FUdR extends the lifespan of the short-lived AP endonuclease mutant in Caenorhabditis elegans in a fertility-dependent manner

The anticancer drug 5-fluorouracil (5-FU) and its metabolite 5-fluoro-2'-deoxyuridine (FUdR) inhibit thymidylate synthase and induce uracil bases in DNA. FUdR is commonly used for inhibiting fertility when measuring the lifespan of the nematode Caenorhabditis elegans. However, it is not known whether DNA damage induced by FUdR affects lifespan. EXO-3 is an apurinic/apyrimidinic endonuclease in C. elegans, and we reported previously that deletion of the exo-3 gene causes reproductive abnormalities and decreased lifespan. In this study, we found that FUdR extended the lifespan of exo-3 mutants. We measured the lifespan of multiple germline mutants to examine whether this lifespan extension effect was dependent on fertility. In the presence of a fem-1 mutation, which causes a deficiency in sperm production, FUdR did not extend the lifespan of the exo-3 mutant. In glp-1 mutants, which do not develop gonads, the exo-3 mutant was not short-lived, and FUdR did not extend its lifespan. These results suggest that the lifespan extension effect of FUdR depends on fertility and the presence of gonads. fem-3 mutants, which do not produce oocytes, had increased lifespan in the presence of FUdR, independent of the exo-3 mutation. It is possible that the fem-3 mutant was susceptible to the lifespan extension effect of FUdR. From these results, we suggest that FUdR affects the lifespan of C. elegans in two ways: by interfering with fertility, which extends lifespan, and by inducing DNA base damage, which reduces lifespan.

INTRODUCTION 5-fluorouracil (5-FU) is an antimetabolite drug used in cancer treatment (Longley et al., 2003;Kim et al., 2009).It induces base damage by being incorporated into DNA and RNA (McNeill et al., 2009).5-fluoro-2′deoxyuridine (FUdR) is a metabolite of 5-FU.It binds to thymidylate synthase and strongly inhibits its activity.Thymidylate synthase is an enzyme that converts dUMP to dTMP.This is the sole synthetic pathway for thymine in the cell.Therefore, DNA replication and cell growth are strongly inhibited in the presence of 5-FU and FUdR.In addition, FUdR and 5-FU treatment increase uracil misincorporation into DNA, because inhibition of thymidylate synthase causes accumulation of dUMP.
DNA base damage such as 5-FU and uracil is mainly processed by base excision repair (BER).In this pathway, DNA glycosylases recognize damaged bases in DNA, and excise them by cleaving the N-glycosidic bond to produce an apurinic/apyrimidinic (AP) site.AP endonuclease then incises DNA at the AP site, and the resulting singlestrand break (SSB) is resynthesized by DNA polymerase and DNA ligase (Dalhus et al., 2009).Because misincorporated 5-FU and uracil are converted to AP sites by DNA glycosylases, the number of AP sites is also increased by treatment with 5-FU (McNeill et al., 2009).In the nematode Caenorhabditis elegans, AP endonucleases and mismatch repair process 5-FU to induce checkpoint-mediated autophagy (SenGupta et al., 2013).
FUdR is widely used when measuring the lifespan of C. elegans to suppress worm reproduction and the growth of progeny (Mitchell et al., 1979).It is believed that FUdR does not affect lifespan or cause severe side effects in adult worms because somatic cell division does not occur 202 Y. KATO et al. in the adult stage (Sulston and Horvitz, 1977).FUdR significantly reduces the time and effort required to measure lifespan.However, recent studies have revealed that FUdR extends the lifespan of some mutant worms, including tub-1 and gas-1 mutants, but the mechanism has not been elucidated (Aitlhadj and Stürzenbaum, 2010;Van Raamsdonk and Hekimi, 2011).In addition, it was reported that inhibition of DNA synthesis by FUdR improves protein homeostasis and increases stress resistance, extending healthspan via a fertility pathway that regulates sexual development (Angeli et al., 2013).Therefore, studies on the side effects of FUdR on worm lifespan are needed.
Here, we aimed to verify the effect of FUdR on lifespan from the viewpoint of DNA damage.Because 5-FU induces DNA base damage (McNeill et al., 2009), we first hypothesized that FUdR adversely affects the lifespan of BER gene mutants.To date, FUdR has not been shown to induce DNA damage in C. elegans.We previously reported that deletion of the exo-3 gene, but not the apn-1 gene, results in decreased lifespan in the absence of FUdR in a ung-1-dependent manner (Kato et al., 2015).EXO-3 and APN-1 are AP endonucleases (Shatilla et al., 2005;Schlotterer et al., 2010;Zakaria et al., 2010;Yang et al., 2012), and UNG-1 is a uracil DNA glycosylase in C. elegans (Nakamura et al., 2008).In this study, we measured the lifespan of the exo-3 mutant in the presence of FUdR, and found that FUdR extended the lifespan of the mutant.To investigate the mechanism of lifespan extension by FUdR, we conducted lifespan analyses using germline mutants.We focused on fem-1, glp-1 and fem-3 as genes involved in fertility (Nelson et al., 1978;Barton et al., 1987;Priess et al., 1987).In fem-1 mutant worms, which do not produce sperm, lifespan extension by FUdR was not observed.In glp-1 mutant worms, which do not develop gonads, the exo-3 mutant was not short-lived, and FUdR treatment did not result in lifespan extension.FUdR extended the lifespan of fem-3 mutant worms (which do not produce oocytes) independent of the exo-3 mutation.We suggest that there are both positive and negative effects of FUdR on worm lifespan.
Lifespan measurement To synchronize the worms, embryos were prepared by the standard alkaline bleach method, and eggs were hatched overnight in M9 buffer without food.L1 worms were grown at 25 °C for 2.5 days.Adult worms were transferred to an NGM plate containing 50 μM FUdR, and their lifespan was measured by transferring worms daily to fresh plates using a platinum pick.As it was assumed that the worms were damaged by picking, we conducted all measurements at 25 °C to finish the experiments in a short period.Worms that did not respond to touch were counted as dead (Kato et al., 2015).Mean lifespans were calculated from the results of three independent experiments, and are presented as means ± SD in figures.For statistical analysis, Student's t-test was carried out, and data were considered significantly different when the P value was < 0.05.

