Metabolism of Zearalenone in the Rumen of Dairy Cows with and without Application of a Zearalenone-Degrading Enzyme

Abstract

The mycotoxin zearalenone (ZEN) is a frequent contaminant of animal feed and is well known for its estrogenic effects in animals. Cattle are considered less sensitive to ZEN than pigs. However, ZEN has previously been shown to be converted to the highly estrogenic metabolite α-zearalenol (α-ZEL) in rumen fluid in vitro. Here, we investigate the metabolism of ZEN in the reticulorumen of dairy cows. To this end, rumen-fistulated non-lactating Holstein Friesian cows (n = 4) received a one-time oral dose of ZEN (5 mg ZEN in 500 g concentrate feed) and the concentrations of ZEN and ZEN metabolites were measured in free rumen liquid from three reticulorumen locations (reticulum, ventral sac and dorsal mat layer) during a 34-h period. In all three locations, α-ZEL was the predominant ZEN metabolite and β-zearalenol (β-ZEL) was detected in lower concentrations. ZEN, α-ZEL and β-ZEL were eliminated from the ventral sac and reticulum within 34 h, yet low concentrations of ZEN and α-ZEL were still detected in the dorsal mat 34 h after ZEN administration. In a second step, we investigated the efficacy of the enzyme zearalenone hydrolase ZenA (EC 3.1.1.-, commercial name ZENzyme^®, BIOMIN Holding GmbH, Getzersdorf, Austria) to degrade ZEN to the non-estrogenic metabolite hydrolyzed zearalenone (HZEN) in the reticulorumen in vitro and in vivo. ZenA showed a high ZEN-degrading activity in rumen fluid in vitro. When ZenA was added to ZEN-contaminated concentrate fed to rumen-fistulated cows (n = 4), concentrations of ZEN, α-ZEL and β-ZEL were significantly reduced in all three reticulorumen compartments compared to administration of ZEN-contaminated concentrate without ZenA. Upon ZenA administration, degradation products HZEN and decarboxylated HZEN were detected in the reticulorumen. In conclusion, endogenous metabolization of ZEN in the reticulorumen increases its estrogenic potency due to the formation of α-ZEL. Our results suggest that application of zearalenone hydrolase ZenA as a feed additive may be a promising strategy to counteract estrogenic effects of ZEN in cattle.

Keywords: degradation; feed additive; hydrolase; metabolism; mycotoxin; rumen; zearalenone.

Figure 1

Enzymatic degradation of zearalenone by zearalenone hydrolase ZenA. Modified from [21,22].

Figure 2

Metabolization and enzymatic degradation of zearalenone in a simulated rumen environment. (A) shows concentrations of zearalenone (ZEN) and its metabolites α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), hydrolyzed ZEN (HZEN) and decarboxylated HZEN (DHZEN) in reactors incubated with ZEN. (B) shows concentrations of ZEN and its metabolites in reactors incubated with ZEN and ZenA. Symbols (red diamond—ZEN; yellow circle—α-ZEL; blue diamond—β-ZEL; green triangle—HZEN; white circle—DHZEN) indicate median of four replicates and error bars indicate interquartile range. If a compound was detectable but below the limit of quantification (LOQ), the concentration in the respective sample was assumed to be LOQ/2 for calculation of median and interquartile range. If a compound was below the limit of detection in all four replicates at a given time point, no symbol is depicted. HZEN and DHZEN were not detected in reactors incubated with ZEN (A) at any time point. Asterisks indicate a significantly higher concentration (p < 0.05) in treatment ZEN compared to ZEN+ZenA or in treatment ZEN+ZenA compared to ZEN for the respective sampling location and time point.

Figure 3

Scheme of the reticulorumen of a dairy cow. Red symbols in the shape of reaction tubes indicate sampling locations.

Figure 4

Metabolization and enzymatic degradation of zearalenone in the reticulorumen of dairy cows. Subfigures in the top row show concentrations of ZEN and its metabolites in reticulum (A), dorsal mat layer (B) and ventral sac (C) after feeding ZEN-contaminated concentrate (“ZEN”; experimental days 1 and 2). Subfigures in the bottom row show concentrations of ZEN and its metabolites in reticulum (D), dorsal mat layer (E) and ventral sac (F) after feeding ZEN-contaminated concentrate supplemented with ZenA (“ZEN+ZenA”; experimental days 3 and 4). Symbols (red diamond—ZEN; yellow circle—α-ZEL; blue diamond—β-ZEL; green triangle—HZEN; white circle—DHZEN) indicate median of four replicates and error bars indicate interquartile range. If a compound was detectable but below the limit of quantification (LOQ), the concentration in the respective sample was assumed to be LOQ/2 for calculation of median and interquartile range. If a compound was below the limit of detection in all four replicates at a given location and time point, no symbol is depicted. HZEN and DHZEN were not detected in any reticulorumen location after ZEN treatment (A–C). β-ZEL was not detected in any reticulorumen location after ZEN+ZenA treatment (D–F). α-ZEL was not detected in ventral sac after ZEN+ZenA treatment (F). Asterisks indicate a significantly higher concentration (p < 0.05) in treatment ZEN compared to ZEN+ZenA or in treatment ZEN+ZenA compared to ZEN for the respective sampling location and time point. Abbreviations: ZEN-zearalenone; α-ZEL-α-zearalenol; β-ZEL-β-zearalenol; HZEN-hydrolyzed ZEN; DHZEN-decarboxylated HZEN.

Figure 5

Concentrations of zearalenone and its metabolites in feces of dairy cows after feeding ZEN-contaminated concentrate (“ZEN”; experimental day 1) and after feeding ZEN-contaminated concentrate supplemented with ZenA (“ZEN+ZenA”; experimental day 3). Symbols (red diamond—ZEN; yellow circle—α-ZEL; blue diamond—β-ZEL; green triangle—HZEN) indicate median of four replicates and error bars indicate interquartile range. If a compound was detectable but below the limit of quantification (LOQ), the concentration in the respective sample was assumed to be LOQ/2 for calculation of median and interquartile range. If a compound was below the limit of detection in all four replicates at a given time point, no symbol is depicted. DHZEN was not detected in any sample. Abbreviations: ZEN—zearalenone; α-ZEL—α-zearalenol; β-ZEL—β-zearalenol; HZEN—hydrolyzed ZEN; DHZEN—decarboxylated HZEN.