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The Journal of Experimental Biology 212, 785-789 Published by The Company of Biologists 2009 doi:10.1242/jeb.023663

Ametabolic embryos of Artemia franciscana accumulate DNA damage during prolonged anoxia

Alexander G. McLennan Cell Regulation and Signalling Division, School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, UK e-mail: [email protected]

Accepted 6 January 2009

SUMMARY Encysted embryos of the Artemia franciscana are able to survive prolonged periods of anoxia even when fully hydrated. During this time there is no metabolism, raising the question of how embryos tolerate spontaneous, hydrolytic DNA damage such as depurination. When incubated at 28°C and 40°C for several weeks, hydrated anoxic embryos were found to accumulate abasic sites in their DNA with k=5.8ϫ10–11 s–1 and 2.8ϫ10–10 s–1, respectively. In both cases this is about 3-fold slower than expected from published observations on purified DNA. However, purified calf thymus DNA incubated under similar anoxic conditions at pH 6.3, the intracellular pH of anoxic cysts, also depurinated more slowly than predicted (about 1.7-fold), suggesting that cysts may in fact accumulate abasic sites only slightly more slowly than purified DNA. Upon reoxygenation of cysts stored 4 under N2 for 30 weeks at 28°C, the number of abasic sites per 10 bp DNA fell from 21.1±4.0 to 9.8±2.0 by 12 h and to 6.2±2.1 by 24 h. Larvae hatched after 48 h and 72 h had only 0.59±0.17 and 0.48±0.07 abasic sites per 104 bp, respectively, suggesting that repair of these lesions had largely taken place before hatching commenced. Thus, unlike bacterial spores, Artemia cysts appear to have no specific protective mechanism beyond what might be afforded by chromatin structure to limit spontaneous depurination, and rely on the repair of accumulated lesions during the period between reoxygenation and hatching. Key words: Artemia, anoxia, DNA damage, depurination.

INTRODUCTION One form of molecular damage that would clearly have to be Embryos of the brine shrimp Artemia franciscana are able to arrest prevented or repaired before development could properly resume development as gastrulae, form thick-walled cysts and enter a stage is DNA damage. DNA is known to undergo a number of spontaneous of diapause, which is characterized by low metabolic activity and hydrolytic reactions, including depurination (and to a lesser extent high stress tolerance. Diapause can be broken by a number of depyrimidination) and cytosine and adenine deamination (Lindahl, environmental factors, including desiccation. Dry cysts display a 1993). Abasic sites (AP sites, apurinic/apyrimidinic sites) arise remarkable resistance to ionising and non-ionising radiation, through the hydrolysis of the N-glycosylic linkage between the bases extremes of temperature and other environmental insults and high and sugars in DNA and RNA and are both potentially mutagenic viability can be maintained for many years (Clegg and Conte, 1980). and lethal (Boiteux and Guillet, 2004; Lhomme et al., 1999; Yu et In this dehydrated state, damage to proteins, membranes and other al., 2003). They have been estimated to occur in mammalian cells macromolecular structures is prevented by the presence of a high at 37°C and pH7.4 at a rate of up to 10,000 per cell per generation level of trehalose and a number of stress proteins (MacRae, 2003). (Lindahl and Nyberg, 1972). Using data from that paper on the This stress resistance is shared by a number of other anhydrobiotic temperature and pH-dependence of DNA depurination, an empirical , including rotifers and tardigrades; however, Artemia rate constant for depurination of 8.4ϫ10–11 s–1 can be estimated for embryos have the almost unique ability to survive for extended Artemia DNA at 23°C and pH6.3, the intracellular pH of anoxic periods in the fully hydrated state in the absence of molecular oxygen cysts (Busa et al., 1982). With a genome size of 2.9ϫ109 bp (Clegg, 1992; Dutrieu and Chrestia-Blanchine, 1966; Stocco et al., (Rheinsmith et al., 1974), this corresponds to 40,000 bases lost per 1972). Even after four years of continuous anoxia at 20–23°C, a day or 0.25% of the total genomic code per year if uncorrected. hatch rate of at least 60% can be achieved when oxygen is restored. This rises to 0.5% and 2.5% at 28°C and 40°C, respectively, During this time, all metabolism appears to be at a complete but temperatures that could be experienced at least temporarily by cysts reversible standstill (Clegg, 1997; Hontoria et al., 1994). This lack in their natural habitat. In an actively metabolising cell, AP sites of metabolism means that any accumulated molecular damage, such arising spontaneously and by enzymatic removal of damaged bases as spontaneous molecular hydrolysis, could not be repaired by are efficiently and continuously repaired by the process of excision energy-requiring systems. This damage must either be prevented or repair. However, this requires energy in the form of ATP for ligation repaired once oxygen is restored. When anoxic embryos are and dNTPs for base replacement, both of which would be quickly reoxygenated, the onset of hatching is delayed and the hatch rate depleted in the absence of restorative metabolism. Thus, to prevent reduced: the longer the period of anoxia, the greater the delay and a catastrophic genetic loss, Artemia embryos must somehow severely the slower the rate. For example after four years of anoxia, hatching restrict depurination or else allow AP sites and other hydrolytic does not begin until about 120h after reoxygenation, compared with damage to accumulate and then rely on efficient post-anoxia repair. the 16–20h observed when dry cysts are rehydrated directly in To investigate these alternatives, we have measured the number of oxygenated seawater (Clegg, 1997). AP sites in DNA purified from anoxic cysts stored at 28°C and

