Differential effects of genotoxic stress on both concurrent body growth and gradual senescence in...

Aging Cell

(2007)

6

, pp209–224 Doi: 10.1111/j.1474-9726.2007.00278.x

© 2007 The Authors

209

Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007

Blackwell Publishing Ltd

Differential effects of genotoxic stress on both concurrent body growth and gradual senescence in the adult zebrafish

Stephanie B. Tsai,

1,2

Valter Tucci,

3

Junzo Uchiyama,

1

Niora J. Fabian,

1,4

Mao C. Lin,

1

Peter E. Bayliss,

1

Donna S. Neuberg,

5

Irina V. Zhdanova

3

and Shuji Kishi

1,6

1

Department of Cancer Biology, Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, MA, USA

2

Division of Graduate Medical Sciences, Boston University School of Medicine, and

3

Department of Anatomy and Neurobiology, Boston University School of Medicine, Boston, MA, USA

4

Department of Biology, College of Arts and Science, Boston University, Boston, MA, USA

5

Department of Biostatistical Science, Dana-Farber Cancer Institute and Department of Biostatistics, Harvard School of Public Health, Boston, MA, USA

6

Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA

Summary

Among vertebrates, fish and mammals show intriguingdifferences in their growth control properties with age.The potential for unlimited or indeterminate growth in avariety of fish species has prompted many questionsregarding the senescent phenomena that appear duringthe aging process in these animals. Using zebrafish as ourmodel system, we have attempted in our current studyto examine the growth phenomena in fish in relation tothe onset of senescence-associated symptoms, and toevaluate the effects of genotoxic stress on these pro-cesses. We observed in the course of these analyses thatthe zebrafish undergoes continuous growth, irrespectiveof age, past the point of sexual maturation with graduallydecreasing growth rates at later stages. Animal popula-tion density, current body size and chronological age alsoplay predominant roles in regulating zebrafish growthand all inversely influence the growth rate. Interestingly,the induction of genotoxic stress by exposure to ionizingradiation (IR) did not adversely affect this body growthability in zebrafish. However, IR was found to chronicallydebilitate the regeneration of amputated caudal fins andthereby induce high levels of abnormal fin regeneration

in the adult zebrafish. In addition, by resembling andmimicking the natural course of aging, IR treatments like-wise enhanced several other symptoms of senescence, suchas a decline in reproductive abilities, increased senescence-associated ββββ

-galactosidase activity and a reduction inmelatonin secretion. Our current data thus suggest thatduring the lifespan of zebrafish, the onset of senescence-associated symptoms occurs in parallel with continuousgrowth throughout mid-adulthood. Moreover, our presentfindings indicate that genotoxic DNA damage may playa role as a rate-limiting factor during the induction ofsenescence, but not in the inhibition of continuous,density-dependent growth in adult zebrafish.Key words: aging, genotoxic stress, growth, melatonin,regeneration, reproduction, senescence, zebrafish.

Introduction

The elucidation of the genetic mechanisms regulating growth,

aging and senescence will greatly enhance our understanding

of some of the most fundamental properties of higher organisms.

Animals display remarkable variability in growth and size,

both within and between species. Among vertebrates, fish and

mammal species show intriguing differences in their growth

control properties during the aging process (Finch, 1990; Patnaik

et al

., 1994; Armstrong & Smith, 2001), with certain fish species

maintaining unlimited or indeterminate growth potential

(Fine

et al

., 1984; Finch, 1990; Patnaik

et al

., 1994; Reznick

et al

.,

2002). This raises the important question of how senescence

phenomena are manifested in these organisms during the aging

process when the animals themselves keep growing. Several

lines of observation now suggest that body growth and the

eventual body size of many teleost fish species largely depends

upon the population density of these animals (Lorenzen &

Enberg, 2002). Hence, in high-density environments, even

with adequate levels of nourishment, older fish remain small.

However, once smaller fish of any age are introduced into a

less dense living space, they appear to have the capability to

undergo further growth to larger body sizes. Thus, one might

speculate that the effect of population density on growth might

also impact the organismal aging process and the onset of

senescence-associated symptoms.

Body growth and its related senescence-associated processes

need to be observed and characterized in any usable animal

model system as they are fundamental processes in higher

organisms. To address still-unanswered questions on growth and

aging, we adopted the zebrafish (

Danio rerio

) model system.

Correspondence

Shuji Kishi, MD, PhD, Schepens Eye Research Institute, Department of

Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston,

MA 02114, USA. Tel.: 617 912 0200; fax: 617 912 0101;

e-mail: [email protected]

Accepted for publication

14 December 2006

Genotoxic stress and zebrafish aging, S. B. Tsai

et al.

© 2007 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007

210

Zebrafish are teleosts of the cyprinid family the class of

ray-finned fish and have emerged in recent years as a powerful

animal model in biomedical science and developmental biology,

providing a robust and tractable genetic system in which to

investigate biological mechanisms. However, few studies to

date have investigated the gerontology of this organism, which

has great potential to give insight into organismal aging and

associated diseases common to vertebrates (Gerhard

et al

.,

2002; Kishi

et al

., 2003; Keller & Murtha, 2004; Kishi, 2004,

2006). Future studies that uncover the fundamental timing of

both growth and senescence can take advantage of the genetic

approaches and fundamental genomics that are possible in

zebrafish. Moreover, rapid increases in zebrafish resources will

greatly assist such studies, but will necessitate core aging research

in this animal such as that undertaken in our current report.

The principal goals of this study were therefore aimed at elucidat-

ing the characteristics of growth and size control in zebrafish

during the aging process to elucidate baseline information on

normal senescence phenotype sequelae. We utilized fish of

approximately young- and mid-adult age to examine the middle

segment of zebrafish lifespan, prior to the occurrence of stochastic

senescent changes in older fish but presumably at their onset.

We followed various senescence-associated processes in our

experiments that pertained to growth, repair and maintenance,

and fitness, and also those that might be affected by age or

other senescence-inducing stresses, such as genotoxic stress.

It has been shown previously that zebrafish retain a remark-

able regenerative capability in several tissues (Poss

et al

., 2002b;

Keating, 2004). Fin regeneration occurs via a process involving

blastema formation and an intricate interplay and exchange

between epithelial and mesenchymal cells. This elaborate

process requires careful regulation and control of the inter-

changes, dedifferentiation and differentiation of the cells involved

(Akimenko

et al

., 2003). The caudal fin of the zebrafish normally

executes partial regeneration at 1 week post-amputation and

the general shape and morphology of the original fin is

largely reconstituted. This is followed by complete regeneration

generally within 2–3 weeks in young adult zebrafish. Hence, an

age-dependent decline in such a regenerative capability may

be a hallmark of senescence and we chose this process for our

initial experimental observations. In addition, many different

vertebrates in general demonstrate a reproductive schedule that

peaks in young adults and then declines with increasing age,

either due directly to a deterioration in body condition or in-

directly to a diminished capacity to fight other competing suitors

(Bercovitch

et al

., 2003; Ricklefs

et al

., 2003). Several studies have

also documented the negative effects of IR exposure on repro-

ductive functions (O’Farrell

et al

., 1972; Erickson & Martin, 1984;

Jagetia & Krishnamurthy, 1995; Neel, 1999). We therefore also

assessed the reproductive schedule in our current zebrafish studies.

Senescence-associated

β

-galactosidase (SA-

β

-gal) staining

was previously described as an assay tool to detect senescent

cells

in vivo

and

in vitro

(Dimri

et al

., 1995)

.

