The Mutagenic Effects of Ultraviolet Radiation on Living Cells

1 Introduction

The Sun has always been considered a source of life, sometimes a God. However, there has been little thought given to the fact that it also has the potential to destroy life. This potential is mostly due to the emission of UV light, which interacts with living cells causing damage.

This investigation was carried out with the following purposes :

 Ÿ to determine if UV light has mutagenic effects, both from the analysis of the results obtained from exposing living cells to UV radiation, and from research done on past investigations.

The living cells used for the investigation were bacteria, which were exposed to UV light under different conditions, to investigate the effects this kind of radiation had on them, specifically looking for mutations. Bacteria were used because they have a short generation period, and are easy to handle, making them good to investigate, as opposed to the use of larger organisms such as animals. A low but significant amount of mutations was expected to be found as a result of exposure to UV light.

I found this topic particularly interesting because it was an opportunity to study an area totally new for me and which is of great importance, because I found from preliminary research that UV effect was potentially dangerous and that there is little real public awareness of the possible consequences of UV exposure. It was as well an opportunity to do a complementary study to other studies on the ozone layer, since it reflects the possible consequences of damaging it.

2 UV and DNA

2.1 How UV radiation affects living cells

UV light causes damage to cells in different ways, the most important being the damage to nucleic acid. This damage is mainly caused by the formation of pyrimidine dimers, which occurs when two adjacent pyrimidine bases (cytosine or thymine) are linked together in a way which changes the shape of the DNA double spiral. This doesn't allow for the DNA to be copied, which means that one pyrimidine dimer is enough to kill a cell.

Although pyrimidine dimers cause most of the DNA damage, there is another form of damage of relative importance called the 6-4 lesion, because of how the DNA is deformed, which also arises from the interaction of UV photons and two pyrimidine bases. This form of damage may cause mutations.

2.2 Repair of Damaged DNA

Because UV photons are abundant in our environment, it would seem that all living cells should be dead by now. However, organisms have developed several mechanisms to repair the damage caused by UV photons. These mechanisms are common to most living organisms, since they were developed in early stages of life on Earth, and have been conserved throughout the evolution of the species. This is favourable for the research purposes of this investigation, since it can be assumed that cells of living organisms other than the bacteria used in the experiments will respond to UV radiation in a similar manner.

The most simple repair mechanism is called photoreactivation, which uses the energy of a light photon to break apart the bond which formed a pyrimidine dimer. The protein responsible for this process is photolyase. This process occurs in the cells of all living organisms, except placental mammals. Nevertheless, photolyase is still found in these mammals, since it is used in excision repair, another method of DNA repair. In this method, the damaged sector of the DNA is physically cut out, and then replaced by a new, re-synthesised portion, copied from the other strand. Although this method requires several proteins, it has the advantage that it can mend damages other than pyrimidine dimers. It is illustrated in figures 2.2.1 through 2.2.4.

fig 2.2.1 : A 'healthy' DNA strand. The coloured sections represent nucleotide bases, and the rods connecting them represent the invisible bonds which hold the helices together.

fig 2.2.2 : A damaged DNA strand. A lesion has been produced, altering the shape between the red and yellow nucleotides.

fig 2.2.3 : The damaged section has been removed by the action of proteins which have identified an abnormal structure.

fig 2.2.4 : The genetic code in the 'healthy' spiral has been reproduced. It will be 'glued' by the action of protein ligase, and the DNA will now look as that in fig 2.2.1. 

Although excision repair is very effective and repairs most UV damage, sometimes this repair can be defective, the main cause being that occasionally, dimers or other DNA lesions are near each other on opposite strands of DNA, so when excision repair is attempted, the cell won't know how to copy the damaged region, so a mistake which leads to a mutation is made. This mutation, however, is most of the times better than not repairing the lesion at all. This repair mechanism is denominated error-prone repair, and is illustrated in figures 2.2.5 through 2.2.7.

fig 2.2.5 : This strand has two lesions close to one another.

fig 2.2.6 : The damaged section in one of the spirals is removed. A new DNA portion is synthesised, reproducing the equivalent section in the other spiral. However, a part of this section is damaged, so a new code that fits the gap is produced.

fig 2.2.7 : The other spiral is repaired copying the new code. If the code is not identical to the original, a mutation has occurred, as in this case.

