog feeds, especially corn, sorghum, rice, wheat, oats, barley, and millet; they are a good source of carbohydrates, fiber, protein, fat, minerals, and vitamins ^[18][19][41,42]. However, cereals present an important risk for the health of dogs because they are vulnerable to contamination by A. flavus both in the field and in storage ^[20][21][22][43,44,45]. In some CDF formulations for dogs, pumpkin seeds, chia, quinoa, and even some legumes such as lentils are included among the ingredients due to their high protein and mineral content. However, these ingredients can also be contaminated by some forms of AF ^[23][24][25][46,47,48]. Furthermore, the incorporation of both potatoes and sweet potatoes in the manufacturing of premium types of dog feeds is used as a source of carbohydrates and fiber. It is reported that the presence of A. flavus and high concentrations of AF can be found in potato tubers ^[26][27][49,50].

Fruits in CDF are used as a natural source of fiber; papaya is one of the main fruits that are included, however, it may have A. flavus in the postharvest, which has effects on its nutritional value, and it may also have AF concentrations ^[28][51]. Blueberries are used as antioxidants, although there are reports of A. flavus contamination and concentrations of AFB₁, AFB₂, y AFG₁ ^[29][52]. Orange and coconut are other ingredients that are included in some dog feeds, but they can also be contaminated by FA-producing fungal microflora ^[30][31][32][53,54,55].

Dairy and meat products as well as eggs are added to dog feed as an important source of proteins and fats. However, secondary AF metabolites such as AFM₁, AFM₂, AFP₁ may be found, which can also contaminate these feed ingredients ^[33][56]. The AF residues can be located in by-products of animal origin used in the manufacturing of CDF, such as liver, kidneys, muscle, meat, milk, and egg. The residual compounds in eggs, milk and meat are derived from the biotransformation of the original AF ingested in the feed of animals used for food and remain in the dog that ingests the CDF ^[34][57].