RESULTS
FUdR extends the lifespan of the exo-3 mutant worm To examine whether FUdR affected the lifespan of C. elegans via DNA damage induction, we measured the lifespan of exo-3 mutant worms in the presence and absence of FUdR.We found that the lifespan of shortlived exo-3 mutants was restored, and indeed extended, by FUdR (Fig. 1, A and B).FUdR increased the mean lifespan of exo-3 mutants by 50% (in the absence and presence of FUdR, the mean lifespan of exo-3 mutants was 9.73 days and 14.52 days, respectively, and the P value from a t-test was < 0.05) (Fig. 1C).Furthermore, exo-3 mutants had a significantly longer lifespan than N2 worms on FUdR-containing plates (mean lifespan was 14.52 days and 10.74 days, respectively, and the P value from the t-test was < 0.05).

DISCUSSION
FUdR as a DNA damage inducer in C. elegans Whether FUdR induces DNA base damage in C. elegans is not known, and further investigation is needed to reveal if FUdR affects worm lifespan via induction of DNA base damage.Contrary to our initial expectation, FUdR extended the lifespan of the exo-3 mutant (Fig. 1B).This result suggests that FUdR affects worm lifespan via the BER pathway.Our result conflicts with a previous report that 5-FU induces base damage (McNeill et al., 2009).We measured the level of AP sites in C. elegans by an aldehyde reactive probe assay, but found no significant increase in AP sites caused by FUdR treatment (data not shown).Inhibition of DNA synthesis and induction of base damage are conflicting effects of FUdR.It is possible that in the presence of a higher concentration of FUdR (for example, 50 μM in this study), DNA synthesis is strongly inhibited so that misincorporation of 5-FU and uracil rarely occurs.Lower concentrations of FUdR might induce base damage in C. elegans.It has been reported that lower concentrations of 5-FU ( < 5 μM) induce AP endonuclease-and mismatch repair-mediated autophagy in C. elegans (SenGupta et al., 2013).Another possible reason why we did not detect any difference in the number of AP sites following FUdR treatment is that FUdR-mediated DNA damage is dependent on DNA replication.In adult worms, only germline cells are dividing (Sulston and Horvitz, 1977).Since an involvement of fem-1, glp-1 and fem-3 in base damage has not been reported, we speculate that the sensitivities of these mutants to FUdR-induced base damage are nearly identical.
Effect of reproductive inhibition on lifespan In C. elegans, it is recognized that lifespan is related to reproduction.For example, killing the germline precursor cells Z2 and Z3 in larvae increases longevity (Arantes-Oliveira et al., 2002).The glp-1 mutant, in which the formation of gonads is prevented, also lives longer (Berman and Kenyon, 2006).We previously reported that deletion of the exo-3 gene causes abnormality in reproduction, including decreased self-brood size (Kato et al., 2015).In this study, we found that the exo-3 mutant has a short lifespan dependent on production of oocytes and gonads.When worms lacked oocytes (fem-3;exo-3) or gonads (glp-1;exo-3), exo-3 mutants were not short-lived (Figs.3A and  4A).These results suggest that deletion of the exo-3 gene caused decreased lifespan via reproduction (especially production of oocytes).Lifespan extension by FUdR was not observed when worms were deficient in sperm or gonads (Figs. 2B and 3B).This suggests that lifespan extension by FUdR was related to worm reproduction and fertility.Because FUdR extended the lifespan of oocyte-less fem-3 mutants independently of mutations in exo-3 (Fig. 4B), it is possible that fem-3 mutants were susceptible to the lifespan extension effect of FUdR.FUdR is very useful when measuring worm lifespan.However, it may not be suitable for measuring the lifespan of mutants that show abnormal reproduction.

