What made you want to research this issue?
Our group has conducted research on fetal programming by maternal occupational exposures for more than two decades, in order to study whether these may pose a risk for women working during pregnancy. The fetal programming hypothesis proposes that environmental factors may affect fetal development so as to prepare the child for the conditions awaiting in life after birth. Since 2006, a large part of our research has focused on the potential for fetal programming by inhaled nanosized particles.
During the last decade it has become apparent that many of the offspring’s organs may be affected by maternal particle exposure, e.g. the central nervous, cardiovascular, and immune systems. Since reproductive function is such an important part of life, we wanted to know if the reproductive function of the male offspring could also be affected by maternal airway exposure to particles. In Denmark, fertile women have a very high employment rate and therefore, adverse effects of prenatal occupational exposures constitute a special area of interest for us.
Could you talk about previous research in this area, how does your study build on this?
Only a few studies have previously investigated this outcome. The first study in this field exposed pregnant mice to nanosized carbon black particles twice during gestation. Testicular structural changes and reduced daily sperm production were observed in the male offspring.
Then our own research group found a reduction in offspring sperm production following maternal exposure to carbon black particles, but in the second generation offspring of the male germline, rather than in the first generation males. This made us wonder if maternal exposure could affect not only the first generation, but also the following generations. Therefore, we designed this four generation study.
To what extent can this model be analogous to humans?
Several regulatory guidelines recommend the use of rodent studies in the investigation of the potential for maternal chemical exposures to affect reproductive capacity in the offspring. In this study we used the most relevant route of exposure in humans, i.e. inhalation, at occupationally relevant exposure levels.
Occupational exposure to engineered nanoparticles can occur during production, refinement, bagging and shipping. Certain occupations such as firefighters and welders may also be exposed to high levels of incidental nano particles. Carbon black particles are however commonly used to model exposure to air pollution particles, e.g. from combustion of diesel fuel. Exposure to the latter may occur both at work and in private life.
In our study, exposure was initiated on gestation day four, but in occupationally exposed women, exposure may have started way before pregnancy, so particles could potentially have accumulated in lung tissue and possibly have translocated to be present in the reproductive organs at fertilization. Differences between the human and the rodent placenta, i.e. the organ that constitutes the interface between mother and fetus and are responsible for exchange of nutrients, could also implicate that larger quantities of particles could translocate to the fetus in humans. Finally, humans may be more susceptible to toxicological insult than mice. We really lack knowledge on the applicability of standard testing protocols and models for testing particles for reproductive toxicity.
Could you tell us about the experiment design, particularly what the doses represent?
The pregnant mice were exposed for 45 minutes daily from gestation day 4 to 18, i.e. 15 days out of the mouse gestation length of 20 days. The weighted average occupational exposure limit of carbon black particles is 3.5 mg/m3 in Denmark for around 8 hours of exposure. The mice in this study were exposed to an average of 4.6 mg/m3 for the low dose and 37 mg/m3 for the high dose for 45 minutes. This corresponds to the average occupational exposure of 1 hour and 8 hours, respectively. Hence, we performed the exposure to carbon black particles at occupationally relevant exposure levels.
What are the implications of the research and what are the next steps?
The results are reassuring in that we did not observe adverse effects on male fertility after the maternal particle exposure, in any of the four generations of males. It would however be premature to conclude that exposure to carbon particles at the Danish occupational exposure limits does not pose a risk to male reproduction in humans. This is because humans may be more susceptible to toxicological insult than mice and because human exposure would probably begin prior to pregnancy, as described above.
Interestingly, in the first generation of male and female mice from our study, we observed changes in the central nervous system, indicating that toxic effects of carbon black particles on other organ systems may occur at lower levels than male reproductive developmental toxicity. Regarding our next steps, we’ve collected several samples from all generations of offspring, and we are very interested in getting in touch with researchers that would like to explore the epigenetic changes throughout the four generations, that could have been induced by the maternal exposure to particles.