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EXPERT REACTION: Nicotine may harm embryos, even if you're not smoking it

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Exposing embryoid bodies - 'simulated' embryos composed of groups of stem cells that give rise to the brain, heart, liver, blood vessels, muscles and other organs - to a growth fluid dosed with nicotine for three weeks, killed off cells, caused growth defects, increased levels of damaging molecules, and interfered with communication between cells and with normal cell functioning, according to US scientists. The findings suggest nicotine itself is damaging to embryos, even without the toxins associated with burning tobacco, and it's harmful at every stage of development, even before embryos adhere to the uterus wall, the researchers say. However, the authors concede that these 'simulated' embryos don't necessarily reflect the environment inside the body of a pregnant woman.

Journal/conference: Stem Cell Reports

DOI: 10.1016/j.stemcr.2019.01.022

Organisation/s: Stanford Cardiovascular Institute, USA

Funder: National Institutes of Health, USA.

Media Release

From: Cell Press

Nicotine may harm human embryos at the single-cell level

Nicotine induces widespread adverse effects on human embryonic development at the level of individual cells, researchers report February 28th in the journal Stem Cell Reports. Single-cell RNA sequencing of human embryonic stem cell (hESC)-derived embryoid bodies revealed that 3 weeks of nicotine exposure disrupts cell-to-cell communication, decreases cell survival, and alters the expression of genes that regulate critical functions such as heart muscle-cell contractions.

The authors say this stem-cell model offers new insights into the effects of nicotine on individual organs and cells within the developing fetus and can be used to optimize drug and environmental toxicity screening.

"These results are especially important in that they provide a scientific basis for educating the public, especially young women, to keep away from smoking when they are pregnant or considering having a family," says senior author Joseph C. Wu (@StanfordCVI) of the Stanford University School of Medicine. "Nicotine found in products such as tobacco, e-cigarettes, and nicotine gums may have wide-ranging, harmful effects on different organs of a developing embryo during pregnancy."

Maternal smoking during pregnancy is an established risk factor for birth defects such as miscarriage, growth restriction, and premature birth. It is closely associated with long-term adverse neurobehavioral, cardiovascular, respiratory, endocrine, and metabolic outcomes in offspring. Nicotine, the main chemical constituent of tobacco smoking, is primarily responsible for the elevated risk. Unfortunately, the introduction and spread of new tobacco products containing nicotine, such as e-cigarettes, is reversing recent progress toward the reduction of tobacco use.

A large body of research has elucidated the negative effects of nicotine in animals, mainly in rodent models. Animal studies have demonstrated that nicotine exposure during pregnancy has detrimental effects on fetal development. But due to interspecies differences, it remains questionable whether this research can be translated to humans. While other studies have examined nicotine toxicity using human cells through bulk RNA-sequencing analysis, these conventional studies did not allow researchers to investigate effects at the single-cell level. As a result, the effects of nicotine on human embryonic development and the underlying molecular mechanisms remain poorly understood.

To address these limitations, Wu and his collaborators used single-cell RNA sequencing to analyze the effects of 21 days of nicotine exposure on the transcriptomes of a total of 12,500 cells generated from hESC-derived embryoid bodies, which are 3D aggregates of different types of pluripotent cells that give rise to the brain, heart, liver, blood vessels, muscles, and other organs. They found that cell survival decreased, suggesting that nicotine can affect embryo development as early as the preimplantation stage.

Nicotine exposure also decreased the size of the embryoid bodies, increased the level of damaging molecules called reactive oxygen species, and resulted in the aberrant formation and differentiation of the embryoid bodies. Moreover, nicotine exposure altered cell cycling in a broad range of progenitor cells differentiated from hESCs and caused dysregulated cell-to-cell communication, another adverse effect that has not been well studied.

"This is important because we know that smoking and nicotine have been shown to increase the pathological risk in endocrine, reproductive, respiratory, cardiovascular, and neurologic systems that rely on intricate and dynamic interactions amongst multiple cell types for homeostasis and function," Wu says.

The researchers also found that nicotine exposure leads to altered expression of genes implicated in metal toxicity and mitochondrial function, brain malformations and intellectual disability, muscle development and disease, lung disease, and Ca2+-associated arrhythmias that affect the contractility of heart muscle cells.

"A major implication of our study is that we now have validated a new method for evaluating the effect of drugs and environmental toxicity on human embryonic development," Wu says. "But one major limitation is that we are not able to recapitulate the whole-body physiology of a pregnant woman using differentiation of hESCs into embryoid bodies. For example, the influence of exercise, stress, food, or hormonal changes are not captured in this model."

In the future, the researchers plan to further investigate the mechanisms of nicotine-induced fetal birth defects. "We hope this will lead to the discovery of novel biomarkers that can help doctors better prevent, diagnose, and treat these diseases," Wu says. "In addition, we plan to utilize our hESC-derived embryoid body model and single-cell-sequencing technology to investigate the wider effects of other harmful conditions such as air pollution on human embryonic development."

