BIOLOGY OF REPRODUCTION 22, 367—375 (1980)

INTRODUCTION

Women of child-bearing age are frequentlyexposed no potentially munagenic drugs whichmay affect the webb-being of the conceptus.For example, alkybaning agents, purine andpyrimidine analogs and folic acid antagonistsare used extensively no treat many pathologicaldisorders and no prevent rejection of organallografns (Schein and Winokur, 1975; Gerberand Steinberg, 1976a,b). The purine analogs,especially azathioprine, have “¿�becomethe

Accepted October 29, 1979.Received January 17, 1979.I This research was supported by NIH biomedical

general research support grant 5S01-RR05458-13.2 To whom reprint requests should be sent. Present

address: Department of Preventive Medicine, NewYork State College of Veterinary Medicine, CornellUniversity, Ithaca, NY 14850.

clinical yardstick by which effectiveness of new

drugs is measured― (Santos, 1972). Azathioprime induces genetic damage in somatic and

germ cells (Clark, 1975; Generoso en al., 1975).

Chromosomal aberrations induced in somaticcells of fetuses during treatment of pregnantwomen with azathioprine are eliminated by20—32 months after birth (Leb en al., 1971;

Price et al., 1976). However, because germ cells

of the female fetus stop dividing before birth,

genetic damage in these cells may remaininstead of being eliminated. As a result, theconsequences of such damage would not bemanifested until the offspring of treatedmothers reach reproductive maturity.

Although there are several published reports

indicating that azathioprine is embryonoxic andteratogenic in animals and man (Ginhenset al., 1965; Rosenkrannz en al., 1967; Scheinand Winokur, 1975; Gross et al., 1977), little isknown about reproductive function in the

367

Bi-GenerationalEffects of 6-Mercaptopurineon Reproduction in Mice'

T. J. REIMERS,2 P. M. SLUSS, J. GOODWIN and G. E. SEIDEL, JR.

Department ofPhysiology and Biophysics,Animal Reproduction Laboratory,

Colorado State University,Fort Collins, Colorado 80523

ABSTRACT

Effects of the mutagenic, immunosuppressive drug 6-mercaptopurine (6-MP) on reproductivefunction were examined in two generations of mice. CD1/CR mice (G0 generation) received eitherphysiological saline (Group A) or 0.5 (Group B), 1.5 (Group C) or 3.0 (Group D) mg/kg 6-MPdaily during pregnancy. Three mg/kg/day administered no G0 mice reduced mean litter sizes(P<0.01), whereas the other doses had no effect. The surviving offspring (G1 generation) werepaired for breeding with untreated mice an 70 days of age. Although body weights and generalappearance were normal, the interval from the day of pairing of G1 females in Group D (4.9 ±0.6 days) with untreated males to the day of breedingwas longer (P<0.01) than for G, females inGroup A (2.7 ±0.3 days). A smaller percentage of the G, females was pregnant in Groups C(78.8%, P<0.05) and D (22.2%, P<0.01) than in Group A (100%). Ovaries from G1 females inGroup D weighed 2.2 ±0.8 mg, whereas those from G1 females in Group A weighed 8.6 ±0.5 mg(P<0.01). Many ovaries in Groups C and D had fewer follicles, oocytes and corpora lutea than didcontrol ovaries. The mean number of G2 fetuses/pregnant C1 female was lower (P<0.01) in GroupsC (10.6 ±0.4) and D (6.5 ±1.3) than in Group A (13.0 ±0.4). A large percentage ofG2 fetuses inGroups C (25.3 ±2.7%) and D (41.7 ±9.1%) were dead; 12.8 ±2.6% were dead in Group A(P<0.01). Most fetuses died early in gestation. Weights of the live G2 fetuses and placennas werenon different among groups. After G1 males were paired with untreated females, 60% and 55.6%of the females were pregnant in Groups C and D, respectively, whereas 100% were pregnant inGroup A (P>0.05). Testes from G1 males in GroupsC (155.7 ±18.8 mg) and D (104.2 ±11.6 mg)weighed less (P<0.01) than testes from G3 males in Group A (258.0 ±5.5 mg) Many seminiferoustubules in testes of G1 males were devoid of germ cells. The interval from pairing no breeding,litter sizes, percentage of G2 fetuses dead and G2 fetal and placental weights were non affectedwhen G1 males were mated. Our results show that 6-MP non only had direct embryotoxic effectswhen administered no pregnant mice, bun also severely impaired reproductive function of the surviving offspring a full generation after the drug was administered.