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40°C for periods up to 36 weeks and from larvae hatched from these 1h at RT. DNA was then purified by extracting three times with cysts. an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1 saturated with TE buffer, pH 8.0). The DNA was dialysed MATERIALS AND METHODS extensively at 4°C against 10 mmol l–1 sodium citrate pH 5.0, Preparation of anoxic Artemia cysts and calf thymus DNA 100mmoll–1 NaCl then heated at 70°C for various times up to Premium-grade encysted embryos of Great Salt Lake Artemia 90min. Samples were removed every 9min, purified by ethanol franciscana (Kellogg) were obtained from ZMSystems, Winchester, precipitation and redissolved in TE buffer at 200μgml–1. DNA UK. Portions (1.5g) of dry cysts were hydrated in greased, ground- heated in this way for 9min contains two AP sites per 104 bp, etc. glass stoppered tubes in 12ml 0.4moll–1 NaCl that had previously (Lindahl and Nyberg, 1972; Mohsin Ali et al., 2004). been bubbled with O2-free N2 gas for 6h. N2 bubbling was continued during hydration for 4h, then the tubes were stoppered and sealed Assay for AP sites with paraffin wax after carefully flushing the air space with N2. AP sites were assayed using a modification of the aldehyde reactive Tubes were then incubated at 28°C or 40°C. probe (ARP) assay previously described (Asaeda et al., 1998; Calf thymus DNA (Sigma Chemical Co., St Louis, MO, USA) Mohsin Ali et al., 2004). This assay tags the free aldehyde group was dissolved in TE buffer (10mmoll–1 Tris-HCl pH7.5, 1mmoll–1 of AP sites with biotin and these are then detected with high EDTA) and adjusted to 0.8mgml–1. It was then dialysed extensively sensitivity using peroxidase-conjugated streptavidin. ARP-DNA at 4°C against 20mmoll–1 MES-KOH pH6.3, 0.15moll–1 KCl, samples were prepared from cyst, anoxic calf thymus and 1mmoll–1 EDTA and sodium azide finally added to 0.1%. Portions depurinated calf thymus DNA by incubating 50μl (10μg) DNA in (5ml) were transferred to greased, ground-glass stoppered tubes and TE buffer with 50μl 10mmoll–1 ARP (Dojindo, Rockville, MD, bubbled with N2 for 8h. The tubes were then sealed and incubated USA) for 2h at 37°C. Unreacted ARP was removed by sequential as above. Periodically, the tubes were sampled (250μl) under N2. dilution and concentration three times using Microcon 30 centrifugal concentrators (Millipore, Watford, UK). The final ARP-DNA was Preparation of DNA from Artemia cysts and larvae adjusted to 1μgml–1 with TE buffer. A tube of hydrated, anoxic cysts was shaken, allowed to settle and ARP-DNA samples (200 μl) were added to the wells of a any floating material removed by aspiration. The remaining cysts protamine-coated 96-well EIA plate (Bio-Rad, Hercules, CA, USA) were decapsulated as previously described (McLennan and Prescott, and incubated at 37°C for at least 1h (Asaeda et al., 1998). Excess 1984) and finally collected by vacuum filtration. Each tube yielded DNA was removed by suction and the wells washed five times with six 0.5g portions, which were frozen at –20°C. Each portion of 350μl PBS–0.1% Tween (137mmoll–1 NaCl, 2.7mmoll–1 KCl, –1 –1 frozen cysts was transferred to a pre-cooled mortar and liquid N2 4.3mmoll Na2HPO4 7H2O, 1.4mmoll KH2PO4, 0.1% Tween added. After grinding the cysts to a fine powder, 5ml DNAzol 20). Wells were blocked with 350μl SuperBlock buffer (Thermo (Invitrogen, Carlsbad, CA, USA) was added and homogenization Fisher, Loughborough, UK) for 30min at RT, then this was replaced continued briefly. The contents