This method has

since become the most popular biomarker for cellular and even

organismal senescence, although criticisms have arisen regarding

the biological and biochemical specificity of this stain (Yegorov

et al

., 1998; Krishna

et al

., 1999; Severino

et al

., 2000; Cao

et al

.,

2003; Genade

et al

., 2005; Keyes

et al

., 2005; Yang & Hu, 2005;

Herbig

et al

., 2006; Kishi, 2006). We have, however, successfully

employed this method in our former study in which we verified

age-related increases in zebrafish muscles showing positive

SA-

β

-gal staining alongside age-related oxidative protein accu-

mulation (Kishi

et al

., 2003). Moreover, stress-induced increases

in SA-

β

-gal staining have also been demonstrated in cultured

cells upon exposure to oxidative or genotoxic stress (Toussaint

et al

., 2000; Kishi & Lu, 2002), suggesting that SA-

β

-gal staining

may also indicate stress-induced senescence. We thus anticipated

that it would be instructive to employ SA-

β

-gal staining in our

current study. Finally, melatonin is a hormone for which the

circulating levels decline with age in several species (Waldhauser

et al

., 1998; Lahiri

et al

., 2004; Paredes

et al

., 2006). Moreover,

melatonin has been found to act as a free radical scavenger,

an antioxidant and an anti-apoptotic agent (Reiter

et al

., 1994).

In zebrafish, melatonin has also been closely and specifically

linked with the circadian cycle (Cahill, 2002), the promotion of

the sleep-like state (Zhdanova

et al

., 2001), and with increasing

cell proliferation and the accelerated development of embryos

(Danilova

et al

., 2004). Since melatonin is a hormone modulated

by growth, aging, and genotoxic stress, its detection may there-

fore reflect a senescence-associated trait in zebrafish.

Growth and senescence are under strict regulation during

the aging process in higher organisms. Cell size and cell number

directly determine total body mass, and numerous crucial signal

transduction pathways regulate and specify the numbers of

cells that form tissues and organs (Zimmerman & Lowery, 1999;

Mommsen, 2001; Johansen & Overturf, 2005). Genotoxic stress

induced by ionizing radiation (IR), resulting in a DNA damage

response, affects cell cycle and cell division, and therefore sub-

sequently impacts upon early embryonic developmental events

or even induces acute adult lethality and an aberrant histology

in zebrafish (Langheinrich

et al

., 2002; Jarvis & Knowles, 2003;

Traver

et al

., 2004; Bladen

et al

., 2005; Imamura & Kishi, 2005;

McAleer

et al

., 2005). IR has also been shown to ablate hemat-

opoietic cells and cause death of spermatocyte precursors (Traver

et al

., 2004). Although lifespan studies in the medaka (

Oryziaslatipes

) have investigated the presence or absence of IR (Egami

& Etoh, 1969; Egami, 1971; Egami & Eto, 1973), little is known

about the genotoxic effects upon growth and aging in adult

zebrafish. Therefore, we sought to evaluate the effects of

genotoxic stress caused by IR on growth properties and on the

senescence-associated processes described above in zebrafish.

Our multifaceted study presented herein combines various

independent experiments that target all of the specific aspects

of aging described above on two age groups of zebrafish that have

been divided into control and IR-exposed groups. We found

zebrafish growth to be effectively and inversely governed by the

population density of the animals in their living environment and

by their current body size and chronological age. The growth in

these animals was also found to continue indefinitely past sexual

maturation within the timeframe of this study (mid-adult age


et al.


211

of about 3 years old). Thus, older small fish retain a comparable

growth capability compared to younger small fish, but with

gradually decreasing growth rates at later stages. Intriguingly,

we did not observe any adverse effects of IR exposure on body

growth ability in adult zebrafish. However, further analyses

found that over the long-term, IR treatment did induce abnor-

mal regeneration of the caudal fin after amputation and did

enhance other senescence-associated markers such as a reduc-

tion in reproductive ability, increase in SA-

β

-gal activity, and

lower levels of melatonin secretion.

Our present data thus indicate that the onset of senescence-

associated symptoms in zebrafish occurs in parallel with continuous

growth, at least throughout the mid-adult ages as studied here.

Moreover, we show that genotoxic DNA damage is an important

rate-limiting factor in the induction of senescence, but not in

the cessation of continuous growth control in the adult fish.

Results

Density-dependent growth and size control in the adult zebrafish

To validate the density-dependent size control phenomenon in

adult zebrafish, we established 61 tanks (2.75 L capacity each)

containing fish population densities ranging from 15 to 63

wild-type fish per tank. The ages of these fish ranged from 6

to 13 months and these animals had been raised and maintained

under assigned density conditions since the embryo stages. We

measured the body sizes (from the tip of the mouth to the end

of the body wall muscle) of all 2285 fish in our cohort of tanks

at the beginning of the experiments, and of all remaining fish

(55 tanks) again 15 months later with ages varying from 21 to

28 months at that time. It has been reported previously that

zebrafish exhibit spinal curvature with age (Gerhard

et al

.,

2002), but using younger fish at the start of this study, we did

not observe any notable body curvature over time so that our

body size measurements were valid.

For the 15-month period during which we allowed fish

growth, we ensured proper and adequate distribution of food

in proportion to fish population density in each tank, and we

also ensured optimal water circulation and quality conditions

for all tanks. At the beginning of the study, a scatter plot of

the initial body size measurements was generated and demon-

strated a strong inverse correlation (Spearman’s rank correlation;

r

=

−

0.817,

P

< 0.0001) between population density and the

average body size of the fish in each tank (Fig. 1A). Moreover,

a further plot demonstrated no significant correlation (Spearman’s

rank correlation;

r

=

−

0.182;

P

= 0.18) between chronological

Fig. 1 Relationship between body size and tank density. (A) At the beginning of the study, 2285 fish subjects exhibited a strong inverse correlation between the number of fish (density) and average body size per tank. (B) At the end of the study (15 months later), the remaining tanks still showed a strong inverse correlation between tank density and average body size per tank. (C) This density dependence occurred regardless of chronological age at the beginning of the study. (D) At the end of the study (15 months later), the data exhibited a negative correlation between chronological age and growth. (Only 55 tanks were used in the second measurement, six tanks were retained for use in other experiments.)


et al.


212

age and average body size (Fig. 1C). Upon our second measurement

of all remaining fish one year later, we again observed a

strong inverse correlation (Spearman’s rank correlation;

r

=

−

0.927;

P

< 0.0001) between body size and population

density (Fig. 1B). However, we also observed a significant

inverse correlation (Spearman’s rank correlation;

r

=

−

0.495;

P

< 0.0001) between body size and chronological age in these

older fish (Fig. 1D). In order to determine the significance of this

and also to control for the influence upon these measurements

of the strong association between density and size at the

beginning, we opted to analyze the percentage growth values.

We again found an inverse correlation with age (Spearman’s rank

correlation;

r

=

−

0.604;

P

= 0.006) (data not shown), indicating

that older fish grow at slower rates and reach smaller eventual

sizes, even when compared to similarly sized younger fish.

To provide a more complete picture of the percentage growth

and employ all of the possible factors involved, we next performed

statistical modeling. The results of these analyses indicated that

the average body size at the start of the study (Wald test;

P

< 0.0001), the number of fish at the start of the study (Wald test;

P

< 0.0001), the percentage loss in number of fish over the course

of these experiments (Wald test;

P

< 0.0001), and the ages of

the fish (Wald test;

P

= 0.0015), significantly influenced the

percentage growth over the duration of this study as follows:

(Percentage growth) = 118.589

−

3.532*(Size at start)

−

0.53*(Fish number at start)

−

0.646*(Age) + 0.237*(Percentage fish loss)

This model explains 80% of the variability in the percentage

loss of fish over the course of these analyses and also illustrates

the negative influences of (i) beginning size, (ii) number of fish

in tank at the beginning, and (iii) age at the beginning with

size in addition to a positive influence of percentage loss on

percentage growth of these fish. These data thus support the

notion that density conditions strongly and inversely govern the

eventual body size of the zebrafish, but that the initial body size

and chronological age also exerts an inverse effect.