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Expert Reaction

These comments have been collated by the Science Media Centre to provide a variety of expert perspectives on this issue. Feel free to use these quotes in your stories. Views expressed are the personal opinions of the experts named. They do not represent the views of the SMC or any other organisation unless specifically stated.

Dr Alexandra Harvey is a Research Fellow in the Reproductive Biology & Assisted Conception Laboratory at The University of Melbourne

The push towards alternatives to smoking (i.e. nicotine replacement therapies, NRT) are assumed beneficial for women's health, however the study by Wu and colleagues highlights that even brief exposure to nicotine at a critical developmental stage is sufficient to induce adverse changes.

It is well known that exposure of the early embryo to perturbations in its surrounding environment induces persistent alterations that increase an individual’s susceptibility for adult-onset diseases and the study by Wu and colleagues raises similar concerns about the use of NRT.

The report by Wu and colleagues uses embryoid bodies to determine the direct impact of nicotine exposure, independent of impacts on the mother which are likely to compound the adverse effects.

Embryoid bodies are 3D embryo-like structures representative of a stage of development shortly after implantation, generally before a woman knows she is pregnant, during which the precursors to the various different cell types of the body are established.

By assessing gene expression at the single-cell level, the researchers were able to tease apart cell-type specific changes, identifying alterations that have been associated with several diseases.

These findings are significant for the ~12% of Australian women who smoke before they know they are pregnant, particularly those that switch to NRT assuming these products are safer alternatives.

Importantly, the embryoid body model could be employed to screen interventions which may minimise the effects of nicotine exposure and lead to treatment options in those that are unable to overcome the addiction to nicotine before trying to conceive.

Last updated: 02 Apr 2019 12:02pm
Declared conflicts of interest:
None declared.
Gino Pecoraro is Associate Professor of obstetrics and gynaecology at the University of Queensland, a Federal Councillor for the National Association of Specialist Obstetricians and Gynaecologists (NASOG) and a Federal member of the Australian Medical Association (AMA) Board

This study provides yet another reason why couples contemplating starting a family should make sure they are cigarette free.  For the first time, the effects of nicotine have been directly studied on human embryonic cells and confirms animal model derived suspicions of harm.

Everyone knows that smoking when pregnant can lead to problems with the baby’s growth and long-term metabolic adverse effects in the children born to smoking mothers. The mothers also suffer from higher rates of miscarriage and birth prematurity as well as heart and lung issues.

There is now evidence to suggest that nicotine- a chemical found in high concentration in cigarette smoke  - can actually affect the RNA of developing human embryonic stem cells.

As little as 21 days of exposure to nicotine has been shown to affect communication between cells, adversely affect survival of cells and also negatively influence the activation and deactivation of critical genes that are important for such basic functions as making heart muscle cells contract. 

Nicotine-exposed cells have to deal with more damaging free oxygen radicals, may have dysfunctional mitochondria (the powerplants of cells) and abnormalities of other intracellular organelles.

This new method for studying the effects on human cells will be rolled out to find answers on the effects of other drugs and toxins on human embryonic development and it is hoped that information gained in this way will further help doctors prevent and treat conditions caused by toxic exposure during organ development.

Last updated: 01 Mar 2019 3:13pm
Declared conflicts of interest:
None declared.
Dr Ian Musgrave is a Senior Lecturer in the Faculty of Medicine, School of Medicine Sciences, within the Discipline of Pharmacology at the University of Adelaide.

Smoking during pregnancy is linked with several poor outcomes for both the pregnant woman and unborn child. Despite this in 2016 nearly 10% of Australian women smoked during pregnancy. Most of these adverse effects are associated with nicotine.

With the availability of nicotine replacement therapy and nicotine containing vaping fluids (despite the latter being illegal in Australia), pregnant women may be exposed to potentially damaging nicotine concentrations. Despite substantial animal research, it is still unclear the extent of nicotine damage to the human fetus and which organs are at risk.

This research has used stem cells to generate cell clusters that model the developing organs in a fetus, and has looked at gene expression in single cells from these model organs. This provides a snapshot of how nicotine influences the development of these tissues, and privies insight into how nicotine exposure translates into low birthweight, sudden infant death syndrome, craniofacial abnormalities and heart disease.

There are some limitations to this approach, one of which was that the authors used concentrations of nicotine around 30 times or more higher that would be found in a mother’s blood (whether smoking, using nicotine patches or vaping nicotine containing liquids). Nonetheless it can provide insights that may lead to better preventative care, even if it is increasing the awareness that nicotine exposure, not cigarettes alone, can be harmful to a developing child.

Last updated: 28 Feb 2019 12:26pm
Declared conflicts of interest:
None declared.

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