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368 REIMERS ET AL.

untreated offspring of mothers who receivedthis drug during pregnancy. Consequently,

several clinical researchers have expressedconcern about possible bi-generanional effectsof potentially munagenic drugs because of the

unique nature of germ cell development (Leb en

al., 1971 ; Saarikoski and Sepp@l@i,1973 ; Con@enal., 1974; Schein and Winokur, 1975; Gerber

and Steinberg, 1976b, Price en ab., 1976). In apreliminary report (Reimers and Sluss, 1978),we confirmed previous results showing thatadministration of 6-mercaptopurine, the activemetaboline of azathioprine (Elion, 1977), wasembryonoxic in mice. In addition, we showedthat reproductive function of the otherwise

normal, surviving female offspring of treated

mothers was severely impaired. We present herefurther detailed observations on reproductivefunction in male and female offspring of miceexposed no 6-mercaptopurine during in unerodevelopment.

MATERIALS AND METHODS

CD1/CR female mice (G0 generation) were randomly assigned to 4 treatment groups as follows:Group A, 0.15 M NaC1;Group B, 0.5 mg/kg/day 6-MP;Goup C, 1.5 mg/kg/day 6-MP; Group D, 3.0 mglkg/day 6-MP. The drug was dissolved in 0.15 M NaCI andall solutions were adjusted to ‘¿�\‘pH8. Treatments,ranging in volume from 0.1 no 0.2 ml, were injecteds.c. daily beginning 3 days before pairing with untreated males through Day 18 after breeding. Femaleswere paired with males for a maximum of 6 days. Theday of breeding (Day 0) was determined by thepresence of vaginal plugs. Bred G0 females were cagedin groups of 4 until Day 18 when they were cagedseparately to deliver their offspring (G1 generation).Live and dead G1 mice were counted and sexed afterbirth and again 21 days later when they were weaned,separated by sex and permanently identified.

An 70 days of age, the surviving C1 mice wereweighed and then paired with normal, untreated micefor 12 days (“s.'3estrous cycles). Four C1 females, 1from each of the 4 treatment groups, were paired witha single male. However, because of unequal numbers,some groups of 4 females represented only 3 of thetreatments. Ten randomly selected G1 males fromGroups A, B and C and all G1 males from Group Dwere paired with normal, untreated females in a ratioof 1 female: 1 male.

All female mice (G1 females paired with untreatedmales and untreated females paired with C1 males)were examined daily for vaginal plugs. Females wereseparated from the males when vaginal plugs werefound (Day 0) or 12 days after pairing.

All females in which vaginal plugs were found werekilled and laparotomized on Day 18; mice in whichvaginal plugs were non found were killed on ‘¿�\‘Day18after the estimated day of breeding. Ovaries from allC1 females were weighed, fixed in Bouin's fluid,embedded in paraffin, sectioned and stained with

hematoxylin and eosin. The uterine horns wereopened and live and dead fetuses (G2 generation) werecounted. Dead fetuses that were small, amorphousmasses of dark-red decaying tissue were classified as“¿�earlydead. “¿�Those that were partly decomposed butstill recognizable as fetuses were classified as “¿�latedead.―All G1 males were killed at ‘¿�\‘lOOdays of age.Testes were weighed and fixed in Bouin's fluid forhistological examination.

Binomial responses were analyzed by x2 with Yatescorrection (Steel and Torrie, 1960a). Continuousresponses were subjected no analysis of variance withappropriate covariates as described below. Standarderrors were calculated from the error term of theanalysis of variance. The experimental units were theG0 females. Therefore, G1 mice were considered assamples and the C2 fetuses and placennas as subsamples. Dunnett's test (Steel and Torrie, 1960b) wasused to compare treatment means with control means(Group A).

RESULTS

Reproductive Functionofthe G0 Generation

Table 1 shows the direct effects of dailyadministration of 6-MP on breeding and pregnancy in the C0 mice. There were no significanttreatment effects on the percentage of thepaired G0 mice bred, the mean interval frompairing to breeding (analyzed with weight anpairing as a covariate), the percentage of bred

G0 mice that delivered offspring and meangestation lengths (analyzed with total bittersizes as a covariane). The mean number of G,offspring (live + dead) and the mean number oflive G1 offspring/pregnant C0 female in GroupD were significantly (P< 0.01) bess than in

Group A. These data were analyzed with theG0 females' body weights on the day of pairingas a covariate.

