## 4.2.9. Developmental toxicity

Author: Jessica Legradi, Marijke de Cock

Reviewer: Paul Fowler

Learning objectives

You should be able to

- explain the six principles of teratology
- name the difference between deformation, malformation and syndrome
- describe the principle of DoHAD
- indicate what epigenetics is and what epigenetic mechanisms could lead to transgenerational effects

Keywords: Teratogenicity, developmental toxicity, DoHAD, Epigenetics, transgenerational

Developmental toxicity

Developmental toxicity refers to any adverse effects, caused by environmental factors, that interfere with homeostasis, normal growth, differentiation, or development before conception (either parents), during prenatal development, or postnatally until puberty. The effects can be reversible or irreversible. Environmental factors that can have an impact on development are lifestyle factors like alcohol, diet, smoking, drugs, environmental contaminants, or physical factors. Anything that can disturb the development of the embryo or foetus and produces a malformation is called a teratogen. Teratogens can terminate a pregnancy or produce adverse effects called congenital malformations (birth defects, anomaly). A malformation refers to any effect on the structural development of a foetus (e.g. delay, misdirection or arrest of developmental processes). Malformations occur mostly early in development and are permanent. Malformations should not be confused with deformations, which are mostly temporary effects caused by mechanical forces (e.g. moulding of the head after birth). One teratogen can induce several different malformations. All malformations caused by one teratogen are called a syndrome (e.g. fetal alcohol syndrome).

Six Principles of Teratology (by James G. Wilson)

In 1959 James G. Wilson published the 6 principles of teratology. Till now these principles are still seen as the basics of developmental toxicology. The principles are:

1. Susceptibility to teratogenesis depends on the genotype of the conceptus and a manner in which this interacts with adverse environmental factors

Species differences: different species can react different (sensitivities) to the same teratogen. For example, thalidomide (softenon) a drug used to treat morning sickness of pregnant woman causes severe limb malformations in humans whereas such effects were not seen in rats and mice.

Strain and intra litter differences: the genetic background of individuals within one species can cause differences in the response to a teratogen.

Interaction of genome and environment: organisms of the same genetic background can react differently to a teratogen in different environments.

Multifactorial causation: the summary of the above. The severity of a malformation depends on the interplay of several genes (inter and intra species) and several environmental factors.

2. Susceptibility to teratogenesis varies with the developmental stage at the time of exposure to an adverse influence

During development there are periods where the foetus is specifically sensitive to a certain malformation (Figure 1). In general, the very early (embryonic) development is more susceptible to teratogenic effects.

Figure 1: The critical (sensitive) periods during human development. During these periods tissues are more sensitive to malformations when exposed to a teratogen. The timing of period is different for different tissues. Source: https://www.slideshare.net/SDRTL/critical-periods-in-human-development

3. Teratogenic agents act in specific ways (mechanisms) on developing cells and tissues to initiate sequences of abnormal developmental events (pathogenesis)

Every teratogenic agent produces a distinctive malformation pattern. One example is the foetal alcohol syndrome, which induces abnormal appearance, short height, low body weight, small head size, poor coordination, low intelligence, behaviour problems, and problems with hearing or seeing and very characteristic facial features (increased distance between the eyes).

4. The access of adverse influences to developing tissues depends on the nature of the influence (agent)

Teratogens can be radiation, infections or chemicals including drugs. The teratogenic effect depends on the concentration of a teratogen that reaches the embryo. The concentration at the embryo is influenced by the maternal absorption, metabolisation and elimination and the time the agent needs to get to the embryo. This can be very different between teratogens. For example, strong radiation is also a strong teratogen as it easily reaches all tissues of the embryo. This also means that a compound tested to be teratogenic in an in vitro test with embryos in a tube might not be teratogenic to an embryo in the uterus of a human or mouse as the teratogen may not reach the embryo at a critical concentration.

