UVB induced DNA photoproducts and repair pathways

All organisms, including humans, which are regularly or occasionally exposed to natural solar radiation may be subjected to DNA damage. In fact, structural changes in DNA are thought to be among the most important deleterious biochemical consequences of UVR [9,10]. UV-B can cause dimerization of DNA bases, leading to the formation of CPDs and 6-4 PPs (Figure 2). These photo-products block DNA transcription and replication such that only a single distortion of DNA may be sufficient to stop DNA replication. CPDs may hinder cell cycle progress and replication because they obstruct de novo synthesis of cellular components and substances required for growth and cell maintenance. As a consequence, population growth is reduced.

CPDs, especially thymine dimers (TT), are the most abundant lesions formed by natural UV-B [28]. However, 6-4 PPs may have a stronger deleterious effect on DNA transcription than CPDs. The non photoenzymatic repair of CPD and 6-4PP is susceptible to error. Therefore, repair may lead to point mutations in the genome. On top of this, longer wavelengths may result in the photoisomeriz-ation of the 6-4 PP to the Dewar pyrimidinone [28]. The action spectrum of this isomer parallels the 6-4 PP absorption spectrum [29], peaking around 310 nm, but also extending into the UV-A region. There is no information regarding the effect of the Dewar isomer on aquatic organisms. Yet it can be assumed that this form of DNA damage is as disruptive as the 6-4 PP itself. At greater depths for instance, 6-4 PP may no longer be formed, but conversion into Dewar isomers by longer wavelengths in the UV-A region may still occur. Similar to other lesions

Figure 2. Structure of two photoproducts, formed by UV-B exposure to DNA. (A) Cyclobutane pyrimidine dimer (thymine dimer). (B) Pyrimidine (6-4) pyrimidone photo-product (6-4 PP). [Redrawn from Friedberg et al. 38.]

induced by both UV-B and UV-A, such as DNA strand breaks and DNA-protein crosslinks, the Dewar isomer may thus be responsible for at least part of the biological UVR effects observed in aquatic environments [30],

Action spectra and biological weighting functions (B WFs) describe the contribution of a certain wavelength to a biological or chemical response. They may thereby help to predict how changes in the spectral composition of solar radiation alter biological or chemical responses. Action spectra are determined through experimental exposures to monochromatic radiation at different wavelengths, assuming that each wavelength contributes independently to the overall effect. In contrast, BWFs are determined through polychromatic exposures [31,32], for instance under natural irradiance conditions. Because responses to polychromatic exposures are assessed, BWFs are often considered relevant for overall responses to UV, since they incorporate damage as well as repair processes simultaneously. The action spectra of CPD and 6-4 PP formation are similar and can be described using the DNA damage action spectrum of Setlow [33] or pyrimidine dimer induction in alfalfa seedlings [34]. Generally, shorter wavelengths cause more damage than longer wavelengths. Both lesions show strongly increasing induction rates with decreasing wavelength in the UV-B region, especially below 302 nm. At higher wavelengths, however, the induction of 6-4 PPs is found to be much lower than that for CPDs.

Cells and tissues are able to handle DNA damage when conditions are favorable for repair. The main pathways to repair dimerized pyrimidines are nucleotide excision repair and photoreactivation ([35] and Chapter 10). Because nucleotide excision repair is light-independent and can therefore take place in the dark, this form of repair is commonly called "dark repair". Photoreactivation is a

Figure 2. Structure of two photoproducts, formed by UV-B exposure to DNA. (A) Cyclobutane pyrimidine dimer (thymine dimer). (B) Pyrimidine (6-4) pyrimidone photo-product (6-4 PP). [Redrawn from Friedberg et al. 38.]

repair pathway where an enzyme (photolyase) reverses damaged sites in the presence of wavelengths between 350 and 450 nm. Photolyases are categorized on the basis of the chromophore involved in energy transfer. Photolyases in the folate class show maximum activity at 384 nm (UV-A region). Other known photolyases fall into the deazaflavin class with maximum activity at 440 nm (blue light), or in a "non-second-chromophore" class with maximum activity at both 370 and 450 nm. Figure 3 presents action spectra of two photolyases associated with microorganisms.

The rate of photorepair is thus directly related to the intensity of UV-A and/or visible light. Therefore, both the rate of DNA damage induction as well as that of photorepair in plankton is dependent on the prevailing light regime in the water column. CPD induction is caused primarily by UV-B, whereas photorepair of DNA damage depends on the intensity of UV-A and/or visible light. Since the attenuation of UV-B in the water column is much higher than that of UV-A or PAR ([36], Chapter 3), the induction to repair ratio is highly depth-related. For sessile organisms, deeper waters will accommodate more favorable UVR conditions as compared with shallower waters, where damage induction rates may exceed damage removal rates. In contrast, plankton organisms are moved in the upper water layer as a result of vertical mixing and exposure of the cells to the different wavelength bands will strongly fluctuate (Chapter 4). If a simple linear dose-response relationship for the induction as well as the repair of DNA damage were applicable (i.e., if the photochemical law of reciprocity would hold), mean UV-B, UV-A and visible light doses within the upper mixed surface layer (UML) would be sufficient to predict effects on plankton organisms. However, there are indications that this is not always the case, especially for repair processes [37]. Repair rates likely increase with increasing initial levels of CPDs [38]. Moreover, not only the rate of photorepair, but also the induction of the repair system is light dependent [39]. This means that under fluctuating irra-