Reproductive FunctionofG1 Females

Mean body weights of the G, females inGroups B, C and D an the time of pairing withuntreated males were non significantly differentfrom those of G, females in Group A (Table 2).Vaginas of all females were open an the time of

pairing. There were no significant differences

among groups in the percentage of the G,females in which vaginal plugs were observed.However, the interval from the day of pairingwith males no the day on which vaginal plugswere found was longer (P< 0.01) in Group D(4.9 ±0.6 days) than in Group A (2.7 ±0.3days). Body weights of C1 females on the day

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- TreatmentgroupA

B CDDose

(mg/kg/day) 0 0.5 1.53.0Numbertreated13 12 1112Paired

mice bred (%) 69.2 100.0 81.958.3Pairingtobreedinginterval(days,

@ ±SEM) 3.1 ±0.4 2.9 ±0.4 2.8 ±0.4 3.6 ±0.5Bredmicewhichdelivered(%)

100.0 83.3 88.9100.0Gestationlength(days,

@ ±SEM) 19.2 ±0.2 19.4 ±0.1 19.9 ±0.1 19.8 ±0.2Offspring/pregnantfemale(live

+ dead, 3c±SEM) 9.8 ±0.8 9.2 ±0.7 8.9 ±0.9 5.2 ±0.9@Liveoffspring/pregnantfemale

(@±SEM) 9.5 ±0.9 9.2 ±0.8 8.9 ±0.9 3.8 ±1.OaaSignificantiy

less than control mean(P<0.O1).of

pairing were used as a covariate for this Ovaries from G, females in GroupDanalysis.

weighed less (2.2 ±0.8 mg) than those fromG,A

significantly smaller percentage of the females in Group A (8.6 ± 0.5,P<0.01).paried

G, females was pregnant on Day 18 in Ovaries from G1 mice in Group BgenerallyGroupsC (P<0.05) and D (P<0.01) than in appeared histologically normal andresembledGroupA. Likewise, a significantly smaller those from mice in Group A. Therewerepercentage

of the G, females in which vaginal abundant oocytes, follicles and corporalutea.plugswere observed was pregnant in those same However, ovaries from G, mice in GroupsC2

groups (Group C, P<0.05 ; Group D, P<0.01). and D ranged in appearance fromcompletelyTABLE

2. Reproductive characteristics of G, female mice.Treatment

groupA

B CDDose

(mg/kg/day) 0 0.5 1.53.0Numberpaired33 46 339Pairing

weight(g,@ ±SEM) 26.9 ±0.4 28.9 ±0.3 26.2 ±0.5 27.0 ±1.2Females

withobservedvaginalplugs (%) 78.8 80.4 87.955.6Pairing

to breedinginterval(days,@ ±SEM) 2.7 ±0.3 2.7 ±0.2 3.3 ±0.3 4.9 ±0@@aPaired

femalespregnant(%) 100.0 95.7 78•8b22.2cFemales

withobservedvaginalplugs that

were pregnant (%) 100.0 100.0 79•3b40.O'Ovarianweights(mg,

5t ±SEM) 8.6 ±0.5 8.7 ±0.4 7.3 ±0.4 2.2 ±O.8c

BI-GENERATIONAL EFFECTS OF 6-MERCAPTOPURINE 369

TABLE 1. Effects of 6-mercaptopurine on reproduction in G0 mice.

a.Significantly greater than control mean (P<0.01).

bSignificantiy less than control mean (P<0.05).

cSignificantly less than control mean (P<0.01).

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370 REIMERS ET AL.

normal to completely devoid of oocytes andfollicles (Fig. 1). Many of the ovaries contained

large areas of cells which resembled luteal cells.

Table 3 shows sizes of litters produced bythe G, females, the percent of their fetuses (G2generation) that was dead and fetal and pla

cennal weights. Only litters from those G, mice

in which vaginal plugs were observed were

examined because the day of gestation wasaccurately known only in those mice.