The effects of chronological age on growth ability as the ‘biological age’ of the adult zebrafish

We next assessed whether chronological age might have any

discernible effect on the ability of the adult zebrafish to grow

(i.e. ‘biological age’) by analyzing the growth characteristics of

relatively small fish of different ages when moved from condi-

tions of higher to lower population density. We compared fish

of two different chronological ages, 6 and 18 months, which

we designated as ‘young-adult’ and ‘mid-adult’, respectively

(this is based upon our observation that the wild-type AB strain

of fish maintained in our facility seem to live for approximately

3 years on average). All of these fish had been raised from birth

under identical population density conditions (48 fish per 2.75-

L tank). At the start of the experiment, the mid-adult fish were

relatively small due to their upbringing in a high animal density

environment, in comparison with same aged fish in less dense

tanks. However, these mid-adult fish were still somewhat

larger than the young-adult fish (21.25

±

1.712 mm vs. 19.75

±

0.937 mm) from the same population densities. These differ-

ences in size were probably due to the general variance that

would be inherent in fish size, as well as due to the young-adult

fish not having had adequate time to reach their potential size

in the particular density conditions.

We separated these fish into four 2.75-L tanks containing six

fish from one age group (two tanks for each group), to give

them adequate space to grow continuously. Over the course

of 47 weeks, the mid-adult fish exhibited similarly shaped

growth curves, plotting actual body size vs. time, compared to

their younger counterparts (Fig. 2A). Moreover, there were no

significant differences in the actual weekly changes between

Fig. 2 Relationship between chronological age and body growth. (A) The propensity of the zebrafish to grow also depended on their initial size with small influences of chronological age. However, young-adult (6 months of the started age) and mid-adult (18 months of the started age) fish exhibited similar patterns of growth, shown by their actual size at each measurement. The young-adult fish began at smaller sizes, but were similar in size to the mid-adult fish by the end of the study (week 47), supporting the notion that younger fish grow at higher rates, although older fish still exhibit nonstop growth, albeit at a slower rate. (B) Young-adult and mid-adult fish exhibited similar changes of growth from week-to-week.


et al.


213

these two age groups (Fig. 2B). These data suggest that while

the rate of growth may decrease with increased size, growth does

not cease through the mid-adult stage of life (Fig. 2), consistent

with the definition of indeterminate growth in teleosts.

The effects of genotoxic stress on survival and body growth in adult zebrafish

We next sought to observe the growth properties of adult

zebrafish under conditions of genotoxic stress. The 24 young-

and mid-adult fish described in the previous section served as

controls. We then set up another two groups of 12 fish from

the same two clutches used for the controls in an identical tank

configuration (two 2.75-L tanks each with six young-adult fish

and two 2.75-L tanks each with six mid-adult fish). These fish

were irradiated with 20 Gy IR, and both the IR and control

fish were observed for 12 months. At the end of this observa-

tion period, the young-adult fish had identical survival rates

between the control and IR groups, 10/12 (83.33%) (data not

shown), whereas in the case of the mid-adult fish, the IR exposed

groups demonstrated a lower survival rate, 5/12 (41.67%) than

that of the controls, 9/12 (75%). In addition, mid-adult IR fish

exhibited higher mortality levels than young-adult IR fish, 5/12

(41.67%) versus 10/12 (83.33%), respectively (Fisher’s exact

test;

P

= 0.003) (data not shown). Deceased fish were also found

to be healthy up until the point of death, and there was no evidence

of wasting or chronic sickness in terms of the gross morphology

of these animals, although necropsies were not performed.

This suggests a possible age-dependent effect of IR that may

render older zebrafish physiologically more susceptible to the

effects of genotoxicity and thus confer a higher mortality rate.

Focusing on growth, the mean body sizes of the un-irradiated

controls of both age groups did not differ significantly from their

respective irradiated fish at the beginning of the experiment,

just prior to the administration of IR. At the end of the study

(12 months post-IR), however, the average sizes of both the

Fig. 3 The effects of ionizing radiation (IR) upon body growth. (A, B) Within both the mid-adult and young-adult fish groups, the control and irradiated fish began at similar sizes, but irradiated fish surpassed their control counterparts in average size. (C, D) Within both mid-adult and young-adult fish groups, irradiated and control fish demonstrated similar growth patterns throughout the duration of the study.


et al.


214

young- and mid-adult IR fish exceeded those of the respective

control fish (Fig. 3A,B). However, statistical analysis could not

confirm a significant effect of IR on growth, possibly due to

small subject numbers. Nonetheless, neither age nor IR exposure

inhibited growth, since we observed that both the young- and

mid-adult IR fish continued to grow throughout the course of

the study. In addition, there were no significant differences in the

actual weekly changes between these two age groups (Fig. 3C,D)

The effects of genotoxic stress on caudal fin regeneration in adult zebrafish

Fin regeneration involves highly orchestrated processes includ-

ing wound healing, establishment of the wound epithelium,

recruitment of the blastema from mesenchymal cells underlying

the wound epithelium, and differentiation and outgrowth of

the regenerated cells (Poleo

et al

., 2001). We anticipated that

such regenerative ability might decline both with age as well

as with genotoxic stress in zebrafish and conducted a number

of experiments to test this.

We first examined the effects of IR exposure alone on fin

regeneration over the long term. We used 55 healthy control

fish and 87 fish that had been irradiated with 20 Gy of IR 8

months prior to the start of this experiment. All of these fish

were 10 months old and had been propagated from the same

clutch. At this time point (8 months post-irradiation), we ampu-

tated the caudal fins of each animal. Two weeks later, we anes-

thetized each fish and photographed and classified the status

of tail regeneration. We utilized the following classifications: A:

complete regeneration; B: somewhat impaired regeneration;

and C: severely impaired or no regeneration (Fig. 4A–C). Com-

plete regeneration, which is the expected outcome under normal

situations, signifies fins with complete morphological regener-

ation, or a fin shape that is similar/identical to the original fin

and harboring a stripe pattern and coloration complete or near

complete (Fig. 4A). Somewhat impaired fins show incomplete

morphological regeneration with a remaining distorted fin shape

(Fig. 4B). We reserved our classification of severely impaired/no

regeneration for fins that still demonstrated grossly distorted fin

shapes or no regeneration of any kind (Fig. 4C).

We found from our analyses that the control (un-irradiated)

fish exhibited a regenerative distribution skewed toward the

A (53%;

n

= 29) and B (45%;

n

= 25) categories (Fig. 4D). Only

2% of these controls (

n

= 1) showed severely impaired regen-

eration (category C). In contrast, the regenerative distribution

of the irradiated fish differed remarkably from the controls

(Cochran–Mantel–Haenszel test;

P

< 0.0001) (Fig. 4D), with

70% (

n

= 61) of the irradiated fish being assigned to the B

category, and 15% (

n

= 13) classified in each of the A and C

categories. Thus, even at 8 months post-irradiation, IR exposure

appeared to impair fin regeneration capacity. When separating

males and females in this analysis, females appeared to exhibit

poorer regeneration following irradiation (Fisher’s exact test,

P

-value = 0.0004), in comparison with males (Fisher’s exact test,

P

-value of 0.02). However, we were unable to irradiate equal

percentages of females and males, 54% and 46%, respectively,

which may have influenced the sex-separated findings.

To examine the effects of chronological age alone, as well

as in conjunction with IR, we again utilized the fish from the

completed growth studies mentioned above, and amputated

their fins (12 months post-irradiation). As shown in Table 1,

there were clear differences in the distribution of regeneration

between young- and mid-adult non-irradiated (control) fish.