300 400 500 600

wavelength (nm)

300 400 500 600 wavelength (nm)

Figure 3. Absorption spectra of two photolyases (A) Escherichia coli; (B) Arabidopsis sp. [Redrawn from Friedberg et al. 38.]

300 400 500 600

wavelength (nm)

300 400 500 600 wavelength (nm)

Figure 3. Absorption spectra of two photolyases (A) Escherichia coli; (B) Arabidopsis sp. [Redrawn from Friedberg et al. 38.]

diance conditions, as experienced by plankton organisms in the UML, conditions for repair may not always be optimal.

9.3 Penetration of DNA effective UVR in marine waters

In the water column, the downwelling irradiance (Ed) of the solar light field diminishes with depth due to absorption and scattering processes. In optically homogeneous waters, irradiance decreases exponentially and can be described using the vertical attenuation coefficient (Kd):

where Edx(z) is the level of downward irradiance at X at z m depth, £¿#0 the level of downwelling irradiance at X just below the surface and Kd^ the vertical attenuation coefficient of A in m-1. The value of Kd is not the same for each wavelength and depends on chemical, biological and physical parameters, such as the concentration of dissolved organic matter (DOM), pigment concentrations and the amount and type of particles present in the water column [40], For example, the concentration of DOM in the water column strongly influences the Kd in the ultraviolet region whereas phytoplankton cells also absorb blue and red light.

It is essential to collect information on the irradiance conditions in the water column when UVR effects on aquatic organisms are considered. Since UVR effects are strongly wavelength dependent and the spectral distribution changes rapidly with depth, broadband radiometers have limited value. Accurate measurements of irradiance can only be achieved applying scanning or narrowband spectroradiometry. To assess possible biological effects of UV-B at a certain depth, measured irradiance spectra are multiplied by an appropriate action spectrum or BWF to determine the amount of biologically effective irradiance (BEI) at that depth. If required, a biological effective dose (BED) can be calculated by integrating several measurements of BEI over time. The choice for a relevant action spectrum may be dictated by the demand for comparing weighted irradiances from various regions or systems. In addition, it will depend on the biological effect under study. For example, a scientist studying DNA damage in plankton will most likely prefer to use a DNA damage action spectrum or weighting function for calculating biologically effective doses.

Where spectroradiometry is not available, profiling radiometers may provide some information on attenuation of DNA effective wavelengths: it has been observed that the 305 nm channel of the PUV500 profiling radiometer (Biosperi-cal Instruments Inc.) gives a good assessment of DNA effective UV-B (Jeffrey unpublished). A more direct way to determine biologically effective doses is to use a dosimeter which contains small amounts of target molecules or simple organisms. Several types of UVR dosimeters have been developed for field applications. These dosimeters are based on biological material [41-44], chemical reactions [45,46] and biochemical material such as DNA solutions [11,47-50]. There are several advantages of dosimeter measurements compared to spectral radiometer measurements [47,48]. Application of dosimeters allows direct determinations of daily doses at different depths by performing underwater incubations during the whole light period. Also, a dosimeter automatically integrates the UY component of solar radiation meanwhile weighting them according to their biological effectiveness. Finally, a dosimeter is inexpensive, small, robust and portable. On the other hand, although individual dosimeters are low cost, the facilities required to process the exposed dosimeters can be expensive, and the process may be time consuming for a large number of samples

Figure 4 and Table 1 present some examples of attenuation coefficients of DNA effective UV-B in various water types using DNA dosimetry. In open ocean waters, DNA effective UV-B may penetrate to significant depths, giving 1 % levels (of surface irradiance) down to 25 m. For example, Central Atlantic Ocean and Gulf of Mexico waters were shown to be very UVR transparent (Table 1), although UV penetration in the Gulf of Mexico was highly variable depending on location (proximity to shore). Measured Kbd-eff values for open ocean waters correspond with calculated XDNA values reported by Smith and Baker [36] for clear ocean waters with low productivity. Waters off Curaçao, Netherlands Antilles, were also shown to be UV-B transparent, with the only exception being the eutrophic Anna Bay (Table 1). Consequently, shallow coral reef communities may be affected by UV-B, as supported by Lyons et al. [52], who measured induction of DNA damage in coral mucus and in eukaryotic and prokaryotic fractions above a coral reef. In the Gulf of Aqaba, Red Sea, no DNA

CPD-MB'1 CPD-MB'1

Figure 4. Attenuation of DNA effective UV-B in various waters types, measured with DNA dosimeters. (A) open ocean waters, (B) coastal waters. Open ocean (Curacao [50], Gulf of Mexico (Jeffrey, unpublished results) inshore/coastal (Antarctica [89], Jeffrey, unpublished results), Anna Bay [50], Bahia Bustamante [53]), fresh water (Andes, Helbling, unpublished results), Red Sea [95]. y-axes: water depth (m); x-axes: CPD concentrations in dosimeter DNA (CPD.MB-1).

