Immunomodulatory effects of maternal atrazine exposure on male Balb/c mice

Abstract

Atrazine is a widely used herbicide applied to corn, sugar and other crops as a broad leaf weed inhibitor. Using the Balb/c mouse model, we have determined that prenatal/lactational exposure to atrazine alters adult immune function. Pregnant Balb/c dams were exposed subcutaneously for 21 days via time release pellets to 700 microg per day of atrazine beginning between days 10 and 12 of pregnancy. Prenatal/Lactational exposure caused no overt physical malformations in the offspring and had no effect on the number of litters carried to term or the litter size. Upon reaching early adulthood (approximately 3 months of age), the state of their immune system was evaluated. There were no changes in body weight or in the organ to body weight ratio of the spleen. Additionally, no changes were observed in the number of CD8+ T cell, CD4+ T cell, or B220+ B cell subpopulations in the spleen. T cell function was assessed by measuring proliferation and cytolytic activity after in vitro allogeneic stimulation. Male mice which had been prenatally/lactationally exposed to atrazine had an increase in both T cell proliferation and cytolytic activity. The humoral immune response was assessed after immunization with heat killed Streptococcus pneumoniae (HKSP). There was a significant increase in the number of HKSP-specific IgM secreting B cells in the spleen of prenatal/lactational exposed male mice. Inasmuch as atrazine is a widespread environmental contaminant, this immunopotentiation raises concerns that it may potentiate clinical diseases, such as autoimmune disease and hypersensitivity, and needs to be carefully monitored and studied.

Fig. 1.

The effects of atrazine exposure on mouse reproduction. (A) Pregnancy rate of a total of 71 breeding females receiving an atrazine pellet and a total of the 49 breeding females receiving a placebo pellet. (B) The average litter size of a total of 48 breeding females receiving an atrazine pellet and a total of 25 breeding females receiving a placebo pellet. Data are the average of data obtained from two experiments.

Fig. 2.

Number of anti‐HKSP antibody secreting splenocytes in 3‐month‐old offspring treated prenatally/lactationally with atrazine (solid bars) or placebo (open bars). The spleens of animals were removed 14 days after immunization and the number of HKSP‐specific B cells quantified by the ELISpot assay. The data are presented as the number of antibody secreting cells (spots) normalized to 10⁶ B220⁺ cells (A; enumerated by flow cytometry) and the number of antibody secreting cells normalized to 10⁶ splenocytes (B) for each sex. Error bars represent the SEM; *indicates that the values obtained for atrazine offspring were significantly (P < 0.05) different from those obtained for control offspring. The data are representative from 1 of 2 experiments (male treated, n = 14 representing offspring from 13 different dams; male controls, n = 16 representing offspring from 12 different dams; female treated, n = 14 from 12 different dams; female controls n = 15 from 14 different dams).

Fig. 3.

The mixed lymphocyte reaction in 3‐month‐old offspring. Splenocytes harvested from 3‐month‐old offspring of dams that were treated with atrazine or placebo as described in the Methods section were stimulated by co‐culturing with irradiated C57Bl/6 splenocytes. Four days after allogeneic stimulation, the cells from co‐cultures were extracted washed in cRPMI and the level of ³H‐Thy quantified by liquid scintillation methods. Error bars represent the SEM; *indicates that the values obtained for atrazine offspring were significantly (P < 0.05) different from those obtained for control offspring. Data representative of 2 experiments (male treated, n = 14 comprised of offspring from 13 different dams; male control, n = 16 comprised of offspring from 12 different dams; female treated, n = 6 comprised of offspring from 6 different dams; female control, n = 6 comprised of offspring from 6 different dams).

Fig. 4.

Cytotoxic lymphocyte response in 3‐month‐old offspring. Splenocytes harvested from 3‐month‐old offspring of dams that were treated with atrazine as described in the Methods were stimulated by co‐culturing with irradiated C57Bl/ 6 splenocytes. The effector and stimulator cells were mixed at a one to one (1∶1) ratio and then incubated for 5 days. After 5 days, specific lysis was determined by incubating the stimulated effector cells with ⁵¹Cr‐labeled EL‐4 target cells at ratios varying from 100∶1 to 3.12∶1 effector:target cell for 4 h and then assaying the supernatant for ⁵¹Cr‐released. Error bars represent the SEM; ANOVA indicates that the values obtained for atrazine offspring were significantly (P < 0.05) different from those obtained for control offspring (atrazine treated, n = 14 comprised of offspring from 13 different dams; control, n = 16 comprised of offspring from 12 different dams).