Vanillic acid protects mortality and toxicity induced by N-ethyl-N-nitrosourea in mice; in vivo model of chronic lymphocytic leukemia

Abstract

Alkylating agents such as N-Ethyl-N-Nitrosourea (ENU) are ubiquitous within living cells and in the environment. This study designed to evaluate the chemopreventive activity of vanillic acid on ENU-induced toxicity and carcinogenesis in mice as an animal model of chronic lymphocytic leukemia (CLL). The female, Swiss albino mice were divided into three groups each with 7 mice, group I received normal saline, group II, mice received ENU at a dose of 80 mg/kg body weight i.p. to induce CLL on the 31th day of the study, and group III, the mice pretreated with vanillic acid at a dose of 20 mg/kg body weight/day, i.p. up to 30 days and received ENU. The animals were monitored for weight changes and mortality during 120 days, and then were sacrificed for isolation of lymphocytes, as target cells in CLL. Cellular parameters like reactive oxygen species (ROS) formation, malondialdehyde (MDA) production, depletion of glutathione (GSH), mitochondrial membrane potential (MMP) and lysosomal membrane integrity were studied. We found that pretreatment with vanillic acid significantly increased the survival of mice up to 57%, delay in death time (30%) and prevented weight changes after exposure to ENU. In addition, it was found that vanillic acid protected ROS formation, lipid peroxidation mitochondrial dysfunction, and lysosomal membrane destabilization in isolated lymphocytes. These data suggest that vanillic acid exhibited significant protection against ENU-induced toxicity and carcinogenicity, which might be related to the protection of the mitochondria and lysosomes and the reduction of ROS formation and oxidative stress.

Keywords: Cancer chemoprevention; Carcinogenesis; Dietary; Leukemia; Phytochemicals.

Graphical abstract

Fig. 1

Effect of ENU exposure (a single dose of 80 mg/kg) and pretreatment of vanillic acid (20 mg/kg/daily/ for 30 days) on the weight changes (A) and survival rate (B) in treated mice during the experimental period (120 days); Data were collected every week for each treatment group. The number of surviving mice is represented as % control group and weight changes are also presented as gram (g). ENU, N-Ethyl-N-Nitrosourea; VA, Vanillic acid.

Fig. 2

Intracellular ROS formation levels were determined by flow cytometry and representative histograms of ROS fluorescence are illustrated. The fluorescence intensity of different groups was calculated, and the results were presented as areas under the curve of relative light units (RLU). ROS formation increased in the ENU-alone group. However, this increase in ROS formation was significantly inhibited in the ENU exposure after vanillic acid-pretreatment group. *** represents a significant difference in relation to the control group (P < 0.001); ### represents a significant difference in relation to the ENU group (P < 0.001). Values present means ± SEM, n = 5 and were analyzed by one-way ANOVA and Tukey’s multi-comparison test. FL1, Fluorescence channel; ENU, N-Ethyl-N-Nitrosourea; DCFH-DA, 2՛,7́́՛dichlorofluorescin diacetate; VA, Vanillic acid; SEM, Standard error of the mean.

Fig. 3

Effects of ENU and vanillic acid on cellular MDA level (A), GSH (B) and GSSG (C) in isolated lymphocytes obtained from different groups. MDA production increased in the ENU-alone group. However, this increase in MDA production was significantly inhibited in the ENU exposure after vanillic acid-pretreatment group. In the GSH and GSSG results bars do not differ significantly. * represents a significant difference in relation to the control group (P < 0.05); # represents a significant difference in relation to the ENU group (P < 0.05). Values present means ± SEM, n = 5 and were analyzed by one-way ANOVA and Tukey’s multi-comparison test. ENU, N-Ethyl-N-Nitrosourea; MDA, Malondialdehyde; VA, Vanillic acid; GSH, Reduced glutathione; GSSG; Oxidized glutathione; SEM, Standard error of the mean.

Fig. 4

Mitochondrial membrane potential was determined by flow cytometry and representative histograms of the fluorescence intensity of rhodamine 123 are illustrated. The fluorescence intensity of different groups was calculated, and the results were presented as areas under the curve of relative light units (RLU). Exposure to a single dose of ENU (80 mg) alone increased mitochondrial depolarization in mouse isolated lymphocytes. This increase in mitochondrial depolarization was significantly inhibited in mice pretreated with 20 mg/kg vanillic acid before ENU exposure. *** represents a significant difference in relation to the control group (P < 0.001); ### represents a significant difference in relation to the ENU group (P < 0.001). Values present means ± SEM, n = 5 and were analyzed by one-way ANOVA and Tukey’s multi-comparison test. FL1, Fluorescence channel; ENU, N-Ethyl-N-Nitrosourea; VA, Vanillic acid; SEM, Standard error of the mean.

Fig. 5

Lysosomal membrane destabilization was determined by flow cytometry and representative histograms of the fluorescence intensity of acridine orange are illustrated. The fluorescence intensity of different groups was calculated, and the results were presented as areas under the curve of relative light units (RLU). Exposure to ENU alone increased lysosomal membrane disintegrity in mouse isolated lymphocytes. This increase in lysosomal membrane disintegrity was significantly inhibited in mice pretreated with vanillic acid before ENU exposure. *** represents a significant difference in relation to the control group (P < 0.001); ### represents a significant difference in relation to the ENU group (P < 0.001). Values present means ± SEM, n = 5 and were analyzed by one-way ANOVA and Tukey’s multi-comparison test. AO, Acridine orange: FL1, Fluorescence channel; ENU, N-Ethyl-N-Nitrosourea; VA, Vanillic acid; SEM, Standard error of the mean.