The mean number of G2 fetuses (live + dead)produced/pregnant C, female was significantly

lower in Groups C and D than in Group A(P<0.01). Twenty-five percent and 42% of theG2 fetuses were dead in Groups C and D,

respectively, whereas only 1 3% were dead in

Group A (P<0.01). Almost all of the dead G2fetuses were classified as “¿�earlydead― and asignificantly greater proportion was “¿�early

dead― in Groups C and D (P<0.01) than inGroup A. No differences among groups in thepercentage of G2 fetuses that were classified as

“¿�latedead' ‘¿�were found.

Weights of the live G2 fetuses and placentaswere not affected by treatment. Mean fetalweights ranged from 1.38 to 1.27 g. Mean

placental weights ranged from 98.43 no 119.67mg. Litter sizes (live + dead) were used as a

covariate to an alyze these weigh ts.

Reproductive FunctionofG, Males

Pairing weights of the G, males did not

differ among groups (Table 4). The percentage

of the normal, untreated females in which

vaginal plugs were observed after pairing with

G, males and the interval between pairing and

breeding were not affected by treatment. Body

weights of the G, males on the day of pairing

were used as a covariate to analyze this interval.The percentage of paired females that waspregnant on Day 18 and the percentage offemales with observed vaginal plugs that waspregnant were not significantly affected by

treatment (P>0.05). However, there was almostcertainly a treatment effect because 20 preg

nancies resulted from 20 pairings in Groups Aand B, whereas only 1 1 pregnancies resultedfrom 19 pairings in Groups C and D.

Mean weights of testes from G, males inGroups C and D were less than those in GroupA (P<0.01). Testes from G, males in Group A

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‘¿�**‘¿�* f $1@* ..- __....1..*.*

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1@@@ _ —¿�—@ ,@FIG. 1. Ovary from C1 offspring of a mouse treated with 3.0 mg/kg/day 6-MP. X 110.

. * . *.

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TreatmentgroupA

B CDDose(mg/kg/day)

0 0.5 1.53.0Numberoflittersexamineda

26 37 232Fetuses/pregnantfemale

(live + dead) 13.0 ±0.4 12.8 ±0.3 10.6 ±0.41) 6.5 ±13bFetusesdead(%)12.8 ±2.6 18.0 ±2.2 25.3 ±2.7c 41.7±9.1CFetuses―earlydead―(%)9.8 ±2.5 14.3 ±2.1 23.5 ±2.6' 36.7 ±8.9cFetuses

“¿�latedead―(%) 3.0 ±1.4 3.7 ±1.3 1.8 ±1.6 5.0 ±4.5Fetalweights (g) 1.38 ±0.02 1.38 ±0.02 1.37 ±0.02 1.27 ±0.07Placental

weights (mg) 100.66 ±2.80 98.43 ±2.49 104.02 ±3.28 119.67 ±8.85aLitters

from G1 females in which vaginal plugs wereobserved.b.

Significantly less than control mean(P<0.01).c.

Significantly greater than control mean(P<0.01).appeared

normal. The seminiferous tubules placental weights were affected bytreatmentcontainedthe full complement of germ cells. (Table5).Figures

2 and 3 show sections of testesfromG,males in Groups C and D, respectively.

Whereas some tubules in Fig. 2 appearedDISCUSSIONnormal,others were devoid of germinal dc- Azathioprine and 6-mercaptopurinehavements.Figure 3 exemplifies the extreme been reported no be embryotoxic and nerano

condition in which all seminiferous tubules genic in animals (Ginhens en al., 1965; Rosenwere completely devoid of germ cells. Only krantz en al., 1967; Gross et al., 1977).OurSertoli

cells were present in the tubules and results in mice confirm these reportsbecauseappearednormal (Fig. 4). Neither bitter sizes, 3.0 mg/kg/day 6-MP significantlyreducedpercentage

of C2 fetuses dead, fetal weights nor sizes of litters produced by the treated G0mice.TABLE

