Behavioral effects of long-term oral administration of aluminum ammonium sulfate in male and female C57BL/6J mice

Abstract

Background: Aluminum (Al) is considered to be a neurotoxic metal, and excessive exposure to Al has been reported to be a potential risk factor for neurodegenerative diseases. Al ammonium sulfate is one of the Al compounds that is widely used as a food additive. However, the effects of the oral administration of Al ammonium sulfate on physical development and behavior remain to be examined.

Methods: In this study, we investigated the effects of the administration of Al ammonium sulfate 12-water dissolved in drinking water (0.075 mg/mL) beginning in adolescence on various types of behavior in adult female C57BL/6J mice through a battery of behavioral tests (low-dose experiment; Experiment 1). We further examined the behavioral effects of the oral administration of a higher dose of the Al compound in drinking water (1 mg/mL) beginning in the prenatal period on behavior in adult male and female mice (high-dose experiment; Experiment 2).

Results: In the low-dose experiment, in which females' oral intake of Al was estimated to be 0.97 mg Al/kg/d as adults, Al-treated females exhibited an increase in total arm entries in the elevated plus maze test, an initial decrease and subsequent increase in immobility in the forced swim test, and reduced freezing in the fear conditioning test approximately 1 month after the conditioning session compared with vehicle-treated females (uncorrected P < .05). However, the behavioral differences did not reach a statistically significant level after correction for multiple testing. In the high-dose experiment, in which animals' oral intakes were estimated to be about ten times higher than those in the low-dose experiment, behavioral differences found in the low-dose experiment were not observed in high-dose Al-treated mice, suggesting that the results of the low-dose experiment might be false positives. Additionally, although high-dose Al-treated females exhibited increased social contacts with unfamiliar conspecifics and impaired reference memory performance, and high-dose Al-treated mice exhibited decreases in prepulse inhibition and in correct responses in the working memory task (uncorrected P < .05), the differences in any of the behavioral measures did not reach the significance level after correction for multiple testing.

Conclusion: Our results show that long-term oral exposure to Al ammonium sulfate at the doses used in this study may have the potential to induce some behavioral changes in C57BL/6J mice. However, the behavioral effects of Al were small and statistically weak, as indicated by the fact that the results failed to reach the study-wide significance level. Thus, further study will be needed to replicate the results and reevaluate the behavioral outcomes of oral intake of Al ammonium sulfate.

Keywords: C57BL/6J; aluminum ammonium sulfate; behavioral test battery; mice; oral intake.

Figure 1

Schematic diagram of experimental procedures. Flow diagram of the experimental design for (A) Experiment 1 and (B) Experiment 2

Figure 2

Anxiety‐like and depression‐related behaviors in low‐dose aluminum‐treated C57BL/6J females. A‐D, Light/dark transition test: (A) distance travelled (cm) in the light and dark chambers, (B) latency to enter the light chamber (s), (C) time spent in the light chamber (s), and (D) number of transitions. E‐H, Elevated plus maze test: (E) distance travelled (cm), (F) number of arm entries, (G) entries into open arms (%), and (H) time spent in open arms (%). I, Immobility time (%) in the Porsolt forced swim test. J, Immobility time (%) in the tail suspension test. Values are means ± SEM. *P < .05, which indicates a nominally statistical significance for comparisons between treatment groups

Figure 3

Social behavior in low‐dose aluminum‐treated C57BL/6J females. A‐E, Social interaction test: (A) distance travelled (cm), (B) number of contacts, (C) total duration of contacts (s), (D) total duration of active contacts (s), and (E) mean duration per contact (s). F‐I, Three‐chamber social approach test, which was consisted of sociability test followed by social novelty preference test: (F) time spent in the chamber with the empty cage or the cage containing a stranger mouse (stranger 1) and (G) time spent around the empty cage or the cage containing a stranger mouse (stranger 1) in the sociability test. (H) Time spent in chamber with the cage containing stranger 1 or stranger 2 and (I) time spent around cage containing stranger 1 or stranger 2 in the social novelty preference test. J, K, Home‐cage social interaction test: (J) the mean activity level and (K) mean number of animals detected over 3 d

Figure 4

Working memory, reference memory, and fear memory in low‐dose aluminum‐treated C57BL/6J females. A, Correct responses (%) in the T‐maze test. B, Spontaneous alternation (%) in the Y‐maze test. C, Time spent around the target hole in the probe trial 1 and 35 d after the last training in the Barnes maze test. D‐H, Fear conditioning test: (D) freezing (%) in the conditioning, (E, G) context test 1 and 28 d after the conditioning, and (F, H) cued test 1 and 28 d after the conditioning. Values are means ± SEM. The asterisk indicates a nominally significance for comparisons between treatment groups (P < .05)

Figure 5

Anxiety‐like and depression‐like behaviors in high‐dose aluminum‐treated C57BL/6J mice. A‐D, Light/dark transition test: (A) distance travelled (cm) in the light and dark chambers, (B) latency to enter the light chamber (s), (C) time spent in the light chamber (s), and (D) number of transitions. E‐H, Elevated plus maze test: (E) distance travelled (cm), (F) number of arm entries, (G) entries into open arms (%), and (H) time spent in open arms (%). I, Immobility time (%) on days 1 and 2 in the Porsolt forced swim test. J, Immobility time (%) in the tail suspension test. Values are means ± SEM

Figure 6

Social behavior in high‐dose aluminum‐treated C57BL/6J mice. A‐E, Social interaction test: (A) distance travelled (cm), (B) number of contacts, (C) total duration of contacts (s), (D) total duration of active contacts (s), and (E) mean duration per contact (s). F‐I, Three‐chamber social approach test, which was consisted of sociability test followed by social novelty preference test: (F) time spent in the chamber with the empty cage or the cage containing a stranger mouse (stranger 1) and (G) time spent around the empty cage or the cage containing a stranger mouse (stranger 1) in the sociability test. (H) Time spent in chamber with the cage containing stranger 1 or stranger 2 and (I) time spent around cage containing stranger 1 or stranger 2 in the social novelty preference test. J, K, Home‐cage social interaction test: (J) the mean activity level and (K) mean number of animals detected over 3 d. Values are means ± SEM. The asterisk indicates a nominally significance for comparisons between treatment groups (P < .05)

Figure 7

Working memory, reference memory, and fear memory in high‐dose aluminum‐treated C57BL/6J mice. A, Spontaneous alternation (%) in the T‐maze test. B, Time spent around the target hole in the probe test 1 and 30 d after the last training in the Barnes maze test. C‐G, Fear conditioning test: (C) freezing (%) in the conditioning, (D, F) context test 1 and 30 d after the conditioning, and (E, G) cued test 1 and 30 d after the conditioning. Values are means ± SEM. The asterisk indicates a nominally significance for comparisons between treatment groups (P < .05)