Sometimes, when DNA is being replicated in a eukaryotic chromosome, a pyrimidine dimer which has not been removed yet blocks the process. The replication is continued further down. This leads to a gap forming in one of the daughter nucleic acid molecules where the dimer blocked the replication process. This gap must be filled before the cell divides, otherwise, a strand will miss information. This gap is filled by genetic recombination with the other daughter DNA molecule or chromosome, containing similar information. However, this information is not always quite what it should be, so a change in the outcoming strand can be produced. This change may cause a mutation when the lesion is then repaired. Figures 2.2.8 through 2.2.12 illustrate a case of recombinational repair.

fig 2.2.8 : The DNA strand has been split in order to be duplicated. However, a lesion which hadn't been repaired is present in one of the spirals.

fig 2.2.9 : The two strands are replicated. However, during the process, the section containing the lesion in one of the strands was skipped, as it couldn't be copied.

fig 2.2.10 : The two strands recombine: the section containing the gap and a similar section of the 'healthy' strand are cut out.

fig 2.2.11 : The section containing the gap is discarded, and the similar section replaces it. A new gap in the donor molecule is formed.

fig 2.2.12 : The similar section is glued by protein ligase to the damaged molecule. The gap in the donor strand is filled by replication. In this opportunity, the gap in the damaged strand has been filled by the correct code. When each strand is separated into different cells, one will be 'healthy' and one will still have a lesion, like the original one (see fig 2.2.2). However, the lesion can be repaired either by photoreactivation or excision repair, since one of the spirals contains the 'correct' information. If this information weren't correct and excision repair was attempted, a mutant will be formed. 

The aim of the experiments to be carried out was therefore to look for cases of error-prone repair and recombinational repair when incorrect information fills the gap, as well as cases of 6-4 lesions.

2.3 UV sources

The ultraviolet spectrum is actually very large, covering the ranges UV-A, UV-B, and UV-C, that is, the wavelengths in the range 400-180 nm. Photons in the UV-C range have the shortest wavelengths and therefore the most energy, UV-B has longer wavelengths and less energy, and UV-A has the least energy. This means that, in the same amounts, UV-C will cause the most damage, followed by UV-B, followed by UV-A. However, mainly thanks to the ozone layer, no UV-C reaches the surface of the Earth from the sun, little UV-B, and more UV-A. The graph shows, in arbitrary units, the damage produced and the amounts present at the surface of the Earth for each range.

Fig 2.3.1 : The graph shows how damage produced increases going for UV-A to UV-C, but also that presence decreases in this direction.

 But as well as the Sun, there are other sources of UV light. Indoor lamps, specially fluorescent lamps, can emit significant amounts of UV radiation, as much as 6% of their total irradiance. In the case of the fluorescent lamps, the UV breakthrough is mainly due to a poor phosphorous coating, which doesn't convert all the UV light produced by the gas inside into useful visible light. In the case of regular tungsten filament lamps, the great amount of energy present is enough to release photons in the UV spectrum.

3 The Experiments

In order to analyse the results of UV exposure on a bigger scale, and to arrive at results which could fulfil the purposes of the investigation, several exposures of bacteria to UV sources were made. These were carried out in two different stages.

3.1 The First Stage

The purpose of the first stage was to become familiar with the necessary techniques and laboratory methods for culturing bacteria, as well as getting a general idea of what results would be obtained in the following stages and which alternatives to future problems or questions should be taken into account.

Therefore, in this stage, cultures of Escherichia coli bacteria were exposed to Sunlight for different periods of time, to investigate how exposed areas were affected by the light, and to identify possible mutants. These possible mutants were then isolated and tested to verify if they were indeed mutants. E.coli appears as smooth red colonies in MacConkey agar, as a consequence of the fermentation of glucose. These colonies, if mutated, could change to rough colonies or change colour.

The E.coli were obtained from a culture of poultry litter in MacConkey nutrient agar. Once suspended, its concentration was calculated using a 10-fold dilutions method. The results of the dilutions were as follows :

Table 3.1.1 : Results of the dilution series for the suspension of E.coli.

Knowing this value, 1.8x105 bacteria/cm2 in three Petri dishes with MacConkey agar were exposed to sunlight for periods of 20, 40, and 60 minutes. The method used was to cover the dishes with aluminium foil, with windows of area 4cm2 which allowed the sunlight to reach the bacteria. In this way, it was easy to check with the unexposed, control area. The dishes were placed in a black box, to avoid reflection and make every situation equal in this aspect.

