At what stage of embryonic fetal development will teratogenic drugs medicines causing fetal malformation impact the development of the limbs?

Animal

The hexavalent form of chromium is a potent teratogen, primarily affecting bone formation. However, trivalent chromium was not found to be teratogenic. Animal studies also show an increase in the risk of cancer after exposure to chromium(VI) compounds.

Human

Chromium(III) is an essential nutrient that helps the body use sugar, protein, and fat. Chronic liver and kidney damage due to long-term exposure of chromium(VI) has been reported. However, chronic low-level exposure to chromium does not appear to produce measurable renal damage. Dermal exposure to chromium compounds can cause irritant dermatitis and skin ulcerations (chrome holes). Breathing high levels of chromium(VI) can cause irritation to the nose, such as runny nose, nosebleeds, and ulcers and holes in the nasal septum. Inhalation of chromium(VI) compounds is also associated with lung cancer, and these compounds are classified as human carcinogens.

Animal

Animal studies have shown cadmium to be a teratogen and a reproductive toxin; however, the results of mutagenesis experiments are equivocal. Cadmium produced local sarcomas in a number of rodent species when the metal, sulfide, oxide, or salts were administered subcutaneously. Intramuscular injection of cadmium powder and cadmium sulfate also produced local sarcomas. Injection of cadmium chloride into the ventral prostate resulted in a low incidence of prostatic carcinoma. Exposure via inhalation of cadmium chloride produced a dose-dependent increase in lung carcinomas in rats.

Human

Chronic exposure to cadmium through any route will have adverse effects on the heart, lungs, bones, gonads, and especially the kidneys. The principal long-term effects of low-level cadmium exposure are generally chronic obstructive pulmonary disease, emphysema, and chronic renal tubular disease. Cardiovascular and skeletal effects are also possible. The initial symptoms of chronic inhalation exposure are those associated with metal fume fever (e.g., fever, headache, chest pain, sore throat, coughing, and rhinitis). Metal fume fever is most often associated with inhalation of zinc oxide but may occur following exposure to other metals such as cadmium. Although inconclusive, there is evidence that the cadmium burden in the body can lead to hypertension.

Since cadmium can displace zinc, its accumulation in the testes can suppress testicular function. Evidence obtained in the past several years appears to relate cadmium to prostate cancer in young men who work with cadmium. Additional investigation, such as epidemiological studies with a larger cohort, needs to be performed to investigate this apparent association of cadmium with prostate cancer.

Skeletal changes due to cadmium accumulation are probably related to calcium loss, which can be influenced by diet and hormonal status. These skeletal changes include osteomalacia (softening of bone resulting from loss of minerals) and pseudofractures. In Japan, people who ate fish contaminated with cadmium experienced skeletal changes, especially in their backs. This very painful effect was called the ‘Itai-Itai’ (‘ouch-ouch’) disease. Postmenopausal women with low calcium and vitamin D intake were apparently most susceptible.

Since the kidneys are the main depot for cadmium, they are of the greatest concern for cadmium toxicity. Cadmium interferes with the proximal tubule's reabsorption function. This leads to abnormal actions of uric acid, calcium, and phosphorus. Aminoaciduria (amino acids in the urine) and glucosuria (glucose in the urine) result; in later stages, proteinuria (protein in the urine) results. When this happens, it is assumed that there is a marked decrease in glomerular filtration. Long-term exposure to cadmium leads to anemia, which may result from cadmium interfering with iron absorption.

Cadmium metallothionein has also been studied extensively. This metalloprotein is high in the amino acid cysteine (∼30%) and is devoid of aromatic amino acids. Metallothionein itself may function to help detoxify cadmium. For some experimental tumors, cadmium appears to be anticarcinogenic (e.g., it reduces the induction of tumors). While cadmium is not genotoxic, International Food Additives Council classifies it as a human carcinogen.

