In the Laboratory
Testing for Ultraviolet Toxicity Using Fungi
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Marcelo Vital and Patricia Esperón* Cátedra de Biologia Molecular, Facultad de Quimica, General Flores 2124, Montevideo CP 11800, Uruguay; *
[email protected] A semester-long molecular biology course with theoretical and laboratory modules has recently been incorporated into the curriculum. For a laboratory project in this course, a bioassay for the assessment of effects of a physical agent, the UV radiation, was designed. Effects of chemical agents upon organisms have already been described in this Journal (1, 2). It is well known that the sun emits large quantities of energy, with ultraviolet radiation composing 2% of the radiation. UV radiation is divided into three segments: UV-A (320–400 nm), UV-B (290–320 nm), and UV-C (100–290 nm). Although UV radiation is important for industrial and medical application, exposure to this type of radiation produces deleterious effects in organisms (3). Furthermore the negative effects of UV radiation have been potentially enhanced by the decrease in the stratospheric ozone layer (4). This long-term depletion results in the increase in the quan-
DNA
tity of solar ultraviolet radiation that reaches the Earth. The short-wave, high-energy UV-C radiation is almost totally absorbed by the earth’s atmosphere. The UV-B radiation is the primary component of sunlight that affects biological activities. Although other molecules (i.e., proteins) may also be affected, DNA is the most vulnerable. The death of the organism is the most dramatic consequence of this type of radiation (Figure 1). The UV radiation acts only on compounds that absorb it directly. In the cell, it affects compounds that have organic ring structures such as purines and pyrimidines; for example, it causes hydration of cytosine across the double carbon bond. The most important types of UV-B-induced DNA damage are cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4´) pyrimidone photoproducts (Figure 2A). This dimerization may be the primary mutagenic effect induced by UV light and produces a distortion in the DNA helix (Figure 2B). These photoproducts interfere with proper DNA replication, causing reduced cell viability and even cell death (5). In addition to these direct effects on DNA, indirect actions of UV light via absorption by metabolic intermediate compounds are also possible. It is known that UV radiation affects the human health. Acute and chronic skin damage occurs as a consequence of solar UV-A and UV-B radiation exposure (6). The connection between sunlight and skin cancer has been discussed in this Journal (5). The UV-B exposure also induces cataract formation after low-dose radiation (7). A most unusual facet of UV-induced mutations is the discovery by Kelner (8) that their effect can be reversed by exposing the cells to visible light containing wavelengths in the blue spectrum. An enzyme, photolyase, removes dimers and repairs the DNA molecule in a process known as photoreactivation (9). Experimental Procedure
DNA damage
or DNA repair
or cell death
DNA mutation
Figure 1. Relationship between UV radiation and cellular damage and the consequences.
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The experiment was performed using Ascomycetes. These microorganisms, routinely employed in our laboratory, have several advantages; they are not pathogenic and grow easily in vitro. The possibility to distinguish colonies based on color allows the recognition of different mutants. The cells were grown in malt extract agar for 48 hours. Then, the spores were collected and suspended in 5 mL of 104 dilution of Tween 80. Each spore suspension was transferred to a glass Petri dish to be irradiated. An aliquot of the spore suspension was removed just before the irradiation was performed. This aliquot was used to calculate the cell count at zero time (t0). Another four aliquots were removed from the Petri dish at successive time intervals of UV exposure. Each sample was serially diluted as a way to obtain isolated colonies when they were plated by duplicate on the culture medium. Then, those plates with
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In the Laboratory A
Table 1. Protocol Working Conditions Working Conditions
Strain
Distance/ cm
Power/ W
1
Aspergillus nidulans
15
15
2
Aspergillus nidulans
15
30
3
Aspergillus nidulans
8
15
4
Penicillium digitatum
8
15
isolated fungus colonies were chosen and the quantity of spores was counted. All treatment and manipulation during and after UV irradiation were carried out in dark to avoid photoreactivation. The survival rate for each exposure time was calculated as Percent Survival = (At兾A0)(100%) where At is the quantity of spores at different exposure times and A0 is the quantity of spores at t0. The quantity of spores at different exposure times versus time was plotted and the dose–response curve was drawn. The laboratory project was performed in classes of 15 students divided into groups of 3 or 4 students. Each group performed the same protocol, changing some parameters such as strains, dosage of UV radiation, and distance to the UV lamp as shown in Table 1. For this laboratory experiment, three, two-hour laboratory periods were required. However, the experiment could be performed as a one-week or as a multiweek project. The different steps of the protocol are shown in Figure 3. B
Hazards This experiment presents no unusual hazards. There are no irritant compounds and the microorganisms are not pathogenic. However, gloves and safety goggles should be worn all the time. Direct exposure to UV light may cause skin cancer and ocular damages. Do not be exposed directly to this type of radiation. Results and Discussion The results of the experiments carried out under different working conditions are detailed in Table 2. Spores were recollected from a 15-cm diameter Petri dish with confluent growth and suspended in 5 mL of 104 dilution of Tween 80. We have previously calculated that this suspension yields a concentration of approximately 106 spores兾mL. As expected, the spore-surviving rate decreased with the increase of UV exposure time (Table 2). The quantities of spores at different exposure times were plotted versus time and the dose–response curves were drawn (Figure 4). After the experiments were concluded, it was deemed interesting to understand which reactions were taking place at the molecular level. Comparing the dose–response curves, we observed that one curve had an initial shoulder, followed by a decline that
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Figure 2. Thymine dimerization induced by UV light. (A) The two primary lesions produced by UV are the cyclobutane pyrimidine dimer (left) and the (6-4´) photoproduct (right), where P represents the phosphate group and S the sugar. (B) Helical conformation distortion of β-DNA due to thymine dimerization. The dashed box highlights the dimerization.