None of the younger fish (0/9) were assigned to category C

(severely impaired), whereas 3/9 (33.3%) of the older fish fit

into this category. When examining the IR effects again in this

group of fish, we found that 5/9 (55.6%) of the younger con-

trols (0 Gy) and 1/10 (10.0%) of the 20-Gy-irradiated fish fit

category A (normal), whereas 0/9 (0%) and 3/10 (30.0%),

respectively, fit category C. Taken together, our results suggest

Fig. 4 Fin regeneration states and the effects of ionizing radiation (IR) and age upon fin regeneration. Representative images of caudal fin regeneration states. (A) During normal regeneration (within 2 weeks), the tail is morphologically reproduced in size and shape, but may still lack complete pigmentation. (B) In somewhat impaired regeneration, the shape remains distorted, particularly at the distal tail end. (C) Severely impaired fin regeneration results in segments along the incision that exhibit no regenerative capacity, as indicated by the small black arrows. Large black arrows in A, B and C indicate amputated regions of the caudal fins. (D) Within the same age group (10 months), most non-irradiated fish exhibit mostly normal or somewhat impaired regeneration, whereas IR fish exhibit a more bell-shaped distribution. For the IR exposure, fish were subjected to IR 8 month prior to the start of the regeneration experiments.


et al.


215

that negative age effects and negative IR effects occur, even at

12 months post-irradiation, upon fin regeneration.

Appearance of senescence phenotypes as a result of age progression and genotoxic stress in adult zebrafish

To examine the onset of senescence in zebrafish, both during

natural age progression and following irradiation, we examined

reproductive abilities, SA-β-gal staining levels in the skin and

melatonin levels in brain. All of these three aspects are known

to change during the aging process.

Effects of aging and IR on reproductive capabilities

To gauge the changes in reproductive ability in our fish subjects,

we performed two independent experiments. In the first of

these (Table 2), we focused upon the effects of chronological

aging with two differently aged groups of fish. Both the

younger group (7 months old) and the older group (20 months

old) comprised 32 fish (16 males and 16 females) which were

randomly mated within their own age groups, once every 1 to

2 weeks. The numbers of laid embryos were recorded at each

mating for a total of 15 trials over 4.5 months. We observed

that the younger fish consistently laid larger clutch sizes

(Wilcoxon test; P = 0.0009), independent of the duration of

mating period (Table 2). When the duration, as well as the

output per female, was factored into the analysis, the young

fish likewise showed higher efficiency (eggs laid per female per

hour of mating) in almost every trial (Wilcoxon test; P = 0.001)

(Table 2). When the duration, as well as the output per female,

was factored into the analysis, the young fish likewise showed

higher efficiency (eggs laid per female per hour of mating) in almost

every trial (Wilcoxon test; P = 0.001) (Table 2). However, the

percentage of surviving eggs at each mating did not differ between

the old and young fish (Wilcoxon test; P = 0.461) (Table 2).

In a separate experiment, we examined the effects of IR upon

fecundity in 14-month-old fish comprising 60 control (30 males

and 30 females) and 72 IR-treated (36 males and 36 females)

fish at 8 months post-irradiation. We mated control fish with

each other and irradiated fish with each other on five occasions

over a 1-month period and found a striking reduction in clutch

sizes of irradiated fish compared with control fish (Table 3).

After the third trial, the IR fish no longer laid any eggs and,

thus, irradiated fish consistently laid smaller and diminishing

clutch sizes and performed at lower reproductive efficiencies

than control fish in all trials. There were, however, not enough

trials to perform meaningful statistical analyses. Nonetheless,

these results suggest a chronic negative effect of irradiation on

reproductive capability. It is noteworthy also that there was no

significant difference in the percentage of surviving embryos at

each mating between IR and control fish (Table 3).

Table 1 Effect of ionizing radiation (IR) on fin regeneration in zebrafish of different chronological ages

Fin regeneration Normal (A) Somewhat impaired (B) Severely impaired (C)

IR (Gy) 0 20 0 20 0 20

Young-adult n 5 1 4 6 0 3

(n = 19) % 55.6 (5/9) 10.0 (1/10) 44.4 (4/9) 60.0 (6/10) 0.0 (0/9) 30.0 (3/10)

Mid-adult n 4 0 2 2 3 2

(n = 13) % 44.4 (4/9) 0.0 (0/4) 22.2 (2/9) 50.0 (2/4) 33.3 (3/9) 50.0 (2/4)

Total n 9 1 6 8 3 5

(n = 32) % 50.0 (9/18) 7.1 (1/14) 33.3 (6/18) 57.1 (8/14) 16.7 (3/18) 35.7 (5/14)

To observe fin regeneration following ionizing radiation (IR), 55 zebrafish were designated controls (unirradiated), and 87 fish were exposed to 20 Gy IR, 8 months prior to fin amputation. All fish were 10 months old, from the same clutch and kept under standard optimal conditions over the 8-month post-IR period. Each fish was then subjected to caudal fin amputation (approximately 50% of the tail fin cut longitudinally with a clean razor), and placed back into original tanks on circulation. At 2-weeks post-amputation, all fish were anaesthetized again to assess fin regeneration states. The fish were categorized according to whether fin regeneration appeared (A) normal, (B) somewhat (slightly) impaired, or (C) severely impaired, as shown in Fig. 4 (A–C).

Table 2 Effect of age on reproduction in zebrafish of different chronological ages

Total egg number Eggs surviving Percentage survival Reproductive efficiency

Young-adult

457 (min 121; max 814) 399 (min 109; max 711) 89.7 (min 68.1; max 100) 13.3 (min 3.89; max 21.8)median (range)

Mid-adult

170 (min 17; max 556) 126 (min 17; max 534) 91.4 (min 70.4; max 100) 3.9 (min 0.6; max 15.2)median (range)

The first reproductive study (Table 2) looked solely at chronological age, comparing mating success of young-adult and mid-adult fish groups. At each mating (a total of 15 matings over 4.5 months), each age group of fish was separated into two mating tanks (2 sets of 8:8/male:female) to ensure adequate space. The ranges of observations over all matings are indicated: minimum number (min) and the maximum number (max), for each parameter.

Genotoxic stress and zebrafish aging, S. B. Tsai et al.


216

Senescence-associated ββββ-galactosidase staining with age and IR

We next employed SA-β-gal staining to assay for senescent cells

at the cellular level in two independent experiments to look at

SA-β-gal staining in fish of differing ages. We were able to

quantitatively analyze the levels of SA-β-gal staining by gener-

ating high-resolution digital photographs, which enabled

selection and calculation of the percentage (staining intensity)

of stained pixels in each image. We observed significant differences

in the SA-β-gal intensities between 18-month- and 36-month-

old fish and between 26-month- and 31-month-old fish

(between both younger and respective older fish groups, Wil-

coxon test; P = 0.005) (Fig. 5A). Next, we again utilized the fish

of both age groups from the growth, reproduction and fin

regeneration experiments described above to assess SA-β-gal

staining levels. Mid-adult control fish consistently stained darker

than young-adult controls, and IR treatment significantly enhanced

SA-β-gal staining intensities in both mid- and young-adult fish

(Fig. 5B,C–F). Thus, IR and age both appear to induce higher

intensities of SA-β-gal staining in zebrafish.

Melatonin levels with age and IR

As part of our ongoing studies of age-related alterations in the

circadian rhythms in zebrafish, we performed another experi-

ment examining both the central (brain) and peripheral (muscle

tissue) melatonin levels in the middle of the dark period in fish

of three different ages and of both sexes. Comparison between

the melatonin levels in (i) youngest (12-month-old), (ii) middle-

aged (24-month-old), and (iii) oldest (38-month-old) zebrafish

showed progressive age-dependent declines in melatonin

levels in both brain (P < 0.001) and muscle tissue (P < 0.05)

(Fig. 6A,B). Gender comparisons showed no significant differ-

ences in melatonin levels. Additionally, we compared night-time

brain melatonin levels in adult zebrafish of 12 and 24 months

of age in an independent experiment incorporating genotoxic

stress. A group of fish from the same clutch as the younger

fish group (12 months old) was irradiated and we therefore

designated three groups of fish: (i) control (12 months old), (ii)

younger + IR (same age and clutch as control), and (iii) older

(24 months old). We found significantly decreased average

levels of melatonin in the older fish (P = 0.025). Irradiation

6-months prior to this in younger zebrafish (12-month-old) also

resulted in a dramatic reduction in melatonin levels, comparable

to those documented in older fish (P = 0.016) (Fig. 6C). These

results therefore indicate that reduced melatonin levels may

signify a senescence-associated phenotype resulting from natural

aging, as well as from genotoxic stress, in adult zebrafish.