were transferred to a tube, 100μl by 100μl of a 1:2000 dilution of streptavidin–peroxidase polymer proteinase K (Sigma Chemical Co., 10mgml–1 in water) added and (Sigma Chemical Co.) in SuperBlock buffer containing 0.5% the tube incubated with rolling at room temperature (RT) for 2–3h. Tween. After 30min at RT, the conjugate was removed and the After centrifugation (13,000g, 5min), 2.5ml of 100% ethanol wells washed 10 times with 350μl PBS–0.5% Tween as follows: was added to the supernatant, the mixture shaken gently for 1min 4ϫ1min; 1ϫ5min; 4ϫ1min; 1ϫ5min, followed by one wash with and the DNA (heavily contaminated with orange lipid) removed by 350μl PBS–0.1% Tween. spooling into 2.8ml 10mmoll–1 Tris base. Once the DNA had Peroxidase substrate was freshly made by adding 20 μl of –1 dissolved, it was extracted three times with an equal volume of 35mgml o-phenylenediamine and 0.5μl 30% H2O2 per ml of –1 –1 phenol:chloroform:isoamyl alcohol (25:24:1 saturated with TE buffer (51mmoll Na2HPO4, 24mmoll citric acid). 160μl of this buffer, pH8.0). 1/10 volume 3moll–1 sodium acetate pH5.2 was was added to each well and incubated for 30min at 28°C with –1 added to the final aqueous layer followed by 2.5 volumes of cold occasional shaking. After adding 40 μl 4 mol l H2SO4, the 100% ethanol. After gentle mixing, the DNA was allowed to absorbance was measured at 495nm. precipitate at –20°C for at least 10min. The DNA was washed twice in 70% ethanol, once in 100% ethanol, dissolved in 1ml TE buffer RESULTS and adjusted to 200μgml–1 with TE. The final yield was typically The number of AP sites was measured in DNA purified from cysts 0.4mg DNA per 0.5g portion of hydrated cysts. stored under N2 for up to 36 weeks at 28°C or up to seven weeks To prepare DNA from larvae, anoxic cysts (1.5g wet mass) were at 40°C. The number of AP sites per 104 bp DNA was estimated by hatched and the swimming larvae separated from unhatched cysts comparison with heat-depurinated calf thymus DNA standards and other material by attraction to a light source in a separator box prepared under conditions where the level of depurination has (Persoone and Sorgeloos, 1972). After collection by vacuum previously been calculated (Lindahl and Nyberg, 1972; Mohsin Ali filtration through a small piece of cheesecloth, larvae were frozen et al., 2004). Figs1 and 2 show that AP sites did accumulate with in liquid N2 and weighed. DNA was then prepared as described time at both temperatures, with calculated rate constants for above, with appropriate volume adjustments. depurination of 5.8ϫ10–11 s–1 (28°C) and 2.8ϫ10–10 s–1 (40°C). In each case, the rate was 3-fold slower than the predicted rates of Preparation of depurinated DNA standards 1.7ϫ10–10 s–1 (28°C) and 8.4ϫ10–10 s–1 (40°C), suggesting that there Depurinated calf thymus DNA was prepared as previously described might be some form of protection in the cyst. However, when (Asaeda et al., 1998; Mohsin Ali et al., 2004). Briefly, RNAase A samples of purified calf thymus DNA stored under N2 at (Sigma Type II) was added to a 0.8mgml–1 solution of calf thymus physiological ionic strength and pH6.3, the intracellular pH of DNA (Sigma Chemical Co.) in TE buffer to final concentration of anoxic cysts (Busa et al., 1982), were compared, they accumulated 100μgml–1 and incubated for 1h at 37°C. Existing abasic sites were AP sites with k=9.4ϫ10–11 s–1 (28°C) and 5.2ϫ10–10 s–1 (40°C), –1 removed by addition of NaBH4 to 100mmoll and incubation for 1.6–1.8-fold slower than predicted in both cases. Given the likely

