Acrylamide Production in Autoclaved Rodent Feed

Abstract

Sterilization of rodent feed by steam autoclaving is a common practice in many research institutions. Often we only consider the beneficial effects of this process-the reduction of microbial contamination-and forget that the high temperatures and pressures can have negative effects on diet quality. The purpose of our study was to assess both the physical and chemical changes to a standard rodent feed autoclaved at multiple sterilization temperatures and the effects of the treated diets on mice. Pelleted NIH31 rodent feed was autoclaved at 4 sterilization temperatures (230, 250, 260, and 270 °F). Feed pellet hardness and the acrylamide concentrations of the diets were tested and compared with irradiated NIH31 feed. Study diets were fed to mice for 28 d, after which tissue samples were collected for analysis of acrylamide, glycidamide (the active metabolite of acrylamide), and genotoxicity. Both feed pellet hardness and acrylamide concentration increased with increasing sterilization temperatures; however, neither affected feed intake or body weight gain. Plasma acrylamide and glycidamide were significantly elevated only in mice fed NIH31 diet autoclaved at 270 °F compared with the irradiated feed, whereas urine acrylamide and glycidamide metabolites were significantly elevated in most autoclaved diets. Liver DNA adducts, which correlate with genotoxicity, were significantly elevated in all autoclaved diets compared with the irradiated diet. Institutions that autoclave their animal diets should carefully consider the temperatures necessary to achieve feed sterilization and the type of studies in which these autoclaved diets are used.

Figure 1.

Chemical structures of acrylamide and glycidamide.

Figure 2.

Daily feed intake (g; mean ± SEM) in mice fed NIH31 study diets through day 26. IR, irradiated pelleted feed (control); A230, pelleted feed autoclaved at 230 °F; A250, pelleted feed autoclaved at 250 °F; A260, pelleted feed autoclaved at 260 °F; A270P, pelleted feed autoclaved at 270 °F; A270G, ground feed autoclaved at 270 °F; AA-low, irradiated ground feed spiked with 200 ppb acrylamide; AA-high, irradiated ground fee spiked with 1000 ppb acrylamide. All groups contained 10 mice except irradiated (n = 15), A270P (n = 15), and A270G (n = 9).⁺, P < 0.005 compared with irradiated control group. Note that A270G, AA-low, and AA-high were fed as ground diets, whereas all others were pelleted. Ground feed waste likely resulted in the increased variability within the acrylamide groups.

Figure 3.

Mean body weight (g) in mice fed NIH31 study diets through day 26. Error bars indicating the SEM (range, 0.233 – 0.7089 g) were removed for clarity. All groups contained 10 mice except irradiated and A270P (n = 15 in each group). Body weight did not differ between groups at any time point. IR, irradiated pelleted feed (control); A230, pelleted feed autoclaved at 230 °F; A250, pelleted feed autoclaved at 250 °F; A260, pelleted feed autoclaved at 260 °F; A270P, pelleted feed autoclaved at 270 °F; A270G, ground feed autoclaved at 270 °F; AA-low, irradiated ground feed spiked with 200 ppb acrylamide; AA-high, irradiated ground fee spiked with 1000 ppb acrylamide.

Figure 4.

Plasma acrylamide concentration (nM) from mice fed NIH31 study diets. Data are given as mean ± SEM (n = 10) for all groups except A270P (n = 15). IR, irradiated pelleted feed (control); A230, pelleted feed autoclaved at 230 °F; A250, pelleted feed autoclaved at 250 °F; A260, pelleted feed autoclaved at 260 °F; A270P, pelleted feed autoclaved at 270 °F; A270G, ground feed autoclaved at 270 °F; AA-low, irradiated ground feed spiked with 200 ppb acrylamide; AA-high, irradiated ground fee spiked with 1000 ppb acrylamide. *, P < 0.05; ‡, P < 0.001; and §, P < 0.0001 compared with irradiated control group.

Figure 5.