Discussion

Our current data suggest a concomitant occurrence of con-

tinuous growth and a gradual onset of senescence-associated

symptoms during the aging process in zebrafish up to the

mid-adult ages studied here. Genotoxic stress, in the form of

IR exposure, also seemed to induce senescence-associated

characteristics in this model organism. However, while age pro-

gression and IR both appear to induce senescence-associated

symptoms, neither inhibited body growth, as fish of both ages

in this study continued to grow through at least the mid-adult

ages and long after IR treatment. This may imply an independ-

ent regulation of growth and senescence in zebrafish, similar

to that in other fish species exhibiting indeterminate growth

(Mommsen, 2001; Gerhard et al., 2002). Alternatively, specific

rate limiting factor(s), or thresholds for growth regulation may

trigger senescence induction. This will necessitate further

investigation of whether chronologically older small fish demon-

strate any symptoms of senescence occurring in parallel with a

persistent small size.

In our first broad look at growth and the onset of patterns of

senescence-associated characteristics in zebrafish, we conducted

a multifaceted experiment combining various independent

studies to target multiple biomarkers in each group of fish.

Due to the pleiotropic nature of the aging process, which sub-

sequently causes systemic catastrophe in organisms, we cannot

definitively characterize the state of senescence using only a few

traits. Hence, we considered several parameters commonly

linked to aging and related to common, fundamental functions

of living organisms including growth, homeostatic cellular main-

tenance of specific factors (SA-β-gal staining and melatonin

levels), wound healing and tissue repair (fin regeneration), and

reproductive fecundity. We then assessed these parameters in

the face of genotoxic stress by IR.

Table 3 Effect of ionizing radiation (IR) on reproduction in young-adult zebrafish

Total egg number Eggs surviving Percentage survival Reproductive efficiency

Control

1964 (min 1604; max 2187) 1725 (min 1415; max 1905) 87.8 (min 87.1; max 88.2) 19.2 (min 15.7; max 21.2)median (range)

Irradiated

median (range) 218 (min 66; max 886) 151 (min 54; max 722) 81.5 (min 69.3; max 81.8) 1.4 (min 0.5; max 6.7)

The second reproductive study (Table 3) looked at IR effects, comparing a control group (unirradiated) to an irradiated group of fish. Subjects (IR and control) were only mated three times over one month because the IR group ceased to mate successfully after the third mating, no longer producing any eggs. The control group consisted of 30 males and 30 females, who were placed into 10 mating cages containing 3 males and 3 females per cage at each mating. The IR group comprised 36 males and 36 females, who were divided into 12 mating cages containing 3 males and 3 females per cage at each mating. The ranges of observations are indicated as the minimum number (min) and the maximum number (max) for each parameter.



217

Growth properties of zebrafish

Most organisms exhibit determinate growth (i.e. a point at

which growth no longer occurs). However, indeterminate

growth may occur when it offers advantages in terms of fitness

or reproductive prowess (Reznick et al., 2002; Congdon et al.,2003). It has been hypothesized that zebrafish also possess

indeterminate growth with persistent proliferative capability of

cells in multiple tissues throughout life (Gerhard et al., 2002;

Kishi et al., 2003). Our current study supports this notion, as the

growth profiles of the fish followed over the duration of our

experiments never stopped, with small mid-adult fish retaining

their capability to grow continuously along with small young-

adult fish. This indicates similarities in ‘biological age’, in spite

of the differences in chronological age, and may represent an

evolutionary adaptation to compensate for onset of senescence-

associated symptoms with age, which we describe in following

sections. However, the oldest fish studied here were mid-adult

aged. This age confers the advantage of preceding the poten-

tially confounding effects of stochastic senescent changes at

later stages. On the other hand, further studies of older fish as

well as of gender differences will also be needed to provide

more definitive definitions of indeterminate growth in zebrafish.

Many factors inevitably play a pivotal role in modulating such

a complex process as growth regulation. Indeed, we observed

here that given adequate nourishment, the available living space

and/or population density conditions governed the final sizes

of the fish. Given that our fish showed an inverse relationship

between fish density and fish body size even at the beginning

of our experiment, we suspect that this effect is intrinsic, even

from the early larval age since our fish were raised in their

assigned density conditions from the larval and juvenile stages.

In any case, to adjust for any possible confounding effects of

the observed initial relationship, we looked at percentage growth

and likewise found a strong inverse relationship between

percentage growth and density. While the exact mechanisms

are unknown, we postulate that animal density may trigger the

inhibition of body mass growth and also affect other processes

by stimulating hypothetical ‘space’ sensors in zebrafish that

monitor the surroundings. This would be analogous to normal

cells that cease dividing in response to contact inhibition.

Alternatively, high-density conditions may stimulate some

form of communication between fish in close proximity to each

other, just as insects use pheromone release, although this

Fig. 5 Senescence-associated β-galactosidase (SA-β-gal) activity changes in the skins of adult fish with age and ionizing radiation (IR). (A) Two differently aged groups of fish were stained in two independent experiments. The older fish (31 or 36 months) in each experiment exhibited a greater percentage of positive SA-β-gal stained skin by pixel imaging than younger fish (18 or 26 months). Analysis was performed using high-resolution digital imagery, as shown in C–F. (B) In both young-adult (18 months) and mid-adult (30 months) fish, IR fish stained with a higher intensity of SA-β-gal than the control fish, potentially due to the induction of a greater number of senescent cells by irradiation. (C–F) Representative SA-β-gal staining in the skin is shown for adult fish with or without IR treatment. (C) Young-adult control fish (18 months) without IR (IR-). (D) Mid-adult control fish (30 months) without IR (IR-). (E) Young-adult irradiated fish (IR+ 20 Gy). (F) Mid-adult irradiated fish (IR+ 20 Gy). Relative intensities were scanned and processed by computer pixel analysis.



218

remains to be determined. We also found two other factors that

significantly governed final body size and growth. First, while

fish growth did not stop throughout the duration of our study,

we did find that older ages resulted in decreased rates of growth

when controlling for size, consistent with many processes that

are known to slow down with age in living organisms. Second,

we recognized an inverse relationship between fish current

body size and growth, with fish starting at larger sizes growing

at slower rates. This indicates that both age and body size can

affect growth propensity, along with density, and our statistical

modeling strongly supported these relationships.

Given animal density’s profound ability to modulate growth,

future studies might also look at the effects of density on

numerous other growth-related or senescence-related symp-

toms, such as the weight of fish, diminished reproductive

functions and gender differences, regenerative capabilities, and

others. As a caveat here, we calculated densities based upon

the number of fish per tank volume. While this serves as a

reasonable rough measure of density conditions, it may be

productive in the future to calculate density based on even

more descriptive measures, such as weight or even body mass

index. Establishing the density-dependence of zebrafish growth

allowed us, however, to execute subsequent experiments in this

study. Transferring fish raised in high density tanks to lower

density conditions effectively induced fish growth, enabling us

to observe growth properties in relation to chronological age

and IR exposure. Interestingly, neither age nor IR treatment

seemed to affect growth properties over the short and long-

term (0–47 weeks) after radiation exposure. However, survival

rates showed that mid-adult irradiated fish fared worse than

younger-adult fish, which may be the result of several causes.

