NanoTIO2 (UV-Titan) does not induce ESTR mutations in the germline of prenatally exposed female mice
© Boisen et al.; licensee BioMed Central Ltd. 2012
Received: 9 November 2011
Accepted: 14 May 2012
Published: 1 June 2012
Particulate air pollution has been linked to an increased risk of cardiovascular disease and cancer. Animal studies have shown that inhalation of air particulates induces mutations in the male germline. Expanded simple tandem repeat (ESTR) loci in mice are sensitive markers of mutagenic effects on male germ cells resulting from environmental exposures; however, female germ cells have received little attention. Oocytes may be vulnerable during stages of active cell division (e.g., during fetal development). Accordingly, an increase in germline ESTR mutations in female mice prenatally exposed to radiation has previously been reported. Here we investigate the effects of nanoparticles on the female germline. Since pulmonary exposure to nanosized titanium dioxide (nanoTiO2) produces a long-lasting inflammatory response in mice, it was chosen for the present study.
Pregnant C57BL/6 mice were exposed by whole-body inhalation to the nanoTiO2 UV-Titan L181 (~42.4 mg UV-Titan/m3) or filtered clean air on gestation days (GD) 8–18. Female C57BL/6 F1 offspring were raised to maturity and mated with unexposed CBA males. The F2 descendents were collected and ESTR germline mutation rates in this generation were estimated from full pedigrees (mother, father, offspring) of F1 female mice (192 UV-Titan-exposed F2 offspring and 164 F2 controls). ESTR mutation rates of 0.029 (maternal allele) and 0.047 (paternal allele) in UV-Titan-exposed F2 offspring were not statistically different from those of F2 controls: 0.037 (maternal allele) and 0.061 (paternal allele).
We found no evidence for increased ESTR mutation rates in F1 females exposed in utero to UV-Titan nanoparticles from GD8-18 relative to control females.
Mutations in male and female gametes may lead to detrimental inherited effects in subsequent generations. Human exposure to particulate air pollution (PAP) has been shown to adversely affect germ cells in males . Moreover, animal studies have demonstrated that inhalation of PAP can induce mutations in the male germline [2–6]. Airborne particles in the nanometer range deposit deep in the airways. These particles are cleared very slowly and a small fraction may translocate into the bloodstream [7, 8]. Inhaled nanoparticles (NPs) are potent inducers of pulmonary inflammation and oxidative stress, which may affect the fetus indirectly during maternal exposure [9–11].
As a model of NP exposure we tested nanosized titanium dioxide (nanoTiO2) UV-Titan, which is used in the production of paints [9, 12, 13]. Large quantities of nanoTiO2 are used globally in a wide range of products. TiO2 was previously believed to be inert, but inhaled TiO2 has now been classified as possibly carcinogenic to humans by the International Agency for Research on Cancer . TiO2 toxicity depends on particle size, crystalline form and surface modifications . Pulmonary exposure to nanoTiO2 causes inflammation in rodents [9, 16] and we recently found that a single UV-Titan instillation induced an inflammatory response in mice after 1 day [12, 17]. In addition, UV-Titan particles remained in lungs 4 weeks after inhalation, causing long-lasting inflammation  .
Expanded simple tandem repeat (ESTR) loci in mice exhibit high spontaneous mutation rates enabling the study of induced germline mutations following environmental exposures. Radiation, air particulates, and a number of chemicals have been shown to increase ESTR mutations in male germ cells [2, 5, 18, 19]. Very limited data exist on induced mutations in female germ cells, which have previously been considered highly resistant to genotoxicity . However, oocytes could be vulnerable during stages of active cell division, i.e. during fetal development [20, 21]. A recent study showed that prenatal exposure to 1 Gy of acute irradiation on GD12 resulted in a 1.94-fold increase in ESTR mutations in the offspring of irradiated female mice [22, 23].
We hypothesized that prenatal exposure to NPs will affect female germline ESTR mutation frequency during stages of active cell division, similar to what has been found for male germline cells . The present study investigates TiO2 nanoparticle-induced effects on female germline DNA by exposing pregnant female mice (P) to nanoTiO2 or clean filtered air via inhalation and subsequently mating their offspring (F1) with unexposed males. The observed F1 female germline ESTR mutation frequency was calculated by comparing allele size in the F2 offspring to their mother’s allele size to quantify repeat gains and losses.
