The Toxicity of Dimethyl Sulfoxide Combined with the Alkylating Agent MNU on Mice: Focusing on Mortality and Activity Impairment

Abstract

Objective: Dimethyl sulfoxide (DMSO) is known to enhance the absorption of chemicals. This study investigated the effects of DMSO in combination with the alkylating agent, N-methyl-N-nitrosourea (MNU), on the activity and mortality of mice, providing foundational data for solvent mixture use and health impact assessment.

Methods: Twelve male SPF C57BL/6J mice were divided into three groups (n=4): DMSO group (D), DMSO combined with low-dose (L), and high-dose MNU group (H). Mice in D received intraperitoneal injections of pure DMSO solution at 12 mL/kg. The L or D group was injected with a DMSO solution containing 40 mg/kg or 60 mg/kg MNU at 12 mL/kg, respectively. Mice were monitored for changes in body weight, activity levels (walking, body stretching, fur condition), and mortality at various time points.

Results: Pre-injection, all groups showed no statistical differences in weight or DMSO dose, while the MNU injection dose was significantly different (H>L>D). Within 2 hours post-injection, all mice except one in the D group exhibited reduced mobility, hunched posture, and lethargy. Mortality progressed rapidly, with one D and two L mice dying by 12 hours, followed by additional deaths (one D, two L, and two H mice) at 24 hours. By 36 hours, all mice in L and H groups had died, while one D mouse recovered normal activity. At the 48-hour endpoint, only one D mouse survived with normal behavior. No significant differences in weight, activity levels and mortality changes were observed in surviving mice throughout the study period.

Conclusion: DMSO has certain toxicity, and when combined with alkylating agents such as MNU, it can lead to reduced activity and an increased mortality rate in mice. It is recommended to closely monitor the mice during the use of such reagents and to establish appropriate research observation protocols.

Keywords: MNU; dimethyl sulfoxide; mice; mortality; physiological effects; toxicity.

Graphical abstract

Figure 1

Body Weight of Each Group of Mice Before Injection. Bar graph showed the body weight of mice in the DMSO, low-dose MNU, and high-dose MNU groups before the injection. There were no significant differences in body weight between the groups (The confidence interval of the Bootstrap results included zero). D: the DMSO control group; L: the low-dose MNU group; H: the high-dose MNU group. N.S.: no statistical difference.

Figure 2

Volume of DMSO Injected in Each Group of Mice. Bar graph illustrated the volume of DMSO administered to each group (DMSO, low-dose MNU, high-dose MNU). The injection volume was consistent across all groups, at 12 mL/kg body weight, with no significant differences (The confidence interval of the Bootstrap results included zero). D: the DMSO control group; L: the low-dose MNU group; H: the high-dose MNU group. N.S.: no statistical difference.

Figure 3

MNU Dose Injected in Each Group of Mice. Bar graph showed the dose of MNU injected in the DMSO (D), low-dose (L) and high-dose (H) MNU groups. The high-dose group (60 mg/kg) received a significantly higher MNU dose compared to the low-dose group (40 mg/kg) and the DMSO group (0 mg/kg) (The confidence interval of the Bootstrap results did not include zero). D: the DMSO control group; L: the low-dose MNU group; H: the high-dose MNU group. None: not any quality of MNU; **: a significant difference existed.

Figure 4

Number of Each Group of Mice at Different Time Points and the Survival Curves. (A) Number of each group of mice at different time points. (B) The survival curves of each group of mice at different time points. The number of mice in each group with the survival curve displayed the number of surviving mice at various time points after injection in the DMSO, low-dose MNU, and high-dose MNU groups. Mortality increased over time, with the low-dose and high-dose groups experiencing complete mortality by 36 hours. The DMSO group displayed a smaller mortality, with one mouse surviving at 48 hours. T1: before injection; T2: at 12 hours post-injection; T3: at 24 hours post-injection; T4: at 36 hours post-injection; T5: at 48 hours post-injection. D: the DMSO control group; L: the low-dose MNU group; H: the high-dose MNU group.

Figure 5

Changes in Body Weight of Each Group of Mice After Injection. Line graph showed the changes in body weight over time in the DMSO, low-dose MNU, and high-dose MNU groups after injection. No significant differences in body weight changes were observed among the groups during the observation period. Timepoint 1 (T1): at the time of purchase (ie, before the 3-day pre-adaptation feeding).; T2: before injection (i.e., after the 3-day pre-adaptation feeding); T3: at 24 hours post-injection; T4: at 48 hours post-injection. D: the DMSO control group; L: the low-dose MNU group; H: the high-dose MNU group.

Figure 6

Changes in Activity Scores of Each Group of Mice After Injection. Line graph illustrated the changes in activity scores (measuring mobility, posture, and alertness) over time in each group after injection. Significant reductions in activity were observed in the low-dose and high-dose MNU groups, with severe declines noted by 24 hours. The DMSO group showed some recovery by 36 hours, though further decline was observed at 48 hours. T1: before injection; T2: at 2 hours post-injection; T3: at 24 hours post-injection; T4: at 36 hours post-injection; T5: at 48 hours post-injection. D: the DMSO control group; L: the low-dose MNU group; H: the high-dose MNU group.

Figure 7

Representative Images of the Activity Levels and Mental Status of Mice at Different Time Points in Each Group. Red arrows indicated mice with reduced activity and a hunched posture, while blue arrows highlighted mice with a good mental status, increased activity, high alertness, and the ability to move freely. Black arrows indicated the dead mice. T1: at 2 hours post-injection; T2: at 24 hours post-injection; T3: at 36 hours post-injection; T4: at 48 hours post-injection. D: the DMSO control group; L: the low-dose MNU group; H: the high-dose MNU group.