Older age, in itself, may render fish more susceptible to the

effects of IR, with the decline of various systems including

muscle fiber structures (Greenlund & Nair, 2003), nervous,

endocrine and immune functions (Goya & Bolognani, 1999;

Hannestad et al., 2004), and cell metabolic regulation (Rattan,

1996; Donati et al., 2001). We also suspect that these cata-

strophic effects may be consistent with shortened lifespan in

irradiated fish, accompanied by ‘accelerated senescence’.

The molecular mechanisms behind these observations should

thus be evaluated in further studies, but we offer here a first

broad longitudinal assessment and characterization of growth

properties in zebrafish. In humans, growth regulation is often

closely linked to levels of senescent cells, and thus, to patho-

physiological aging and to genetic diseases such as progeroid

syndromes (Rodriguez et al., 1999; Taylor & Byrd, 2005). Further

details regarding the mechanisms underlying growth, senescence

and aging in zebrafish will lead to a greater understanding of

these prominent diseases, as well as the natural course of aging

in vertebrates.

Fin regeneration

Zebrafish have an impressive regenerative capability that higher

vertebrates lack and can regrow their caudal fin after amputa-

tion. Various studies on regeneration have revealed numerous

ordered and complex processes behind this orchestrated

process, including strict and careful regulation and reprogram-

ming of the cell cycle, followed by distinct sequential differen-

tiation and organization of the cells (Johnson & Bennett, 1999).

Given the integrated involvement of cell de-differentiation,

Fig. 6 The impact of age and ionizing radiation (IR) upon melatonin levels. (A, B) Melatonin levels were evaluated in fish of three age groups in each gender: younger (12 months), middle (24 months) and older (38 months). (C) Brain melatonin levels were measured in young-adult control fish (12 months), young-adult IR fish (12 months: 20 Gy at 6 months of age) and mid-adult fish (24 months).



219

re-differentiation, movement, and reorganization, any environ-

ment or insult that disrupts cell-cycle regulation or growth

would lead to an impairment of the regenerative processes

(Johnson & Weston, 1995; Poss et al., 2002a). Therefore, we

expected that age and IR exposure might disrupt regeneration

in zebrafish and indeed observed that successful fin regenera-

tion declined with age in older fish which exhibited a higher

incidence of impaired fin formation. Numerous other age-related

observations in repair and maintenance processes have been

documented, such as declines in hepatocyte regeneration due

to decreased growth hormone secretion and transcription factor

Foxm1b expression (Krupczak-Hollis et al., 2003), diminished

regenerative capacity of muscle cells due to fewer proliferative

resident precursor cells (‘satellite cells’) (Conboy et al., 2003),

impaired wound healing due to less efficient cytokine signal

transduction and altered balance of proteins (Ashcroft et al.,2002) and general declines in organ function due to impaired

homeostasis as a result of accumulating degenerative cells and

decreased regenerative capacity (Knapowski et al., 2002). We

may now add decline in zebrafish fin regeneration to this exten-

sive list. IR exposure also markedly impaired fin regeneration,

even at 8–12 months post-exposure. We speculate that IR insult

effectively induced long-term growth arrest or terminal senes-

cence status in the cells involved in regeneration, particularly

stem cells, resulting in an inability to regenerate effectively.

Combining the effects of both age and IR experimentally,

older irradiated fish showed a greater prevalence of severely

impaired regeneration than younger irradiated counterparts,

implicating either an age-related increase in IR susceptibility

to regenerative impairment, or otherwise, a synergistic effect

of age and IR. Similarly, greater negative IR effects have been

observed in aged rats with an inferior ability to repair IR-induced

8-oxo-2′-deoxyguanosine DNA modifications, possibly leading

to accumulation of oxidative DNA damage (Kaneko et al., 2003).

Additionally, age-dependent damage accumulation in many

macromolecules including nucleic acids, proteins, lipids, and

carbohydrates are enhanced by IR. Therefore, the pathways

by which aging-and IR-induced symptoms presumably manifest,

seem likely to coincide or act in parallel.

Reproduction

Vertebrate animals tend to demonstrate reproductive peaks

during the young adult stage, which then declines with increased

age (Bercovitch et al., 2003; Ricklefs et al., 2003). Larger body

sizes can offset this decline by allowing animals to brood larger

numbers of offspring (Congdon et al., 2003). Given the sup-

position that guppies (Poecilia retculata) exhibit reproductive

senescence and extended post-reproductive lifespan (Reznick

et al., 2006), it was not surprising to find consistent decreased

clutch sizes in older mid-adult zebrafish compared with young-

adult fish. Taken together, this supports the hypothesis of an

evolutionary antagonism between the inevitable decline in

reproductive function with age and continued growth. How-

ever, parity in the percentage survival of eggs between these

age groups demonstrated that older (mid-adult) fish continue

to lay clutches of eggs, albeit smaller, with equal viability to those

of younger (young adult) fish, and that genomic protective

mechanisms remained intact at all ages studied.

Various studies investigating reproduction and IR have revealed

several findings. For example, one study documented increased

structural chromosome anomalies in mice sperm after radiation

exposure, leading to subsequent sperm elimination to prevent

fertilization of unviable gametocytes (Alavantic & Searle, 1985).

Kuwahara et al. obtained similar findings in the medaka fish

(Kuwahara et al., 2003), where post-β-irradiation, medaka

exhibited a ‘reset’ mechanism that eliminated all existing

spermatogenic cells except for spermatogonial stem cells. In

our current study, IR exposure also appeared to negatively affect

reproduction. However, this negative effect persisted even many

months post-treatment when spermatocytes and sperma-

togenesis should presumably have recovered if zebrafish

spermatogonial stem cells retained their function. Instead, we

observed dramatic and progressive decreasing clutch sizes of

IR fish in three successive matings, after which IR fish no longer

laid any eggs. This finding poses the question of the status of

the spermatogonial cells in zebrafish. We also considered

possible impairment in oogenesis, as significant chromosome

damage induced by irradiation of immature oocytes in mice has

been reported (Griffin & Tease, 1988). Other possible explana-

tions include early exhaustion of oogenesis, premature ovarian

failure, or possible primordial germ cell senescence, since fish

species live post-reproductively despite continuing primary

oogenesis (de Bruin et al., 2004; Reznick et al., 2006). One should

also consider behavioral changes in response to genotoxic

stress. We have observed that IR-exposed, as well as aged fish,

show decreased physical locomotor activity and cognitive

functions (Yu et al., 2006). Thus, aging-and IR-induced changes

in behavior and movement may also modify reproductive

success, since the act requires social interaction and physical

movement.

In short, we find both age- and IR-related declines in repro-

ductive fecundity (‘reproductive senescence’), which manifest

as drastically decreased clutch sizes. In contrast, however, we

noted an essentially equivocal survival rate for all of the eggs

successfully laid, regardless of the age or IR exposure of parental

fish. This reveals an uncompromised integrity of the gametogenic

processes by robust defense mechanisms that eliminate un-

viable gametocytes. Further studies of the molecular details and

behavioral influences of IR and age on zebrafish with gender

differences will help determine the underlying mechanisms of

these reproductive observations.

