Airway exposure to multi-walled carbon nanotubes disrupts the female reproductive cycle without affecting pregnancy outcomes in mice

Abstract

Background: The use of multiwalled carbon nanotubes (MWCNT) is increasing due to a growing use in a variety of products across several industries. Thus, occupational exposure is also of increasing concern, particularly since airway exposure to MWCNTs can induce sustained pulmonary acute phase response and inflammation in experimental animals, which may affect female reproduction. This proof-of-principle study therefore aimed to investigate if lung exposure by intratracheal instillation of the MWCNT NM-400 would affect the estrous cycle and reproductive function in female mice.

Results: Estrous cycle regularity was investigated by comparing vaginal smears before and after exposure to 67 μg of NM-400, whereas reproductive function was analyzed by measuring time to delivery of litters after instillation of 2, 18 or 67 μg of NM-400. Compared to normal estrous cycling determined prior to exposure, exposure to MWCNT significantly prolonged the estrous cycle during which exposure took place, but significantly shortened the estrous cycle immediately after the exposed cycle. No consistent effects were seen on time to delivery of litter or other gestational or litter parameters, such as litter size, sex ratio, implantations and implantation loss.

Conclusion: Lung exposure to MWCNT interfered with estrous cycling. Effects caused by MWCNTs depended on the time of exposure: the estrous stage was particularly sensitive to exposure, as animals exposed during this stage showed a higher incidence of irregular cycling after exposure. Our data indicates that MWCNT exposure may interfere with events leading to ovulation.

Keywords: Developmental toxicity; Estrous cycle; Female; Fertility; Multi-walled carbon nanotubes; Nanomaterials; Ovulation; Pregnancy; Reproductive toxicity.

Fig. 1

Design of Experiment 1. Vaginal smears were obtained once a day for 14 days from 50 females. Here after, 25 females were randomly chosen for exposure to 67 μg MWCNT by intratracheal instillation, and 25 received vehicle only. Vaginal smears were obtained for 2 weeks more, where after the experiment was finalized and vaginal smears evaluated by a person blinded to exposure status

Fig. 2

The absolute values for cycle length before, during, and after exposure to 67 μg of MWCNT by instillation for controls (a) and exposed (b) females are shown (control group n = 19–22, exposed group n = 21–23)

Fig. 3

Cycle lengths before, during, and after exposure, obtained from the mixed model SAS analysis. The cycle during exposure was significantly longer than the cycles before exposure. The cycle immediately after exposure was significantly shorter than both the cycles before exposure and the exposed cycle. No effects were observed in the vehicle exposed animals. Values are given as mixed model estimate average ± SEM. (**: p = 0.001 compared to the cycle before to exposure; ##: p < 0.001 compared to the exposed cycle, n = 20–23)

Fig. 4

Cumulative littering curves relative to time to delivery of litter. Littering curves were obtained by registration of the day of delivery of the litter relative to start of cohabitation with a mature, unexposed male. Females were exposed to vehicle or 2 μg, 18 μg, or 67 μg of MWCNT by instillation, 1 day prior to cohabitation

Fig. 5

Expression of Bdnf (a), Igf-1 (b), and Tnfα (c) 8 weeks after exposure to vehicle or 2 μg, 18 μg, or 67 μg of MWCNT by instillation. Values are given as average ± SEM (*: p < 0.05)