Lab animal study suggests smoking during pregnancy places descendants at risk for asthma

NIH supported research indicates even great grandchildren of pregnant smokers may be at risk

For this research conversation, NICHD’s Dr. Tonse Raju spoke with grantee Dr. Virender Rehan on his study of rats given nicotine during their pregnancies.  (Nicotine is a key component of tobacco smoke, and the researchers gave the substance to the animals to simulate smoking.)  Other studies have shown that children born to women who smoked during pregnancy are more likely to develop asthma than are children born to non-smokers.  In this study of rats as substitutes for pregnant smokers, Dr. Rehan and his colleagues found evidence that the increased risk of asthma was conveyed through generations, to the animals’ grandchildren and even great grandchildren.

NICHD Research Conversations are audio interviews with NICHD scientists and grantees, on the latest NICHD-supported scientific findings. Listen to this Research Conversation at (MP3 - 1.6 MB).


Mr. Robert Bock: Welcome to the National Institutes of Health. I'm Robert Bock, Press Officer for the NIH's Eunice Kennedy Shriver National Institute of Child Health and Human Development, the NICHD. Thank you for joining us for today's Research Conversation.

Our host is Dr. Tonse Raju, of the NICHD's Pregnancy and Perinatology Branch. Today, Dr. Raju will interview Dr. Virender K. Rehan of the Los Angeles Biomedical Research Institute at Harbor–UCLA Medical Center, in Los Angeles. Dr. Rehan was the lead researcher for a study of lab animals, the results of which indicated that smoking during pregnancy could increase the chances that a woman's descendants will develop asthma. Dr. Raju.
Dr. Tonse Raju: Thank you very much, Bob. Asthma is a long-term lung disease that inflames and narrows the airways. Those who have asthma really have significant episodes of wheezing, chest tightness, shortness of breath, and coughing. At least, it has been estimated that more than 25 million people are known to have asthma, and it affects people of all ages. Typically, it begins during childhood. By 2023, some scientists estimate it can reach to as many as 400 million. Currently, it's about 300 million people with asthma, worldwide.

Now, in spite of all that, we do not know why people get asthma. Some experts believe that is the result from a combination of genetic and environmental factors. But, we do know that children who are born to women who smoke, either during pregnancy or even afterwards, are at much higher risk for developing asthma than children who are born to mothers who don't.

So, before we begin, let me say a few words for those among the audience who may be smokers. I know you have been told many times to quit smoking and I'm not here to preach you about that. And I also know that quitting is very difficult but I do want to urge you and recommend you that you should seek help from your healthcare provider to help you quit smoking. And if you're a pregnant woman who smokes, you probably have mentioned this to your healthcare provider. If not, please do so as soon as you can.

So, Dr. Virender Rehan, he's our guest today. He has recently published a nice study using laboratory rats, which indicates that the risk of asthma is not only limited to the children of women who smoke but also goes to grandchildren and even great-grandchildren.

Before we begin, I would like to point out that Dr. Rehan's research has been supported not only by the National Institutes of Child Health but also by our sister institute, the National Heart, Lung, and Blood Institute. So, we are very grateful for the assistance they have provided.

So, Dr. Rehan, welcome to this program. And before you tell us about your study, can you explain what is it about smoking that increases the risk of asthma in children born to mothers who smoke?
Dr. Virender Rehan: Good morning, Dr. Raju, and thanks for having me. As you've just mentioned, there is very clear and convincing evidence of increased predisposition of risk to asthma in infants born to mothers who smoke during pregnancy. Though this association has been known for decades, it's not very clear that, out of thousands of chemicals that are found in cigarette smoke, which one or which ones completely account for this association.

However, a large body of evidence suggests that nicotine might be a key ingredient in cigarette smoke that accounts for childhood asthma following smoke exposure during pregnancy. Supporting this, there is strong experimental and clinical evidence showing that, on exposure of the developing fetus, nicotine crosses the human placenta with minimal biotransformation. It accumulates in fetal blood, amniotic fluid, and several tissues, including the respiratory tract, and it is known to impair lung developmental programming that is consistent with molecular, cellular, structural, and functional changes relative of childhood asthma.
Dr. Raju: Yes.
Dr. Rehan: I think in this context, it may also be important to point out that these effects may not be restricted to nicotine exposure as a result of cigarette smoking alone but may―also are likely to result from the use of nicotine patch during pregnancy, which is often used as a nicotine-replacement treatment.
Dr. Raju: Thank you, Dr. Rehan. I would like to explain now to our listeners that this kind of study is very difficult to do, especially to see what happens to grandchildren and great-grandchildren. You know, it would take a long time to enlist a large number of women who smoke during pregnancy and follow them, follow their grandchildren or great-grandchildren. So, that will take several, several decades. So, that is why you needed to do this study in rats. I know that Dr. Rehan didn't teach the animals to smoke, but please tell us how you conducted your study, Dr. Rehan.
Dr. Rehan: No, you're absolutely right that conducting prospective transgenerational studies in humans is almost impossible in one's lifetime, so thereby forcing us to use animal models to describe and study this phenomenon. And of course, no, we didn't teach these rats to start smoking. So, we used nicotine as a model of exposure to cigarette smoke.

So, our animal model, which has actually been used by many other investigators as well, consists of using first-time pregnant Sprague Dawley rat dams, which we designated as F0 generation. These dams received either placebo or nicotine in a dose of 1 milligram per kilogram, subcutaneously, which actually is equivalent to exposure of a moderately cigarette-smoking pregnant woman.