Plasma glycidamide concentration (nM) from mice fed NIH31 study diets. Data represents mean ± SEM; n = 10 for all groups except A270P (n = 15). Several groups had samples in which some or all sample replicates were below the limit of detection (LOD) of 1.1 nM. Those replicates were assigned a value of LOD/√2 (0.8 nM). The number above each column indicates the number of samples with 1 to 3 replicates below the LOD. IR, irradiated pelleted feed (control); A230, pelleted feed autoclaved at 230 °F; A250, pelleted feed autoclaved at 250 °F; A260, pelleted feed autoclaved at 260 °F; A270P, pelleted feed autoclaved at 270 °F; A270G, ground feed autoclaved at 270 °F; AA-low, irradiated ground feed spiked with 200 ppb acrylamide; AA-high, irradiated ground fee spiked with 1000 ppb acrylamide. *P < 0.05, **P < 0.01, *P < 0.001.

Figure 6.

Urine N-acetyl-S-(2-carbamoylethyl)-cysteine concentration (AAMA, μM) from mice fed NIH31 study diets. All groups represent a single pooled sample except A270P which had 2 pooled samples. Data represents mean of duplicates ± SEM. IR, irradiated pelleted feed (control); A230, pelleted feed autoclaved at 230 °F; A250, pelleted feed autoclaved at 250 °F; A260, pelleted feed autoclaved at 260 °F; A270P, pelleted feed autoclaved at 270 °F; A270G, ground feed autoclaved at 270 °F; AA-low, irradiated ground feed spiked with 200 ppb acrylamide; AA-high, irradiated ground fee spiked with 1000 ppb acrylamide. *P < 0.0001 compared with irradiated control group.

Figure 7.

Urine N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-cysteine concentration (GAMA, μM) from mice fed NIH31 study diets. All groups represent a single pooled sample except A270P which had 2 pooled samples. All samples run in duplicate. Data are presented as the mean ± SEM of duplicate samples. IR, irradiated pelleted feed (control); A230, pelleted feed autoclaved at 230 °F; A250, pelleted feed autoclaved at 250 °F; A260, pelleted feed autoclaved at 260 °F; A270P, pelleted feed autoclaved at 270 °F; A270G, ground feed autoclaved at 270 °F; AA-low, irradiated ground feed spiked with 0.2 ppm acrylamide; AA-high, irradiated ground fee spiked with 1.0 ppm acrylamide. *, P < 0.05; †, P < 0.01; and §, P < 0.0001 compared with the irradiated control group.

Figure 8.

The number of hepatic glycidamide–guanine DNA adducts (N7-[2-carbamoyl-2-hydroxyethyl]-guanine or N7-glycidamide-guanine) per 10⁸ total nucleotides in mice fed NIH31 irradiated or autoclaved diet. Data are given as mean ± SEM (n = 10) for all goups except A230 (n = 8) and A270P (n = 14). IR, irradiated pelleted feed (control); A230, pelleted feed autoclaved at 230 °F; A250, pelleted feed autoclaved at 250 °F; A260, pelleted feed autoclaved at 260 °F; A270P, pelleted feed autoclaved at 270 °F; A270G, ground feed autoclaved at 270 °F; AA-low, irradiated ground feed spiked with 200 ppb acrylamide; AA-high, irradiated ground fee spiked with 1000 ppb acrylamide. †, P < 0.01; ‡, P < 0.001; and §, P < 0.0001 compared with the irradiated control group.

Figure 9.

The number of hepatic glycidamide–adenine DNA adducts (N3-[2-carbamoyl-2-hydroxyethyl]-adenine or N3-glycidamide-adenine] per 10⁸ total nucleotides in mice fed NIH31 irradiated or autoclaved diet. Several groups had samples in which some or all measurements were below the limit of detection (LOD) of 0.16 adducts per 10⁸ nucleotides. Those samples were assigned a value of LOD/√2 (that is, 0.11 adducts/10⁸ nucleotides). Data are given as mean ± SEM (n = 10) for all groups except IR (n = 14) and A270P (n = 15). The number above each column indicates the number of samples per group that were below the LOD. IR, irradiated pelleted feed (control); A230, pelleted feed autoclaved at 230 °F; A250, pelleted feed autoclaved at 250 °F; A260, pelleted feed autoclaved at 260 °F; A270P, pelleted feed autoclaved at 270 °F; A270G, ground feed autoclaved at 270 °F; AA-low, irradiated ground feed spiked with 200 ppb acrylamide; AA-high, irradiated ground fee spiked with 1000 ppb acrylamide. Statistical analysis used a 1-sample t test with hypothetical mean of 0.16. ‡, P < 0.001; §, P < 0.0001 compared with irradiated control group.