Senescence-associated ββββ-galactosidase activity

Cytochemically and histochemically detectable SA-β-gal at

pH 6.0 has been shown to increase during the replicative

senescence of cells in culture and in tissue samples (Dimri et al.,1995), and has subsequently been used most widely as a marker

of cellular senescence in several vertebrate animal systems, both



220

in vivo and in vitro (Cao et al., 2003; Kishi et al., 2003; Genade

et al., 2005; Keyes et al., 2005). Both Genade et al. and our

laboratory have also specifically demonstrated age-related increases

in fish species and confirmed staining of the dermis using tissue

sectioning in killifish (Nothobranchius furzeri) and zebrafish,

respectively (Kishi et al., 2003; Genade et al., 2005). Although

questions remain regarding the physiological relevance of SA-

β-gal staining and its contribution as a biomarker to organismal

aging in mammals (Herbig et al., 2006), we have reproduced

significant correlations between age and staining intensity with

stress-induced enhancement in several independent experi-

ments of two different fish species (Kishi et al., 2003; Kishi, 2006;

Valenzano et al., 2006). However, we also note that SA-β-gal

intensity increases following multiple stress insults. Nonetheless,

this is biologically intuitive given that the origin of SA-β-gal

activity presumably derives from lysosomal β-galactosidase

(Kurz et al., 2000; Lee et al., 2006), and the cellular lysosomal

content increases in aging cells due to the accumulation of

nondegradable intracellular macromolecules and organelles in

autophagic vacuoles (Brunk & Terman, 2002). Thus, lysosomal

β-galactosidase induction could represent a general adaptive

response to cellular senescence.

In this study, we again noted age-dependent increases in

SA-β-gal staining, supporting previous observations, and we

also characterized the effects of genotoxic stress on changes in

SA-β-gal staining in adult zebrafish many months after IR expo-

sure. We found that SA-β-gal not only appeared a good marker

for aging in vivo, but also for IR stress-induced changes. The

significantly increased staining intensities in IR-exposed fish,

concurring with other aging-related symptoms, suggests that

stressful conditions may induce cells to enter senescence in a process

that is not necessarily only chronological age-related. The specific

molecular mechanisms underlying SA-β-gal activity at pH 6.0 still

require further investigations. However, recent evidence for

lysosomal β-galactosidase as the molecular origin of SA-β-gal,

suggests that SA-β-gal may be a surrogate marker for increased

lysosomal number and/or activity rather than a specific marker

of senescence. Therefore, expression of SA-β-gal in both replicative

senescence and stress-induced senescence may result from a link

of lysosomal functions to cellular senescence and organismal aging.

Brain melatonin levels

Whereas an age-dependent decline in brain melatonin levels,

the principal circadian hormone, has been well documented in

higher vertebrates (Miguez et al., 1998; Karasek, 2004), it has

not been established in lower vertebrates, including zebrafish.

Previously observed decreases in melatonin levels with aging

may result from several age-dependent changes including

reduction in sympathetic innervation of the pineal gland with

age, alterations in the level of melatonin precursor (serotonin),

and calcification and degeneration of the pineal gland (Tang

et al., 1985; Carneiro et al., 1993; Schmid et al., 1994). These

phenomena have been described in other vertebrates, including

humans (Touitou, 2001), as well, and may reflect common

age-related physiological changes typical of aging progression.

Melatonin, acting via specific G-protein-coupled melatonin

receptors located in multiple tissues, can affect major intra-

cellular signaling pathways, e.g. cAMP-dependent signaling

(Sugden et al., 2004). Thus, inadequate levels would likely offset

some of the temporal and physiological balances of intracellular

signaling pathways. We found in our current experiments that

nighttime melatonin levels in zebrafish declined with chrono-

logical age, in line with observations in other animals. This

shows the great potential of zebrafish as a model system for

studying the actual mechanisms underlying the age-related

reduction in melatonin, and its impact on physiological func-

tions. In the broader picture, this may translate into better

understanding of changes in melatonin levels and circadian

rhythm in higher vertebrates, including humans. In addition,

irradiated zebrafish also showed reductions in brain melatonin

levels, which persisted even more than 6 months following

treatment. This finding suggests that various insults, such as

genotoxic stress and perhaps chemical exposure among others,

may likewise propagate a decline in melatonin levels. Ahlersova

et al. (1998) similarly observed a reduction in pineal and circu-

lating melatonin in rats repeatedly exposed to increasing

dosages of IR and attributed these effects to changes in key

enzymes involved in melatonin synthesis and/or the metabolism

of its precursor, serotonin. However, further studies are clearly

needed to understand the mechanisms responsible for both

the acute and delayed effects of genotoxic stress on melatonin

production and metabolism in zebrafish. Lastly, given that the

reduced melatonin levels in our IR fish were comparable to those

measured in the aged zebrafish brain, we further suspect that

irradiated fish may serve as a valid senescence-associated

phenotype.

Conclusions

Our present study serves as an initial overview of the progression

of growth and aging in zebrafish. We surveyed several basic

processes common to all living organisms that are generally or

potentially affected by the aging process. We also examined

whether IR exposure would affect growth and aging, in order

to investigate the potential of using zebrafish as an IR-induced

aging model. A number of noteworthy caveats to this approach

include the characteristic lifespan in various zebrafish strains

and the pathological changes stimulated by aging and IR in

zebrafish, both of which are still largely unknown. Moreover,

we have investigated only some physiological aspects of the

effects of IR on adult zebrafish. Hence, in future studies that

incorporate targeted molecular and histopathological methods,

it will be necessary to characterize tissue-specific effects and

sensitivities in detail, particularly in the context of our method

of IR administration, at the cellular and molecular levels.

In summary, we find that adult zebrafish exhibit comparable

abilities to grow in a density-dependent and size-dependent

fashion regardless of chronological age and that this phenom-

enon is undeterred in the long term by IR exposure. At the same



221

time, these animals exhibit signs of senescence with age, char-

acterized by a decline in reproductive fecundity and low central

and peripheral melatonin levels, impaired fin regeneration and

increased SA-β-gal activity. These phenotypes are consistent

with an increased accumulation of senescing cells in vivo and

alterations of an integrative function of the circadian system in

zebrafish. IR exposure also appears to enhance these parameters,

even months after the exposure event, supporting the possibility

of the further use of this organism as an aging model system.

Further studies will be needed to elucidate the mechanisms

underlying both these events and the responses to aging and

IR insult. However, the information provided by our current

results should further establish the groundwork that is necessary

to realize the full potential of zebrafish as a model organism in

aging research.

Experimental procedures

Fish husbandry

Zebrafish of the wild-type AB strain were maintained with a

14:10 h light/dark cycle and fed living brine shrimp twice per

day. Brine shrimp was given using 1 mL pipettes at an amount

of about 0.75 mL brine shrimp per 20 fish. Flake food was also

given a few days per week semiquantitatively according to the

number of fish in the tanks. A continuously cycling Aquatic

Habitats™ system was used to maintain water quality (Apopka,

FL, USA) which completely replaces the water in each tank every

6–10 min. Each tank is a baffle/tank system that ebbs water in

a circular motion to ensure flushing and water turnover. Ultra-

violet (UV) sterilizers (110 000 microwatt-s cm−2) were employed

to disinfect the water and prevent the spread of disease in the

recirculating system. The water temperature was maintained at

28 ± 0.5 °C. The system continuously circulated water from the

tanks through Siporax™ strainers, through a fiber mechanical

filtration system, and finally into a chamber containing foam

filters and activated carbon inserts. Water quality was tested

daily for chlorine, ammonia, pH, nitrate and conductivity, and

also was under real-time computer monitoring with alarms to

signal potential fluctuations. The general health of each fish was

also observed on a daily basis, and abnormal looking or acting

fish were quarantined into isolated tanks unconnected from

the general circulation. The water in these quarantined tanks

was treated with methylene blue. If and when fish recovered,

they were returned to their original tanks on the general

circulation.

Quantitative analysis and statistics

Data processing and statistical analyses were performed using

Statistical Package for the Social Sciences (SPSS) version 14.0.

This software was used to generate each of the scatter plots,

tables and graphs shown in the text and perform statistical

tests where appropriate. More advanced statistical analyses

were performed at the Department of Biostatistical Science in

the Dana-Farber Cancer Institute and Harvard School of Public

Health.