5. The four manifestations of deviant development are death, malformation, growth retardation and functional deficit

A teratogen can cause minor effects like functional deficits (e.g. reduced IQ), growth retardations or adverse effects like malformations or death. Depending on the timing of exposure and degree of genetic sensitivity, an embryo will have a greater or lesser risk of death or malformations. Very early in development, during the first cell divisions, an embryo will be more likely to die rather than being implanted and developing further.

6. Manifestations of deviant development increase in frequency and degree as dosage increases, from the no-effect to the 100% lethal level

The number of effects and the severity of the effects increases with the concentration of a teratogen. This means that there is a threshold concentration below which no teratogenic effects occur (no effect concentration).

Developmental Origins of Health and Disease (DOHaD)

The concept of Developmental Origins of Health and Disease (DOHaD) describes that environmental factors early in life contribute to health and disease later in life. The basis of this concept was the Barker hypothesis, which was formulated as an explanation for the rise in cardiovascular disease (CVD) related mortality in the United Kingdom between 1900 and 1950. Barker and colleagues observed that the prevalence of CVD and stroke was correlated with neo- and post-natal mortality (Figure 2). This led them to formulate the hypothesis that poor nutrition early in life leads to increased risk of cardiovascular disease and stroke later in life. Later, this was developed into the thrifty phenotype hypothesis stating that poor nutrition in utero programs for adaptive mechanisms that allow to deal with nutrient-poor conditions in later life, but may also result in greater susceptibility to metabolic syndrome. This thrifty hypothesis was finally developed into the DOHaD theory.

Figure 2: Standardized mortality ratios for ischaemic heart disease in both sexes (y-axis) and neonatal mortality per 1000 births, 1921-1925 (x-axis). Redrawn from Barker et al. (1986) by Wilma Ijzerman.

The effect of early life nutrition on adult health is clearly illustrated by the Dutch Famine Birth Cohort Study. In this cohort, women and men who were born during or just after the Dutch famine, were studied retrospectively. The Dutch famine was a famine that took place in the Western part of the German-occupied Netherlands in the winter of 1944-1945. Its 3-months duration creates the possibility to study the effect of poor nutrition during each individual trimester of pregnancy. Effects on birth weight, for example, may be expected if caloric intake during pregnancy is restricted. This was, however, only the case when the famine occurred during the second or the third trimesters. Higher glucose and insulin levels in adulthood were only seen for those exposed in the third trimester, whereas those exposed during the second trimester showed a higher prevalence of obstructive airways disease. These effects were not observed for the other trimesters, which can be explained by the timing of caloric restriction during pregnancy: during normal pregnancy pancreatic islets develop during the third trimester, while during the second trimester the number of lung cells is known to double.

The DOHaD concept does not merely focus on early life nutrition, but includes all kinds of environmental stressors during the developmental period that may contribute to adult disease, including exposure to chemical compounds. Chemicals may elicit effects such as endocrine disruption or neurotoxicity, which can lead to permanent morphological and physiological changes when occurring early in life. Well-known examples of such chemicals are diethylstilbestrol (DES) and dichlorodiphenyltrichloroethane (DDT). DES was an estrogenic drug given to women between 1940 and 1970 to prevent miscarriage. It was withdrawn from the market in 1971 because of carcinogenic effects as well as an increased risk for infertility in children who were exposed in utero (link to section on Endocrine disruption). DDT is a pesticide that has been banned in most countries, but is still used in some for malaria control. Several studies, including a pooled analysis of seven European cohorts, found associations between in utero DDT exposure levels and infant growth and obesity.

The ubiquitous presence of chemicals in the environment makes it extremely relevant to study health effects in humans, but also makes it very challenging as virtually no perfect control group exists. This emphasizes the importance of prevention, which is the key message of the DOHaD concept. Adult lifestyle and corresponding exposure to toxic compounds remain important modifiable factors for both treatment and prevention of disease. However, as developmental plasticity, and therefore the potential for change, is highest early in life, it is important to focus on exposure in the early phases: during pregnancy, infancy, childhood and adolescence. This is reflected by regulators frequently imposing lower tolerable exposure levels for infants compared to adults.

