## Toxicological studies assess the potential risk of substances in the environment to human health

Since substances from the external environment can affect the processes taking place in our bodies, it is important to monitor their effects. Toxicological studies are of central importance and their task is to assess the potential danger posed by substances to human health and other living organisms. Currently, this area of activity is also covered by large organisations or authorities. In Europe, this is the European Food Safety Authority (EFSA), while in the USA, it is the Food and Drug Administration (FDA). At the international level, the Organisation for Economic Co-operation and Development (OECD) approves, among other things, the series of tests (a test battery) that substances are required to undergo if they are thought to have a practical use, or that are found at elevated levels in the environment. A test battery is a series of tests that, according to the OECD, all substances with potentially toxic effects must pass. The results of the individual tests indicate whether, to what extent and on which organisms the substance has an adverse effect.

The spot test is used to determine the toxicity of a substance on prokaryotic cells (Figure 12.4). Bacteria (Salmonella typhimurium) are used, which are inoculated onto a complete medium containing all the nutrients necessary for growth. After soaking the cells in the medium, a sterile circle of filter paper is placed in the centre of the dish. Next, the substance to be tested is dropped onto this circle at the desired concentration and can pass freely from the filter paper into the medium. After an incubation period, the so-called growth inhibition zone is determined, i.e., whether the substance caused the cells around the ring to die. If the inhibition zone is not present, the substance has no toxic effect. If there are dead cells around the ring, the larger the inhibition zone, the stronger the effect of the substance and thus the substance is more toxic.

The Ames test is used to determine the mutagenicity (ability to induce the formation of mutations) of the substance under investigation on bacterial cells (Figure 12.5). Here, S. typhimurium bacteria are used which have been modified to have an interrupted gene for the synthesis of the amino acid histidine, so that these bacteria cannot grow on the medium without the addition of this amino acid. The bacteria are exposed to the potentially toxic substance and then allowed to grow on a medium without histidine. If the bacteria grow, it is because of what is called a reverse mutation (either point-substitution or frameshift mutation – the insertion or deletion of a short section, more about mutations in Chapter 5 – Mutations how they arise and what to do with them). This means that due to the mutation, the cells ability to synthesise the amino acid histidine has been restored and the bacterium is therefore able to grow on this medium. Since the tested substance caused the formation of (reverse) mutations, we can speak of its mutagenic effect. When evaluating the result, the more reverse mutants that have grown, the higher the mutagenic effect of the substance. If the tested substance is not added to the cells (such a sample serves as a negative control in the experiment), only a small amount of the so-called spontaneous revertants grow. The reason for this is that the ability to synthesise an amino acid is always restored in the cells with a certain low frequency (spontaneous mutations).

The comet assay is used to detect primary DNA damage (DNA strand breaks) that can be repaired in the cell but, if not repaired, will lead to mutations (Figure 12.6). In this assay, the substance tested is applied to different types of eukaryotic cells at a specific concentration for a specific time. Then the cells are lysed (the cytoplasmic and nuclear membranes are destroyed) to release DNA. In the next step, an electric current is applied which causes the DNA fragments to migrate from the head (intact DNA) to the tail – a comet is formed. Short DNA fragments migrate faster in an electric field than longer fragments, so they cover a greater distance in the same amount of time. The longer the tail of the comet or the more DNA it contains, the more DNA is damaged. This assay is evaluated microscopically after the DNA has been stained with a suitable dye.

In addition to the tests mentioned above, there is also a screening programme for the detection of endocrine disruptors developed by the United States Environmental Protection Agency (the Endocrine Disruptor Screening Program, EDSP). This screening is a two-step process, with the first step focusing on identifying compounds that interact with the endocrine system. Subsequently, all compounds found to interact in this way are also tested in the second step, the aim of which is to identify the adverse effects of these endocrine active substances. In addition, it is important to uncover the relationship between the dose of a substance and its effect and, if necessary, to adopt regulatory measures to guide the use of the substance so that there is no risk to human health.

The CALUX® and REA assay As many of the EDC chemicals have adverse outcomes in humans, it is important to monitor the levels of estrogen and other compounds in food to reduce the risk of contaminations. To do this quickly and effectively receptor-based assays have been designed.

CALUX® assays are a family of bioassays using mammalian cells. The cells are genetically modified so that when a chemical binds the receptor, it results in the transcription of a reporter gene, luciferase. Translation of luciferase results in an enzyme that produces light when its substrate, luciferin, is added to the cells. The amount of light produced is related to the activity of the chemical to which the cells are exposed and can be quantified. Commercial CALUX® assay kits are available for rapid, sensitive and cost effective screening for dioxins, one of the most toxic human-made compounds. Similarly, estrogen levels can be detected using the CALUX® assay as well.

Another reporter gene assay is the REA assay. This is a yeast estrogen bioassay, which is a fully validated method to measure the effects that are directly mediated by the estrogen receptor, as binding to and activation of the estrogen receptor. The REA is a reporter-gene bioassay making use of Saccharomyces cerevisiae yeast cells which were transfected with two DNA constructs (Figure 12.7). The first construct contains the gene sequence for the human estrogen receptor alpha (hER-α), which enables the binding of chemicals containing estrogenic activity. The second construct encodes for the yeast enhanced green fluorescence protein (yEGFP) gene, which is under transcriptional control of a hER-α responsive promotor. If the yeast cells are exposed to a sample containing estrogenic compounds, they will bind to the estrogen receptor. This complex will bind and activate DNA at a specific estrogenic response element (ERE), which will cause a response. In the case of the REA, binding of hER-α to the ERE allows transcription and translation of yEGFP ultimately resulting in a green fluorescent signal that can be measured by fluorescence spectrometry in intact, living, cells. As such, this fluorescent signal is a direct measure for the total estrogenic activity in the sample to which the yeast cells were exposed.

In practice, the REA test has been successfully applied to detect 17beta-estradiol in the urine of calves treated with the hormone in vivo. The findings suggest that this technique could serve as an effective laboratory method for veterinary monitoring of illegal steroid use. In addition, the estrogenic activity observed in rodent feed was attributed to high levels of genistein, daidzein, along with trace amounts of zearalenone. The REA test has also demonstrated its ability to detect 17β-estradiol in animal feed at low concentrations, ranging from 1.15 to 2 μg/kg-1. Another study confirmed that the REA assay is specific for the detection of estrogenic compounds in water. These and similar studies hold significance for human health, as they contribute to predicting and identifying potential risks to public health.