So, we start at about embryonic day six of gestation and continue this exposure up to postnatal day 21, the rat gestation being 22 days. Following delivery at term, then the F1 pups are allowed to breastfeed ad libitum. So, the rats are exposed, for a total of about five weeks, to nicotine, covering most of the pregnancy and the perinatal period. Then, to examine the lung phenotype at postnatal day 21, these pups are subjected to pulmonary function tests, tracheal tension and airway contractility studies, and lungs are examined for molecular, cellular, and structural biomarkers of asthma.
Dr. Raju: Thank you. So, to simplify what you just said, so you took pregnant rats and then gave them nicotine and then tested how bad the lungs would be in their grandchildren and great-grandchildren rats. So, what did you find? And did you find what you had expected?
Dr. Rehan: So, actually, you know, before going to that, I must just elaborate a little bit more on the model. So, at postnatal day 21, as you said, you know, we separate males and females and then we breed them as to generate F2 generation pups, without giving any nicotine in the subsequent generation. And then, F2 pups are studied at postnatal day 21. Then, similarly, F3 pups are generated and those F3 pups are also studied at postnatal day 21.
Dr. Raju: So, by F2 you mean the grandchildren and F3, meaning great-grandchildren. OK. So, what did you find? Can you tell us?
Dr. Rehan: Sure. The key findings of our study include that the following exposure to nicotine in pregnancy, offspring asthma-like phenotype is not restricted only to F1 generation offspring―that is, children and grandchildren. This has been shown previously. But we see, in this study, that it is also transmitted transgenerationally to F3 generation offspring―that is, great-grandkids.
Dr. Raju: So...
Dr. Rehan: These changes are more severe in males, and nicotine-induced increased upper airway contractility was seen actually exclusively in males. And when we looked at the molecular profiling for the specific lung cells, that is, the lung interstitial fibroblasts, they showed changes similar to what we have seen in F1 and F2 generations.
Dr. Raju: So, that’s very interesting. And how could you explain exposure to nicotine while the fetus is inside the womb increases the chances of the offspring’s, well into third generation? Do you have an explanation?
Dr. Rehan: You know, this is actually a key question. You know, this is really to the heart of these studies, or these kind of problems, and gives us an opportunity to explore this fundamental issue underlying the mechanism of transmission of these effects of exposure to environmental toxins during pregnancy―nicotine, in this case―and even, actually, the bigger question of transmission of an acquired trait during an individual's lifetime.
Dr. Raju: Right.
Dr. Rehan: I think this addresses a much broader question. But, just to continue to answer your question specifically in regards to nicotine, I would say, of course, you know, it's very easy to understand that, when a pregnant F0 dam is exposed to nicotine, the developing fetal lungs are directly exposed to nicotine, resulting in an asthma phenotype in first-generation offspring.

However, what is not generally realized is that the germ cells within the developing first-generation fetus, which give rise to second-generation offspring, also get exposed to nicotine when gestating F0 dams receive nicotine. So, just like the first-generation offspring, second-generation offspring can develop asthma as a result of direct exposure of nicotine during gestation.
Dr. Raju: Yes, that's, once again, very intriguing and very much concerning. But you also found a difference between males and females. So, can you explain why it was more severe, the effect, in males?
Dr. Rehan: Certainly. Dr. Raju, again, before I go on to―so, I have explained this transmission up to second generation―that is, F2. But, that is called multi-generational transmission. However, the transmission of this effect beyond the second generation, what we've observed in this study, the true transgenerational effect, since F3 generation and beyond offspring were not directly exposed to nicotine at all.

So, to unravel the mechanisms underlying the transgenerational transmission is the focus of our ongoing studies.
Dr. Raju: Uh-huh.
Dr. Rehan: The evidence so far has suggested that nicotine-induced epigenetic changes, such as DNA methylation and histone acetylation in germs cells, might be the key in this transgenerational transmission. So, as I mentioned that in our previous study we had shown actually the blockage of nicotine-induced gonadal epigenetic changes normalized the lung phenotype, at least up to second-generation offspring. And now I predict in my hypothesis that this will be true for the transgenerational transmission up to third-generation as well.

So, now addressing the question that you asked, why the findings were more severe in males as compared to females: You know, I would say that this is also a very interesting aspect of these studies and I must say that we have seen this phenomenon in every generation that we have examined so far. It is true for generation one, generation two, and generation three―that pups born following exposure to nicotine during F0 generation had much greater severity of asthma in males.
Dr. Raju: Yes. I mean, clearly, this all adds up to the fact that one should quit smoking, and that's the best message, and you have a lot more to learn from the negative effects of nicotine and smoking itself. But the related question may be: What about the father smoking? Does it have any effect other than environmental? Do you think that can effect genetic modification also?
Dr. Rehan: At this moment, I would say that, you know, this is, again, a good question what you have raised and there is epidemiological evidence from case control studies that there is increased risk of childhood asthma even after exposure to paternal smoking. But the risk is not as high as following exposure to maternal smoking.
Dr. Raju: OK.
Dr. Rehan: I must also add that, to my knowledge, the transgenerational asthma risk following exposure to paternal smoking has not been reported so far. But, I have a feeling that it really should not matter whether the fetus is exposed to cigarette smoke from the mother or from the father. If the exposure to cigarette smoke is equivalent, the effect on the fetus should be the same.
Dr. Raju: Right. Yes, you're making an excellent point. And thank you so much for doing this wonderful study because, you know, for those who want to quit, this is additional motivation that they can consider and then request the doctors to help them. So, thank you for doing this study and hope all the best for your future research and for explaining us your interesting and important work.
Dr. Rehan: Thank you so much, again, and thanks for having me.
Dr. Raju: Thank you, Bob.
Dr. Rehan: Thanks, Bob.


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