Fig 3.1.1 : Petri dish covered with aluminium foil with windows.

After the dishes were incubated for 24 hours the effect of the exposures could be clearly seen. The areas which were exposed for 20 and 40 minutes showed a very low concentration of colonies compared to the unexposed area. In the 60 minutes exposures almost no bacteria survived. The shape of the windows could be easily identified. The results of these exposures showed that there is an exponential correlation between exposure time and surviving colonies.

As well as having a great biocidal effect, some of the surviving colonies showed abnormal characteristics. These colonies, which could present mutations, were isolated, cultured and incubated for 24 hours. The characteristics of these colonies and the results of the corresponding cultures are summarised in table 3.1.2 :

Table 3.1.2 : Colonies seeded and results obtained.

The results obtained here were rather confusing, since there seemed to be little relationship between what was cultured and the results. However, since clean area G later showed some colonies, it can be assumed that when colonies A through E were isolated, other invisible bacteria were carried as well. The difference between big and small red colonies can be attributed to the competition for the nutrients in the medium. However, the only explanation for the formation of white colonies is that a mutation in some gene related to the fermenting process has occurred. Since the process by which E.coli ferments glucose is quite complex and involves several steps, from the synthesis of proteins to the actual chemical interaction with glucose, there are many genes coding all this information which could have been mutated.

3.2 The Second Stage

 Having obtained a general idea of the effects of UV light on bacteria and having become familiar with the necessary techniques to work with bacteria, the Second Stage was aimed at obtaining more precise results, as well as investigating the effects of other UV sources. Fluorescent tubes from different brands intended for use in homes were purchased, and bacteria were exposed to these tubes. As well, a Philips 30 W / G30 T8 UV-C lamp intended for research and laboratory purposes was used to investigate the effects of UV light in the absence of visible light, thus cancelling the possibility of DNA repair by photoreactivation, and increasing the possibility of mutations occurring. In this stage, not only E.coli bacteria were used, but also Salmonella enteritidis and other unidentified bacteria taken from the air, to investigate if results could vary between different kind of cells.

I decided that counting bacteria in the original solution was not necessary, since quantitative analysis of mutation occurrence would be so low that it couldn't be calculated. However, determining that mutations indeed occur is enough to conclude that UV is potentially dangerous, since, as it is known, a single mutation causing excessive or uncontrolled growth could lead to cancer.

3.2.1 The Sun as a source of UV Radiation

The following cultures were exposed to sunlight at noon and in the afternoon in order to compare the effects of the angle of the sun on the UV intensity. Different media and concentrations were used as results could show up more clearly in one or another culture. Exposures of 15, 30, 45, and 60 minutes were made, using the aluminium foil as in fig 3.1.1, but this time, the 4 periods were attributed to different windows, not different Petri dishes, covering each window after each period ended. I found this method more favourable for comparative analysis (see picture 5.3.1 and 5.3.2).

E.coli on MacConkey medium x 2 dishes

E.coli x10-1 on MacConkey medium x 2 dishes

E.coli on nutritive agar medium

E.coli x10-1 on nutritive agar medium

The results of these exposures showed once again an exponential correlation between surviving colonies and exposure time, and the germicide effect was found to be much greater at noon than in the afternoon. At noon, almost no colonies survived for any period, while in the afternoon an important amount was present in exposed areas (see picture 5.3.3). Sunlight is therefore much more dangerous when the angle with the earth is greater, that is, when the UV is less filtered by the atmosphere.

Of the surviving colonies, however, none showed any abnormal characteristics.

The same procedure was repeated using a Salmonella culture, and the deleterious effects of sunlight were even more pronounced. As well, the exposed areas presented colonies of a variety of sizes. Several colonies were isolated and cultivated. The results showed once again a mixture of colonies of different sizes.

3.2.2 Fluorescent Tubes as a source of UV Radiation

Using the same E.coli x10-1 suspension, dishes were exposed using the same methods to regular fluorescent tubes at 20 cm, for periods of 30, 60, 90, and 120 minutes (see picture 5.3.4). The tubes used were :

None of these tubes showed any negative effect whatsoever. However, when the more sensitive Salmonella culture was exposed to a 20 W Philips, Made in Brazil, and a 18 W Osram, Made in Germany, a reduction in the concentration of colonies in areas exposed for 2 hours and 4 hours was visible, although this reduction couldn't be appreciated in photographs. This proves that there is UV breakthrough from fluorescent tubes.