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In the Laboratory
became linear. This shoulder could be explained by the presence of a repair mechanism in the cell. It has been observed in yeast and other organisms. In the absence of the repair mechanism, the shoulder vanished and the curves became steeply exponential (10). We also noticed that the inflection
point in the curves might suggest that at low doses of UV, the damage was less important than at higher doses. The DNA repair mechanism is dose-dependent. As was demonstrated by Cox and Parry (10), more than one repair pathway was involved in UV resistance. The arguments that allowed the authors to arrive at this conclusion were the number of loci involved in the UV sensitivity and the observation that different mutants had different sensitivities. This could be the case for A. nidulans since it has two genes, uvsI and uvsC involved in UV mutagenesis (11). The characteristics of the dose–response curves suffered several changes when we analyzed the different results, as shown in Figure 4. Both strains used showed a similar UV radiation sensitivity, and therefore the UV surviving curves, were similar under the same distance and dosage of UV radiation, (plots C3 and C4 in Figure 4). As expected, the 100% of mortality was reached with the longest times of exposure. Percent survival decreased when the distance to the UV tube was shorter as shown by comparison of plots C1 and C3 in Figure 4. The results also let the students corroborate that the mutant frequency increased with the UV dosage but, the rate of mutation decreased and even vanished at higher dosages. Comparing the results obtained after exposing a strain to different dosages of UV radiation, it was observed that the relationship between mutation rate and UV dosage was not linear, as was previously reported by Bos (12).
prelab protocol explanation
spore collection from the Petri dish with confluent growth
transfer to a glass Petri dish
1st period
irradiation
removal of aliquots
preparation of spore solution dilutions
2nd period plating suitable dilutions
100
Survival Rate (%)
counting of surviving spores
calculation of viable spores
3rd period drawing of graphics
C1
80
C4
60
C3 C2
40 20 0 1
0
collection of results from other groups
2
3
4
5
6
7
Time / min Figure 4. Survival curves obtained after UV radiation with four different working conditions (C1, C2, C3, and C4); the working conditions are detailed in Table 1.
Figure 3. Flow chart of the experiment illustrating UV radiation effects.
Table 2. Survival Rates Obtained from Experiments with Different Working Conditions Working Conditions Time/ min
1
2
Spores/ mL
Survival (%)
Spores/ mL
0.0
2.0 x 106
100
6.2 x 106
0.5
6
100
5
2.0 x 10
1.0
6
2.0 x 10
100
5.0
1.4 x 106
70
7.0
5
9.0 x 10
2.0 x 10
4
2.5 x 10 0
45
0
3 Survival (%) 100
Spores/ mL 5.5 x 106
4 Survival (%) 100
2.0 x 10
6
0.4
1.0 x 10
6
18.2
0
5.0 x 105
9.1
0
3
3.3
2.7 x 10
36.4
0.05
Spores/ mL 3.8 x 106
100
6
80
5
12
3.1 x 10 4.6 x 10
2.8 x 105
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3
1.1 x 10
NOTE: The working conditions are detailed in Table 1.
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Survival (%)
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7.3 0.03
In the Laboratory
Conclusions
Acknowledgments
This experiment is useful to demonstrate the effects of UV light and also to generate quantitative results that could be used to show the effects of the UV radiation on the Earth and in organisms. This experiment exposed the students to the manipulation of microorganisms under sterile conditions and quantification of the lethal effects of the UV radiation. In addition, the students had the chance to discuss the radiation effects on living organisms and to think about the importance of preventing those effects. We suggest that the protocol is a good laboratory experiment to be included in a biochemical course because this protocol:
We would like to thank F. Iribarne for critically reading the manuscript. This work was supported by a CSE (Nº 246) grant from the Universidad de la República, Montevideo, Uruguay.
• Involves simple procedures • Can be included in a one-week or in a weekly course • Does not need special equipment • Does not employ expensive reagents and materials • Introduces discussion about topics of medical and environmental relevance
For future courses, we are planning to complement the experimental activities with computational manipulations. Three-dimensional modeling programs would allow students to manipulate and visualize DNA molecule distortions introduced by UV radiation.
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Supplemental Material Instructions for the students and notes for the instructor are available in this issue of JCE Online. W
Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Brower, H. J. Chem. Educ. 1991, 68, 695. Helliwell, S.; Harden, T. J. J. Chem. Educ. 1996, 4, 368. Abney, J. R.; Scalettar, B. J. Chem. Educ. 1998, 75, 757. Braid, C. Envir. Chem. Freeman: New York, 1995. Taylor, J. J. Chem. Educ. 1990, 67, 835. Bhawan, J.; Yaar, M.; Bello, Y.; Lopiccolo, D.; Nash, J. F. J. Am. Acad. Dermatol. 2000, 43, 610. Micheal, R.; Soderberg, P. G. Invest. Ophthalmol. Vis. Sci. 2000, 41, 3539. Kelner, A. Prod. Nat. Acad. Sci. 1949, 35, 73–79. Weinbauer, M. G.; Wilhelm, S. W.; Suttle C. A.; Garza, D. R. App. and Env. Microb. 1997, 63, 2200. Cox, B. S.; Parry, J. M. Mutat. Res. 1968, 6, 37. Chae, S. K.; Kafer, E. Mol. Gen. Genet. 1997, 254, 643. Bos, C. J. Curr. Genet. 1987, 12, 471.
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