Animals and exposure
DNA extraction and mutation analysis
Summary of ESTR mutation rates in F2 offspring of prenatally exposed female C57BL/6 mice
N (F2 offspring)
Mutation rate ± SEM (P valuea)
0.0671 ± 0.0002
0.0305 ± 0.0002
0.0549 ± 0.0004
0.0427 ± 0.0004
0.0610 ± 0.0028
0.0366 ± 0.0030
Female TiO2 exposed
0.0521 ± 0.0004
0.0208 ± 0.0002
Female TiO2 exposed
0.0417 ± 0.0004
0.0365 ± 0.0003
Female TiO 2 exposed
0.0469 ± 0.0107 (P = 0.84)
0.0286 ± 0.0133 (P = 0.79)
Results and discussion
F1 females were prenatally exposed to UV-Titan by maternal inhalation of 42.4 mg UV-Titan/m3 1 hour/day on GD8-18 (Figure 1). 164 and 192 offspring from control and exposed females, respectively, were scored. Thus, a total of 328 and 384 inherited bands were scored per group. The observed mutation rate in germ cells of UV Titan-exposed F1 females was not significantly different from controls (Table 1). The Ms6-hm and Hm-2 mutation rates in control females were similar to those found for females in other studies using the same mouse strain [2, 19]. Furthermore, the number of offspring, sex-ratio and time to birth of the first F2 litter did not differ between groups, suggesting that UV-Titan did not affect viability of the F2 offspring (data not shown). Absence of effect is therefore not due to lower viability of affected offspring. Mutations in ESTRs should not affect offspring fitness since these loci do not have known functions.
ESTR mutations have been suggested to be induced via polymerase pausing resulting from the presence of epigenetic changes or DNA damage such as oxidative stress, strand breaks or adducts elsewhere in the genome rather than by direct DNA damage . We have reported that the inhalation of a total dose of 840 μg UV-Titan per animal at GD8-18 induced persistent inflammation in the lungs of the time-mated P generation (Figure 1) [9, 24]. Furthermore, 476 genes were found to be differentially expressed in the liver of newborn F1 generation females prenatally exposed to UV-Titan. We hypothesize that the transfer of inflammatory cytokines across the placenta may have caused this differential gene expression  since no TiO2 was detected in maternal liver, mother’s milk or offspring liver .
ESTR mutation analysis is a sensitive method, enabling analysis under realistic exposure scenarios. An a priori power analysis showed that group size in the present study provided a 77% chance of detecting a 2-fold increase in ESTR mutations at the 5% significance level. The exposure and the estimated inhaled dose of 840 μg used in this study is comparable to the permissible exposure limit by Danish Regulation and the exposure route (inhalation) is also relevant to environmental exposure . As little as 54 μg UV-Titan can induce inflammation in mouse lungs after one day [12, 17]. Female germ cells enter meiotic prophase on ~ GD13.5 . In the present study female mice were prenatally exposed from GD8-18 ensuring that the period of mitotic germ cell division was targeted; these mothers were exposed to ~458 μg prior to GD13. Consequently, a high degree of inflammation was likely to be present at GD13.5, when oocytes cease to be susceptible to ESTR mutations [21, 22].
In parallel with the present study (in the same laboratory and time period), ESTR germline mutations in male and female mice prenatally exposed to diesel exhaust particles (DEP) by inhalation were quantified . Male germ cell mutation rates were significantly increased following exposure to DEP and may thus be regarded as a positive control for the ability to detect induced ESTR mutation. ESTR mutation rates were not significantly increased in germ cells of females prenatally exposed to DEP. To our knowledge, this is the only other study, which has investigated chemically induced ESTR mutations in prenatally exposed females. A recent study showed that dividing oocytes are susceptible to mutations in vivo. Prenatal exposure to 1 Gy of acute irradiation on GD12 resulted in a 1.94-fold increase in the ESTR mutation rate .
NanoTiO2 can induce DNA strand breaks and carcinogenic effects in vivo[11, 25, 26]. We recently reported that UV-Titan inhalation did not increase DNA strand breaks in the P or F1 generations , suggesting that genotoxic effects in offspring are negligible. Correspondingly, in the study of prenatal DEP exposure by , which showed ESTR instability in male offspring, the exposure also failed to increase DNA strand breaks in liver from newborns . Epigenetic changes have been suggested as the underlying mechanism of ESTR instability [5, 22]. A recent study found DNA deletions in mice prenatally exposed to nanoTiO2. However, the small effective sample size and the very large maternal dose used in the study hamper interpretation. The results on nanoTiO2 induced mutations and genotoxicity are conflicting [10, 15, 26]. The various types of commercially available nanoTiO2 also make it difficult to generalize. It is possible that NPs with very active surface chemistry, which produce more reactive oxygen species (ROS) or a large inflammatory response, could induce germline mutations. In the present study we have only assessed the effects of a single type of TiO2 NP. We are currently investigating the effects of prenatal exposure to nanosized carbon black Printex90, a more efficient generator of ROS than both DEP and nanoTiO2 to further address the question of female susceptibility to NPs. The present study indicates that prenatal exposure to nanoTiO2 does not affect female germline ESTR mutation frequency.
The authors gratefully acknowledge statistical support from Andrew Williams and technical assistance from Michael Guldbrandsen, Gitte Kristiansen and Colin Davis. This work was supported by the Danish Centre for Nanosafety from the Danish Working Environment Research Fund.
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