Animal

Accutane is a potent rat and rabbit developmental toxin (teratogen). Testicular atrophy and evidence of lower spermatogenesis was noted in dogs given isotretinoin for 30 weeks at 20 or 60 mg kg−1 day−1. Fischer 344 rats dosed at 8 or 32 mg kg−1 day−1 for over 18 months had a dose-related raised incidence of pheochromocytoma, an adrenal gland tumor. The relevance in man is unknown since this animal develops spontaneous pheochromocytoma at a significant rate.

Human

Any level of exposure may be teratogenic, so potentially fertile females must not be pregnant or get pregnant within 30 days before, during, and after exposure (see Mechanism of Toxicity). Other effects that often require monitoring are psychiatric disorders, including depression and suicidal thoughts, and benign intercranial hypotension, which can lead to headache, visual disturbances, or nausea and vomiting. These disorders may not stop upon discontinuation and should be evaluated by a professional. Dose-dependent adverse effects on the skin and mucous membranes may include inflammation or cracking of the lips, dry eyes, nosebleeding, irritation of the palpebral conjunctiva, and redness or dryness of the skin. Less common effects on the same organ systems include hair loss, photosensitivity, formation of granular tissue, or dark adaptation dysfunction. Colonization and, rarely, infection by Staphylococcus aureus can also occur. Hyperlipidemia is reported in 25% of treated patients during therapeutic courses of treatment on a systemic level, with the most common effect being increased triglyceride levels. There may also be increased cholesterol levels, raising of low-density lipoprotein levels, or lowering of high-density lipoprotein levels. Long-term treatments can generate several skeletal side effects including joint or lower back pain, bone hypertrophy, ossification at tendinous insertions, and lowered bone density. Children may experience premature closure of the epiphyseals. Tests of sperm count and motility in man have shown no significant changes.

12.09.4.1 Application of Gene Expression Measurements to Teratogen Exposure

A common finding in adverse exposures – whether to toxicants or teratogens – is the reduced penetrance and variable expressivity of outcomes. Even in highly inbred strains, it is rare to find 100% of the exposed individuals affected, and even if possible, individuals may vary in the severity of outcomes. For example, in the FVB inbred strain, streptozotocin (STZ)-induced maternal diabetes produces about 15% of malformed embryos (Pavlinkova et al. 2008, 2009), and although almost all of them exhibit NTDs, some also have heart defects and other malformations (Salbaum and Kappen, unpublished observations). The factors that make certain individuals more susceptible than others despite identical genetic makeup have not been identified, but likely involve threshold phenomena and a quantitative distribution of risk. This is fundamentally different from polymorphisms in natural (human) populations that may be responsible for altering protein function or expression levels, or the action of so-called modifier genes. Instead, inbred strains offer the possibility of identifying nongenetic mechanisms that confer variability of risk to the individuals in the population. Through quantitation of gene expression levels for a single gene in a large number of animal samples (Kruger et al. 2006), we have shown that gene expression levels follow a normal distribution within the population, but also that they can vary by as much as fivefold between individuals in the extreme 10% margins of the distribution. Thus, even in normal embryos, there may be considerable variation in the levels of expression for any gene. Whether these differences in any one gene or a particular constellation of genes increase susceptibility to exposure remains to be investigated. A major technical limitation is presented by the fact that gene expression is typically measured in terminal samples, and thus preexposure parameters and outcome data cannot be collected from the same animal. In adults, it may be possible to remove part of a tissue, or one duplicate, assuming equivalency and lateral symmetry at the molecular level, such as fat pads or limb muscles. However, for developmental exposures, this is infeasible. Once specific targets have been identified, however, reporter transgenes may be amenable to quantitation by whole-body live imaging methods. It is unlikely that technologies to do this on a genome-wide scale would become available soon. Yet, even in the absence of direct methods for observation of response to exposure, it may be possible to derive insight into variability from the existing microarray results.