THE JOURNAL OF EXPERIMENTAL BIOLOGY DNA damage in anoxic Artemia cysts 787

60 hatching, the number of AP sites was measured in samples of cysts and in larvae hatched from cysts stored under N2 for 30 weeks at 50 28°C followed by incubation in oxygenated seawater for up to 72h. 4

b p A marked reduction in AP sites per 10 bp from 21.1±4.0 to 9.8±2.0 4 40 was observed in cysts 12h after reoxygenation, with a further 30 reduction to 6.2±2.1 by 24h, when 4% of the cysts had hatched (Fig.3). However, as only 71% of the cysts hatched by 72h, and 20 this number did not increase significantly beyond this time, the level of DNA damage remaining in ‘24h’ cysts (30% of that in ‘0h’ cysts) AP s ite per 10 10 may represent that specifically in dead and diapause-arrested cysts and so the damage in viable, hatchable cysts may have been fully 0 010203040 repaired by this stage. Unfortunately, insufficient larvae had hatched 4 Time (weeks) by 24h to test this directly; however, the level of AP sites per 10 bp present in larvae hatched after 48 and 72h was much lower Fig. 1. Time-dependent accumulation of abasic (AP) sites in the DNA of (0.59±0.17 and 0.48±0.07, respectively) and was comparable with hydrated Artemia cysts (closed circles) and purified calf thymus DNA (open that found in 48h larvae hatched from cysts that had not been circles) stored under anoxic conditions at 28°C. For each determination, exposed to anoxia (0.60±0.23 per 104 bp, N=6), indicating that full DNA was prepared from two separate 0.5 g cyst or 250 μl calf thymus DNA portions and duplicate ARP-DNA samples made from each of these and repair had taken place by 48h. assayed in triplicate (N=4). Error bars are ± s.d. Comparison of the slopes of the two experimental regression lines using GraphPad Prism software DISCUSSION showed the difference to be extremely significant (P<0.0001). The dotted These results clearly show that ametabolic, encysted Artemia line represents the predicted rate of depurination of DNA at pH 6.3 embryos accumulate AP sites in their DNA during storage in the calculated from the data of Lindahl and Nyberg (Lindahl and Nyberg, hydrated state under anoxic conditions and that most, and possibly 1972). all, of this damage is repaired in the lag period between reoxygenation and hatching. It is likely that other forms of spontaneous DNA damage accumulate and that these and the differences in the precise conditions employed here and in the study probable oxidative damage induced by reoxygenation itself induce of Lindahl and Nyberg upon which the calculated rates are based cell cycle arrest to permit post-anoxia repair before DNA synthesis (Lindahl and Nyberg, 1972), the agreement between both is can resume (Freiberg et al., 2006). The slightly lower than predicted remarkably good for purified DNA. Thus, the cyst DNA may only rate (1.7- to 3-fold) may simply reflect a mild protective effect of be accumulating AP sites at a slightly lower rate (1.7-fold) than chromatin structure. Although one measurement of AP site expected at both temperatures. generation in live cells has yielded a rate very similar to that found The % viability of anoxic embryos stored at 28°C (but not 40°C) was tested by measuring the hatch rate 64h after reintroduction to oxygenated seawater. These were found to be 85, 83, 75 and 67% 30 80 after 0, 8, 20 and 36 weeks anoxia. Although impressive, these rates are considerably lower than those reported by Clegg for San 70 Francisco Bay cysts, which maintained almost full hatchability for 25 two years (Clegg, 1997). However, the cysts were not dried before 60 hatching, which may improve the hatch by helping to break the 20 anoxia-induced diapause, so the unhatched cysts may not be dead 50 b p but still locked in diapause (Abatzopolous et al., 1994; Clegg, 1994). 4 To find evidence that accumulated DNA damage is repaired before 15 40