3.2.3 UV Radiation in the absence of light

As well as the Sun and the Fluorescent tubes, exposures to direct UV light from a special UV-C lamp were made, in a dark environment and at a distance of 20 cm, in order to study the effects that removing the possibility of photoreactivation repair had, since photoreactivation requires photons in the visible light spectrum. When working with this tube, sunglasses with UV filter were used to prevent eye damage and/or keratoconjuntivitis (see picture 5.3.5). Otherwise using the same methods as before, exposures of a E.coli culture were done for 2, 5, 10, and 15 minute intervals. The results, however, showed no surviving colonies in the whole of the dish. This was because of two reasons: exposure periods were too long, and, the source was a long tube, not a point, causing reflection inside the dish by the aluminium foil (see fig 3.2.1).

In order to get more precise results, the exposure periods were reduced to 30, 60, 120, and 300 seconds. The aluminium foil was replaced by dark paper, covering half of each dish (see fig 3.2.2). The results are summarised in table 3.2.1 and fig 3.2.3.

Table 3.2.1 : Results of the exposures of E.coli to the UV-C lamp.

Fig 3.2.3 : The graph shows the results summarised in table 3.2.1.

It can be easily appreciated in the graph that the result for 120 seconds of exposure is anomalous, and that decay of surviving colonies is exponential. However, no colonies with abnormal characteristics were found here either. Nevertheless, when the procedure was repeated using the more sensitive Salmonella, the results showed two things:

The small and big colonies were isolated, from different areas, and were re-exposed to the UV tube for periods of 5, 10, 15, and 30 seconds. The results of these re-exposures revealed no difference from the first exposure, except a more pronounced concentration of either big or small colonies (see picture 5.3.8).

The last exposures carried out were on different unidentified microbes isolated from a culture obtained from particles in the air. One of these formed sporulated colonies. These microbes proved to be much more resistant to the UV light from the UV-C lamp than the E.coli or the Salmonella, especially the sporulated ones (see picture 5.3.9).

3.3 Analysis of results and Relation to DNA damage and repair theory

It is evident from the results that UV light has very strong effects on living cells, which are not necessarily mutagenic. In the case of the Sun, it can be seen that a 15 minute exposure was enough to kill most of the bacteria. The presence of colonies of different sizes gives an indication that mutations could have occurred. However, it is difficult to determine if a mutation has really happened or if nutrients limited growth. Nevertheless, repeated abnormal behaviour, as in the experiment using Salmonella and the UV-C lamp, could be an indication of mutations. In the case of the white colonies obtained in the First Stage, the is no other explanation for this except that some gene related to the process of fermentation of glucose was mutated.

In the case of the Fluorescent tubes, it is clear that because the visible light radiated exceeds UV light, photoreactivation effectively repairs most of the damage. This is also sustained by the fact that, when the UV-C tube was used, Salmonella and E.coli behaved similarly. This means that E.coli, which was unaffected by the fluorescent tubes, relies more on photoreactivation than Salmonella, which was slightly affected by the fluorescent tubes.

This difference in the way in which E.coli and Salmonella repair their DNA damage, leads to the conclusion that different organisms can respond to DNA damage in different conditions, in this way adapting themselves to their environment. The microbes recovered from the air, which are more exposed to UV radiation in their environment than Salmonella or E.coli proved to be more proficient in DNA damage repair.

It is possible that many of the bacteria which were killed by the UV light were actually killed indirectly, if they had been mutated but in such a way that they couldn't survive. This can explain why in some exposures, a relatively high amount of colonies survived, in the case that mutations which lead to killing the bacteria were in these cases harmless, allowing for the bacteria to survive.

From the results of the exposures to the UV-C lamp, the fact that UV-C causes much more damage is corroborated, since exposures of only 5 seconds showed serious effects.

It can be assumed from the results that there is a small but direct correlation between UV damage and mutation, so any source of UV light, even a fluorescent tube, could cause a mutation at any time, this meaning that any source can be responsible for developing skin cancer in animals or some kind of deficiency in smaller organisms.