Epigenetics

For some compounds in utero exposure is known to cause effects later in life (see DOHad) or even induce effects in the offspring or grand-offspring of the exposed embryo (transgenerational effect). Diethylstilbesterol (DES) is a compound for which transgenerational effects are reported. DES was given to pregnant women to reduce the risk of spontaneous abortions and other pregnancy complications. Women who took DES during pregnancy have a slightly increased risk of breast cancer. Daughters exposed in utero, on the other hand, had a high tendency to develop rare vaginal tumours. In the third-generation, higher incidences of infertility, ovarian cancer and an increased risk of birth defects were observed. However, the data available for the third generation is small and therefore possess only limited evidence so far. Another compound suspected to cause transgenerational effects is the fungicide vinclozolin. Vinclozolin is an anti-androgenic endocrine disrupting chemical. It has been shown that exposure to vinclozolin leads to transgenerational effects on testis function in mice.

Transgenerational effects can be induced via genetic alterations (mutations) in the DNA. Thereby the order of nucleotides in the genome of the parental gametocyte is altered and this alteration is inherited to the offspring. Alternatively, transgenerational effects can be induced is via epigenetic alterations. Epigenetics is defined as the study of changes in gene expression that occur without changes in the DNA sequence, and which are heritable in the progeny of cells or organisms. Epigenetic changes occur naturally but can also be influenced by lifestyle factors or diseases or environmental contaminants. Epigenetic alterations are a special form of developmental toxicology as effects might not cause immediate teratogenic malformations. Instead the effects may be visible only later in life or in subsequent generations. It is assumed that compounds can induce epigenetic changes and thereby cause transgenerational effects. For DES and vinclozolin epigenetic changes in mice have been reported and these might explain the transgenerational changes seen in humans. Two main epigenetic mechanisms are generally described as being responsible for transgenerational effects, i.e. DNA methylation and histone modifications.

DNA methylation

DNA methylation is the most studied epigenetic modification and describes the methylation of cytosine nucleotides in the genome (Figure 3) by DNA methyltransferase (DNMTs). Gene activity generally depends on the degree of methylation of the promotor region: if the promotor is methylated the gene is usually repressed. One peculiarity of DNA methylation is that it can be wiped and replaced again during epigenetic reprogramming events to set up cell- and tissue-specific gene expression patterns. Epigenetic reprogramming occurs very early in development. During this phase epigenetic marks, like methylation marks are erased and remodelled. Epigenetic reprogramming is necessary as maternal and paternal genomes are differentially marked and must be reprogrammed to assure proper development.

Figure 3: The methylation of the cytosine nucleotide. One hydrogen atom is replaced by a methyl group. Drawn by Steven Droge.

Histone modification

Within the chromosome the DNA is densely packed around histone proteins. Gene transcription can only take place if the DNA packaging around the histones is loosened. Several histone modification processes are involved in loosening this packaging, such as acetylation, methylation, phosphorylation or ubiquitination of the histone molecules (Figure 4).

Figure 4: (a) The DNA is wrapped around the histone molecules. The histone molecules are arranged in a way that their amino acid tails are pointing out of the package. These tails can be altered for example via acetylation. (b) If the tails are acetylated the DNA is packed less tightly and genes can be transcribed. If the tails are not acetylated the DNA is packed very tight and gene transcription is hampered. Redrawn from http://porpax.bio.miami.edu/~cmallery/150/gene/c7.19.4.histone.mod.jpg by Evelin Karsten-Meessen.

References

Barker, D.J., Osmond, C. (1986). Infant mortality, childhood nutrition, and ischaemic heart disease in England and Wales. Lancet 1 (8489), 1077–1081.