FUdR affects lifespan in two ways
In this study, we suggest that deletion of the exo-3 gene caused a short lifespan via the fertility pathway, and that lifespan is restored by inhibiting oocyte production or gonad development.However, in the presence of FUdR, the lifespan of exo-3 mutants was increased, not just restored, compared with N2 worms (Fig. 1B).This phenomenon cannot be explained by the hypothesis that FUdR extends the lifespan of exo-3 mutants by interfering with fertility.Taking DNA base damage induction by FUdR into account might explain this phenomenon.We hypothesize that FUdR affects lifespan in two opposing ways (Fig. 5).By interfering with fertility in a specific manner, FUdR extends worm lifespan.However, FUdR also reduces worm lifespan by inducing DNA base damage, resulting in AP sites and SSBs.In N2 worms, it seems that FUdR does not affect lifespan because these two effects are almost equal and counteract each other.In exo-3 mutant worms, AP sites are not efficiently converted into SSBs.SSBs are more harmful than DNA base damage and AP sites in the short term, and therefore deletion of the exo-3 gene is advantageous for survival in the presence of FUdR.FUdR extends the lifespan of exo-3 mutants because exo-3 mutants were barely affected by the negative aspects of FUdR treatment compared with the N2 worms.FUdR may only influence specific aspects of nematode fertility.In worms lacking fem-1 function, these aspects may still be present, which explains the slight decrease in lifespan observed in fem-1 mutant worms treated with FUdR (Fig. 2C).The glp-1 mutant tested does not possess gonads; therefore, lifespans were affected by neither the exo-3 mutation nor FUdR (Fig. 3C).It is possible that the fem-3 mutant was susceptible to the lifespan extension effect of FUdR because the mutant was long-lived independent of the exo-3 mutation (Fig. 4C).

Fig. 1 .
Fig. 1.Lifespan of the exo-3 mutant in the presence of FUdR.(A and B) Survival plots of exo-3 (tm4374) mutants in the absence (A) and presence (B) of 50 μM FUdR at 25 °C.Worms were synchronized at the L1 stage and grown at 25 °C.Adult worms were placed on normal NGM plates, or NGM plates containing 50 μM FUdR, and transferred to a fresh plate daily.(C) Mean lifespan of the exo-3 mutant.Data shown are the mean ± SD from three independent experiments.* denotes a significant difference (P < 0.05) as determined by t-test.

Fig. 2 .
Fig.2.Lifespan of fem-1 mutants in the presence of FUdR.(A and B) Survival plots of fem-1 (hc17) mutants in the absence (A) and presence (B) of 50 μM FUdR at 25 °C.When grown at 25 °C, fem-1 (hc17) mutants do not produce sperm.To measure lifespan, worms were synchronized at the L1 stage and grown at 25 °C.Adult worms were placed on normal NGM plates, or NGM plates containing 50 μM FUdR, and transferred to a fresh plate every day.(C) Mean lifespan of the fem-1 mutants.Data shown are the mean ± SD from three independent experiments.n.s., not significant.

Fig. 3 .
Fig.3.Lifespan of glp-1 mutants in the presence of FUdR.(A and B) Survival plots of glp-1 (e2141) mutants in the absence (A) and presence (B) of 50 μM FUdR at 25 °C.When grown at 25 °C, glp-1 (e2141) mutants do not develop gonads.To measure lifespan, worms were synchronized at the L1 stage and grown at 25 °C.Adult worms were placed on normal NGM plates, or NGM plates containing 50 μM FUdR, and transferred to a fresh plate every day.(C) Mean lifespan of glp-1 mutants.Data shown are the mean ± SD from three independent experiments.n.s., not significant.

Fig. 4 .
Fig. 4. Lifespan of fem-3 mutants in the presence of FUdR.(A and B) Survival plots of fem-3 (q20) mutants in the absence (A) and presence (B) of 50 μM FUdR at 25 °C.The fem-3 mutant does not produce oocytes when grown at 25 °C.To measure lifespan, worms were synchronized at the L1 stage and grown at 25 °C.Adult worms were placed on normal NGM plates, or NGM plates containing 50 μM FUdR, and transferred to a fresh plate every day.(C) Mean lifespan of the fem-3 mutants.Data shown are the mean ± SD from three independent experiments.* denotes a significant difference (P < 0.05) as determined by t-test; n.s., not significant.

FUdRFig. 5 .
Fig.5.Model of lifespan extension/reduction by FUdR.FUdR inhibits DNA replication by binding to thymidylate synthase.This interferes with fertility, and ultimately results in lifespan extension.FUdR also induces base damage including 5-FU and uracil.These are converted to AP sites by DNA glycosylase, and the AP sites are then processed by EXO-3.As SSBs are more harmful than base damage and AP sites, this pathway causes lifespan reduction in an exo-3-dependent manner.Thus, FUdR affects lifespan of C. elegans in two different ways, and lifespan is determined by the proportion of these two effects in the presence of FUdR.