4 Conclusions

Although it is very difficult to prove with the results obtained from the exposures, it can be concluded that UV light can have mutagenic effects, since abnormal characteristics were found in some very few surviving colonies. Mutations therefore occur only occasionally, but this very low probability of having a mutation, minimised by the even smaller probability that this mutation is harmful, is multiplied by the huge amount of cells making up one very large organism, and the amount of time which it is exposed to UV radiation throughout its life, building up a great potential danger.

It is very difficult to identify mutant colonies, since it is very unlikely that, if the DNA has been changed, the affected gene would alter some visual characteristic. In order to properly identify mutants, special repair-defective bacteria should be used, such that neither photoreactivation nor excision repair can take place, decreasing the amount of surviving bacteria, but increasing the probability of having mutants. As well, a deeper analysis of the original and resulting bacteria should be carried out, involving more biochemical and molecular analysis. This requires, however, more sophisticated techniques, which also require professional assistance, and deeper knowledge of the area.

It is of vital importance that the problem of the ozone layer be studied in detail, because if the concentration of ozone is decreased to a level that UV-C is no longer completely filtered, then, the dangers of being exposed would increase dramatically, since it could be seen from the use of the UV-C lamp that this kind of radiation has particularly strong effects. It would also be important to improve the quality control of indoor lamps in order to reduce the amount of indoor UV radiation. As well, public awareness campaigns should not only encourage the use of UV protection products, but also inform of the mechanism of UV action and its dangers and consequences, since it would further encourage protection.

5 Appendices

5.1 Personal Experience

I found the Extended Essay experience very enrichning, since I gained knowledge in several areas in which I would have had little opportunities of working. Since Biology isn't one of my IB subjects, it was also a possibility to broaden my area of knowledge.

I also found very surprising and stimulating the fact that many techniques and procedures which I had devised by myself for the purpose of the experiments carried out, were in fact standard procedures in microbiology and UV research. I believe this will be of great importance and significance for future research on other topics, since it represents a skill which can represent significant help when designing new experiments, or a substancial gain in time since less of it needs to be wasted on studying standard reference books.

5.2 Preparation of MacConkey agar

25 grams of MacConkey nutrient were weighed into a conical flask, to which 500 cm3 of water were added (see picture 5.2.1). Once the nutrient had dissolved completely, the solution was sterilised in a steam pressure chamber for 20 minutes (see picture 5.2.2). Once sterile, it was distributed into several Petri dishes, which were then kept at 2-6°C, until they were used. Before being used, they were placed in an incubator for 15 minutes in order to remove any excess humidity.

Picture 5.2.1 : dissolving the nutrient in distilled water.

Picture 5.2.2 : Sterilising the MacConkey solution.

5.3 Photographic Evidence

Picture 5.3.1 : Petri dishes being exposed to sunlight. The black paint removes most reflection, making the situation equal in this aspect for different types of exposures. The two different kinds of media can be appreciated here.

Picture 5.3.2 : analysis is much easier having all the exposure periods in one same dish. I is the shortest period, while IV is the longest.

Picture 5.3.3 : Exposures to sunlight at noon (left) and afternoon (right). At noon, only one bacteria survived in the second period (30 minutes). At the afternoon, more bacteria survived, showing that UV is more dangerous at noon.

Picture 5.3.4 : Dishes being exposed to a fluorescent tube.

Picture 5.3.5 : Working with the UV-C tube. UV filter sunglasses are used for protection of the eyes, which are particularly sensitive to UV radiation.

Picture 5.3.6 : Only seven bacteria (which grew into one colony each) survived 30 seconds of exposure to the UV-C tube.

Picture 5.3.7 : Results of the exposure of Salmonella enteritidis for periods of 20 seconds (left) and 40 seconds (right). Notice that it has resisted UV action very similarly to E.coli (see picture 5.2.6).

Picture 5.3.8 : Two variations of the Salmonella picked up from different exposures. Note the exponential decay of population and the slightly different behaviour.

Picture 5.3.9 : Comparative picture showing exposures of Salmonella (top) for 20" (left) and 40" (right), one kind of bacteria picked up from the air (middle) for 20" (left), 40" (middle) and 5 minutes (right), and another kind of bacteria (bottom) for 20" (left) and 40" (right). Note how much more resistant to UV action the two types of bacteria picked up from the air are, compared to Salmonella.

Picture 5.3.10 : Observing the results of sunlight. The windows can be clearly identified.

Picture 5.3.11 : Isolating specific colonies, in order to culture and analyse them.

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