CPD-MB'1 CPD-MB'1

Figure 4. Attenuation of DNA effective UV-B in various waters types, measured with DNA dosimeters. (A) open ocean waters, (B) coastal waters. Open ocean (Curacao [50], Gulf of Mexico (Jeffrey, unpublished results) inshore/coastal (Antarctica [89], Jeffrey, unpublished results), Anna Bay [50], Bahia Bustamante [53]), fresh water (Andes, Helbling, unpublished results), Red Sea [95]. y-axes: water depth (m); x-axes: CPD concentrations in dosimeter DNA (CPD.MB-1).

Table 1. Vertical attenuation coefficients (Kbd_eff) for DNA effective UVBR in various marine and fresh water systems, measured with DNA dosimeters. Attenuation coefficients were determined from linear regressions of natural logarithmic biologically effective irradiation against depth using the log-linear part of the curve

Table 1. Vertical attenuation coefficients (Kbd_eff) for DNA effective UVBR in various marine and fresh water systems, measured with DNA dosimeters. Attenuation coefficients were determined from linear regressions of natural logarithmic biologically effective irradiation against depth using the log-linear part of the curve

Location

Date

^bd-eff

sd

n

Reference

Gerlache Strait, Antarctica

Oct 1995

0.13

0.014

3

Jeffrey et al., in preparation

Gerlache Strait, Antarctica

Oct 1996

0.28

0.030

6

Jeffrey et al., in preparation

Weddell-Scotia Confluence

Oct 1998

0.14

-

1

Mitchell et al., submitted

Central Gulf of Mexico

Jun 1995

0.19

-

1

Jeffrey, unpublished

Central Atlantic Ocean

Aug. 1996

0.19

0.01

6

Boelen et al„ 1999 [50]

Curaçao open ocean station

Nov. 1996

0.36

0.04

3

Boelen et al., 1999 [50]

Curaçao Buoy 1

April 1998

0.28

0.04

6

Boelen et al., 2001 [94]

Gulf of Aqaba, Red Sea

Sept. 1998

0.46

0.04

3

Boelen et al., 2002 [95]

Coastal Gulf of Mexico (StA)

Sept 1994

0.54

-

1

Aas et al, 1996 [2]

Coastal Gulf of Mexico (StC)

Jun 1995

0.87

-

1

Aas et al., 1996 [2]

Coastal Gulf of Mexico

Sept 1994

0.51

0.040

2

Jeffrey et al., 1996 [11]

Curaçao Anna Bay

Nov. 1996

1.53

0.10

2

Boelen et al., 1999 [50]

Ryder Bay, Antarctica

Jan-Mar 1998

0.66

0.13

5

Buma et al., 2001 [89]

Kongsfjorden, Spitsbergen

June 2001

0.91

0.30

7

van de Poll, unpublished

Lago Moreno Este, Andes

Jan. 1999

0.74

-

1

Helbling unpublished

damage could be detected in dosimeter DNA below 15 m. Nevertheless, UV-B penetrated to significant depths in the Gulf of Aqaba, giving 1% depths of biological effective UV-B around 10 m. Attenuation coefficients were comparable to those measured by Regan et al. [47] in the clear oligotrophic coastal waters off Lee Stocking Island, Bahamas but slightly higher than those measured in the clear coastal waters off Curaçao [50].

Eutrophic coastal areas typically have much lower transparency for DNA effective UV-B (Table 1). In an enclosed bay in the Antarctic the presence of a phytoplankton bloom caused high Kbd.eff values giving rise to shallow 1 % depths for DNA effective radiation, between 5.4 and 9.6 m. In the temperate Bahia Bustamante, Argentina, attenuation of DNA effective UV-B was very rapid due to the presence of phytoplankton cells. iCbd.eff could not be calculated here due to low resolution of data in the upper meters [53]. In an Arctic fjord (Kongsfjorden, Spitsbergen) attenuation of DNA effective UV-B was very rapid due to the input of sediment-containing melt water from the surrouding land. The mean Kbd_eff over a four week period was as high as 0.91 (van de Poll, unpublished results). In summary, DNA effective wavelengths may reach a substantial fraction of the primary producers in oligotrophic systems such as open tropical marine waters, and in coral reef areas. Moreover, aquatic organisms inhabiting more eutrophic systems, characterized by higher productivity, may also experience high DNA effective irradiance. Pelagic organisms circulating in the upper meters of the water column or sessile organisms living in littoral or sub-littoral zones may be affected.

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