Fish growth and size measurements

For all body size measurements, fish were placed into 5 mL of

0.5% tricaine solution mixed with 100 mL of fish water from

the circulating system. This ensured that the solution was of suf-

ficient temperature and electrolyte balance for fish. The subjects

were left in the tricaine solution for between 30 s and 2 min

until they flipped sideways, indicating unconsciousness. Care

was taken to try and adapt the time to the variance in individual

fish tricaine sensitivities. The fish were then individually trans-

ferred to a Petri dish, measured with a ruler placed against the

clear bottom of the dish, and then returned to a 2.75 L tank

of fresh fish water to allow for recovery. The body length meas-

ured spanned from the tip of the mouth to the end of the body

wall muscle. After the fish had revived, they were returned to

their original tank and put back on circulation.

To study density-dependent growth, we took cross-sectional

measurements of each fish maintained in the 61 tanks under

study, with densities ranging from between 15 and 63 animals

per tank. All fish were the wild-type AB line from the same

parental matings, with ages varying from 9 to 16 months at

beginning of the study, at the first measurement. Measure-

ments of 55 tanks (from original 61 tanks) were taken again

15 months later with ages varying from 21 to 28 months (six

of the tanks had been used in other studies).

To study age-related and IR-related growth properties, we

used two tanks containing the youngest and the oldest age

groups that had completed sexual maturation, 6 months (young

adult) and 17 months (mid-adult) old at the start of the experi-

ment raised at identically high population densities (48 fish/

2.75 L tank), such that the fish were all of similar size. We

divided the fish into eight groups of six fish (four young-adult

and four mid-adult groups) and transferred these into eight

separate tanks. Fish from two of the tanks from each age group

were then full-body irradiated with 20 Gy of irradiation in glass

jars (holding each group of six at once). The dosage of 20 Gy

was determined to be the appropriate dosage based on the

observation that 30 Gy was lethal, while 10 Gy did not produce

discernible effects on survival and growth. This concurs with the

report of Traver et al. (2004), which determined the sublethal

dosage to zebrafish to be 25 Gy. The remaining four tanks

served as the control fish. These tanks were classified as follows:

(i) mid-adult control, (ii) young-adult control, (iii) mid-adult

20 Gy fish, (iv) young-adult 20 Gy fish.

We observed the fish growth over the next 47-week period.

Based on our extensive experience with zebrafish, we calculated

that the space provided (2.75 L/6 fish) was more than adequate

to sustain uninhibited growth for the duration of this study. Dur-

ing the period of growth in this experiment, we ensured optimal

standard living conditions and adequate nutrition. We noted the

survival rate daily after IR exposure and monitored the general

health of the fish and the effects of the IR. Post-IR, we measured



222

the fish every 2 weeks until week 14, and then measured the

fish every 4 weeks until week 47.

Fin amputation

To examine fin regeneration following IR exposure, 55 zebrafish

were designated as controls, whereas 87 fish were exposed to

20 Gy IR, 8 months prior to amputation. All fish were from the

same clutch and were kept under optimal standard conditions

over this 8-month period. Each of the fish was then subjected

to caudal fin amputation. First, the fish were placed into a

diluted tricaine solution as outlined above, then individually

placed on a clean cutting surface and approximately 50% of

the tail fin was cut longitudinally with a clean razor. They were

then placed back into their original tanks on circulation after

revival. By 2-weeks post-amputation, all of the fish were anes-

thetized again to assess their fin regeneration states by digital

photography. The fish were categorized according to whether

fin regeneration appeared (A) normal, (B) slightly (somewhat)

impaired, or (C) severely impaired.

The control and irradiated fish (both young-adult and mid-

adult) from the growth studies also underwent fin amputation,

after the growth analyses had been completed to determine the

effects of chronological age upon regeneration, as well as to

provide for supporting data for the impact of IR on this process.

Reproductive studies

Two differently aged groups of similarly sized zebrafish were

also used to study the effects of aging upon reproduction. The

old-adult groups of fish were 23 months old and the young-

adult groups were 7 months old at the first mating. Each age

group consisted of 16 healthy males and 16 healthy females.

The fish were maintained under standard husbandry conditions

between matings. At each mating, to ensure adequate space,

each group was separated into two mating tanks (2 sets of 8 : 8/

male : female). Fish were allowed to mate during a 3-h period

on the mating days. The eggs were collected immediately after-

wards and the fish were returned to regular tanks on circulation.

The eggs were then placed in sterile Petri dishes with antibiotic

egg water and stored in an incubator at 28.5 °C overnight. The

next day, the numbers of surviving eggs were tabulated. These

procedures were repeated every 1 to 2 weeks for a total of

21 matings. Reproductive efficiencies were calculated by eggs

surviving/number of female/h.

To study the effects of IR on reproduction, we used 14-month-

old fish from the same clutch that was used in our fin regeneration

studies. One group was designated as the control group and the

other was irradiated with 20 Gy of IR at 6 months prior to the start

of the experiment. The control group consisted of 30 males and

30 females. At each mating, the fish were placed into 10 mating

cages each containing 3 males and 3 females per tank. The IR group

comprised 36 males and 36 females divided into 12 smaller mat-

ing tanks, each again containing three males and three females

per tank. The subsequent mating procedure was performed as

described above, except that the matings were repeated only

five times over a 1-month period. Data for only the first three

trials is shown because the IR fish no longer laid eggs after the

third trial.

SA-ββββ-Gal staining

Zebrafish were anesthetized in tricaine and placed into ice-cold

PBS. The fish were then washed twice in PBS and fixed for 3 days

in 4% paraformaldehyde in PBS. After fixation, the fish samples

were washed four times in PBS, and incubated at 37 °C (without

CO2) for 12–16 h with SA-β-gal staining solution (5 mM

potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2in PBS at pH 6.0) (Dimri et al., 1995). Qualitative analysis was

done by stereo zoom microscopy. To analyze the quantitative

levels of SA-β-gal staining, images were captured by high-

resolution digital photography. These high-resolution digital

images were then processed using Adobe Photoshop version

7.0. The SA-β-gal pixels were selected, filtered and counted, and

the net total number of pixels from each image was determined

by subtracting both the black/brown melanocyte pixels and the

white pixels indicating light reflection from the total number.

The percentage of SA-β-gal stained pixels out of the net total

was then calculated. The filters used were tested against control

unstained fish samples to ensure that the pixels filtered

accurately represented SA-β-gal staining.

Brain melatonin levels

Melatonin levels were evaluated in fish of three age groups:

younger (12 month, n = 17), middle (24 month, n = 15) and

older (38 month, n = 12). Fish were maintained in a 12:12 h

light/dark cycle at 26–28 °C, and then frozen in liquid nitrogen

in the middle of their night, under dim red light (less than 5 lux).

In addition, in order to test the effects of irradiation on brain

melatonin levels, nine young fish were irradiated with 20 Gy of

IR at 6 months of age. Six fish from the same clutch served as the

control group, i.e. underwent all the same procedures as the

IR fish, except for the irradiation. At 12 months of age, these two

groups of fish were also frozen 6 h after lights off and the brain

and muscle tissue was removed and processed. The tissue samples

were sonicated in 96% ethanol and then centrifuged. The super-

natant was removed and diluted to a concentration of 10% in

ethanol. Melatonin was extracted from the diluted supernatant

through C18 columns and measured using radio-immunoassays

(ALPCO, Windham, NH, USA). Pellets were solubilized in 0.1 N

NaOH and assessed for protein content. Melatonin concentrations

in each sample were then adjusted for protein levels.

Acknowledgments

We are very grateful to Thomas Roberts for his support and

continued encouragement. We also thank Kilian Perrem and

Arthur Pardee for critically reading the manuscript. This work

was funded by research grants from the A–T Children’s Project,



223

the Ellison Medical Foundation and the NIA/NIH to S.K., and the

NIMH/NIH to I.Z.

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