* 30 H a tch (%) 80 10

AP s ite per 10 ** 70 20 60 5 b p

4 10 50 40 0 *** *** 0 0122448 72 30 Time after reoxygenation (h) 20 AP s ite per 10 Fig. 3. Loss of AP sites from DNA of cysts and larvae hatched from cysts 10 stored under anoxic conditions at 28°C for 30 weeks followed by incubation 0 in oxygenated seawater at the same temperature. Black bars indicate the 0102030540 0 number of AP sites in cysts after 0, 12 and 24 h incubation in oxygenated Time (days) seawater and in separated larvae hatched 48 and 72 h after introduction to oxygenated seawater. Error bars are ± s.d. (N=4, as in Fig. 1). Asterisks Fig. 2. Time-dependent accumulation of abasic (AP) sites in the DNA of denote t-test significance compared with 0 h cysts (*P=0.01; **P=0.005; hydrated Artemia cysts (closed circles) and purified calf thymus DNA (open ***P=0.001). Grey bars indicate the % hatch of these cysts, determined on circles) stored under anoxic conditions at 40°C. Other details and statistical samples of 250–300 cysts distributed among the wells of a 24-well plate as analysis as for Fig. 1. previously described (Clegg, 2007).

THE JOURNAL OF EXPERIMENTAL BIOLOGY 788 A. G. McLennan for naked DNA of 9000 per day per generation (Nakamura et al., spontaneous hydrolytic DNA damage. In the dehydrated state, water 1998), a 2.3-fold reduction in the rate of acid depurination of replacement by trehalose and glycerol will clearly limit this damage chromatin compared with DNA has also been reported (Duijndam but when hydrated, compaction of the DNA into chromatin, which and Vanduijn, 1975). Furthermore, although the intracellular pH of may exclude the possibility of DNA binding by more specific anoxic cysts has been measured at 6.3 (Busa et al., 1982), the value protective proteins, may be the only mechanism available. Therefore, used to predict the rate of depurination, this may not truly represent developing embryos must rely on efficient pre-hatching systems to the microenvironment of the chromatin. Thus, a small reduction in repair this damage before DNA replication can resume. the expected rate of depurination cannot be interpreted as a specific protective effect. LIST OF ABBREVIATIONS In prokaryotes, DNA-binding proteins such as HU and the ferritin- AP apurinic/apyrimidinic (abasic site) like Dps proteins protect the bacterial DNA from radiation and ARP aldehyde reactive probe oxidative damage (Nair and Finkel, 2004) whereas small, acid- PED pre-emergence development RT room temperature soluble spore proteins are known to provide protection against wet heat and to reduce the rate of DNA depurination in Bacillus subtilis spores by a factor of 20 (Setlow and Setlow, 1994; Setlow, 2007). REFERENCES Abatzopolous, T., Triantaphyllidis, G., Sorgeloos, P. and Clegg, J. S. (1994). 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