12.09.4.2 Coordinate Regulation as Identified by Cluster Analyses

In a wide variety of experimental paradigms subjected to microarray analysis, it is of interest to identify coordinately regulated subsets of genes. This is typically done through cluster analysis (Eisen et al. 1998). The merits of the different cluster algorithms have been discussed elsewhere (Do and Choi 2008; Fan and Ren 2006; Gollub and Sherlock 2006; Quackenbush 2006; Rahnenführer 2005). Figure 1 shows an example of hierarchical clustering of microarray data obtained from our diabetes-exposed embryos. Several clusters of genes can be identified that change in the same direction and exhibit either greater or lesser variability between individual animals. Such clustering may indicate coordinate regulation of these genes, possibly through common signaling pathways or transcriptional mechanisms. Second, those genes in clusters that show variability could be associated with variability of the response of individuals with respect to differences in outcome. While such an analysis of cluster features can identify promising candidates from microarray data in a discovery paradigm, when applied to validated genes their predictive power should be even greater. This is actually borne out by identification of cancer subtypes via microarrays and gene expression profiles (Alizadeh and Staudt 2000; Bittner et al. 2000; Golub et al. 1999; Hu et al. 2006; Ross et al. 2000). In the future, it may be possible to apply similar approaches to teratogen exposure paradigms; predictive power relative to susceptibility will most likely be dependent on the degree of variation between individuals as well as sample number available for a specific exposure.

12.09.4.3 Temporal Trajectories of Altered Gene Expression

As alluded to earlier, toxicological and teratological studies of altered gene expression aim to identify the molecular basis of pathogenicity of a given exposure. Inherently, there are proximal and distal elements to the initial insult, and the two types of responses become even more difficult to dissociate in chronic exposures. Exposure to maternal diabetes during pregnancy is obviously a chronic exposure, although there may be distinct phases of vulnerability in the embryo, depending on tissue and cellular response mechanism. Vulnerability to oxidative stress, for example, may be ongoing and related to physiological thresholds, while altered gene expression itself is expected to make its impact as soon as the product becomes limiting or is present in excess. For the latter, detailed investigations over time should identify the earliest time point of response to the insult. In different molecular pathways, this may follow different timelines; for example, reduction in a critical transcription factor may well have a more immediate effect than lack of relatively stable cytoskeletal or extracellular matrix components. Similarly, proliferating and quiescent cells may respond differently to the same molecular change in expression level. Therefore, to understand how the various pathways affected by altered gene expression contribute to pathogenesis and variations in response to exposure, it is indispensable to consider the temporal trajectories of individual genes (Mitiku and Baker 2007) and the changes in their spatial expression patterns. For the developing embryo, the relevant experimental methods then move away from whole-genome scale to more focused and functional analyses in the respective exposure paradigm.

12.09.4.4 Will Technical Advances Soon Make Microarrays Obsolete?

Recently, massively parallel sequencing technology (Wheeler et al. 2008) has been developed with decreasing price, so that it may soon be possible to profile gene expression by directly measuring the occurrence of specific sequences in the transcriptome of cells or organisms. The advantage over microarrays is that identification of a transcript is no longer dependent on the specificity of hybridization. This eliminates the need for corrections for cross-hybridization, such as mismatched oligonucleotide probes, with their implicit limitations. In fact, transcriptomes of several organisms (Erdner and Anderson 2006; Jongeneel et al. 2005; Torres et al. 2008) and different cell types (Chen et al. 2005; Morin et al. 2008a,b) have recently been characterized by massively parallel sequencing. While this may eventually make the wet-bench aspects of microarray technology obsolete (Coppee 2008), many principles of data analysis will remain similar when the goal is to identify genes that are differentially regulated as a result of pathogenesis, disease, or exposure. However, because direct sequencing enables detection of not only transcripts but also DNA sites occupied by transcription factors (Wederell et al. 2008), it will be possible to elucidate mechanisms of regulation as well as outcomes, such as differential gene expression. This will have particular implications for epigenetic modifications (Charalambous et al. 2007) that are currently difficult to detect at the whole-genome level, but which may play an increasingly prominent role in understanding predisposition to disease (Dolinoy and Jirtle 2008) and susceptibilities to the effects of environmental exposures, such as nutrition (Dolinoy et al. 2007) or teratogens.