4. Reproductive characteristics of C@ male mice.Treatment

groupA

B CDDose

(mg/kg/day) 0 0.5 1.53.0Numberpaired 10 10 109Pairing

weight(g,@ ±SEM) 35.3 ±0.8 35.9 ± 1.0 33.0 ± 0.8 33.5 ±0.5Females

withobservedvaginalplugs (%) 100.0 100.0 100.088.9Pairing

to breedinginterval(days,@ ±SEM) 3.2 ±0.4 2.5 ±0.5 2.3 ±0.5 3.1 ±0.5Paired

femalespregnant(%) 100.0 100.0 60.055.6Females

withobservedvaginalplugsthatwere

pregnant (%) 100.0 100.0 60.062.5Pairedtesticularweights(mg,

@ ±SEM) 258.0 ±5.5 250.5 ±10.3 155.7 ±18.8a 104.2 ±11.6a

BI-GENERATIONAL EFFECTS OF 6-MERCAPTOPURINE 371

TABLE 3. Characteristics of fetuses and placentas produced by C1 female mice. Means ±SEM.

aSignificantly less than control mean (P<0.01).

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372 REIMERS ET AL.

FIG. 2. Testis from G1 offspring of a mouse treated with 1.5 mg/kg/day 6-MP. X 110.

@f@ ‘¿�*‘ .v'

3@ •¿�@‘@

FIG. 3. Testis from a C, offspring of a mouse treated with 3.0 mg/kg/day 6-MP. X44.

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TreatmentgroupABCDDose

(mg/kg/day)00.51.53.0Numberoflittersexamineda101065Fetuses/pregnant

female(live+ dead)13.3 ±0.713.9 ±0.813.8 ±0.912.2 ±1.1Fetuses

dead (%)15.5 ±4.09.7 ±3.719.1 ±4.48.1 ±5.6Fetuses“¿�earlydead―(%)9.4 ±4.05.9 ±3.616.7 ±4.15.9 ±5.6Fetuses“¿�latedead―(%)6.1 ±2.43.8 ±2.32.4 ±2.92.2 ±3.4Fetal

weights (g)1 .43 ±0.031 .42 ±0.031 .45 ±0.041 .36 ±0.42Placentalweights (mg)94.91 ±2.5694.63 ±2.7092.55 ±3.3294.63 ±3.70

BI-GENERATIONAL EFFECTS OF 6-MERCAPTOPURINE 373

FIG. 4. Testis from a G5 offspring of a mouse treated with 3.0 mg/kg/day 6-MP. X 279.

However, the drug did not appear to influencelengths of estrous cycles or estrous behavior

in this generation, since the mean pairing to

breeding interval and the percentage of micebred were not affected. Three mg/kg/dayalso severely impaired reproductive function ofthe treated mothers' offspring (G1 generation).Even though 1.5 mg/kg/day of the drug did not

adversely affect reproduction in the G0 genera

tion, it did have detrimental effects in the G1

generation. Furthermore, the dramatic effects

in the G, generation occurred even though

general appearance and body weights of the G1

mice were normal at the time of breeding.

We observed distinct differences in reproductive function between male and female

TABLE 5. Characteristics of fetuses and placentas produced by G male mice. Means ±SEM.

aLitters from females in which vaginal plugs were observed.

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374 REIMERS ET AL.

offspring of 6-MP treated mothers. Although

gonadal function was impaired in both sexes, as

reflected in gonadal weights and histological

appearance, the resultant reprodu ctive impair

ment was more pronounced in the G, females.

The interval from the day of pairing to the day

of breeding was extended in the G, females butnot in the G1 mates. The proportion of the G,female offspring of mice treated with 1.5 and

3.0 mg/kg/day 6-MP that was pregnant 18 daysafter breeding was dramatically reduced. In

contrast, pregnancy rates were not significantly

different among untreated females paired with

G, males. However, more G, males need to beexamined before a definite conclusion can be

made.Mean litter sizes of the pregnant G, females,

but not pregnant untreated females bred by G1

males, were affected by treatment of their

mothers with 6-MP. Apparently the drug

reduced the number of oocytes which arose

during development from the small pool of

germ cells in the embryo (Mintz, 1960). Thelack of oocytes in sections of the ovariessupports this contention. Many ovaries resembled those from mice and rats exposed toionizing radiation (Palayoor et al., 1977).

Our resultsfurthersuggestthatthe oocytesthat were present in ovaries of G, females

contained genetic damage which was lethally

expressed shortly after fertilization in some ofthe G2 embryos.

The genetic damage was acquired while theG, females' germ cells were differentiating inutero during treatment of the G0 mothers. Itapparently was not eliminated or repaired, as it

probably was in somatic cells, because synthesis

of DNA stops when oocytes arrest in prophaseof the first meiotic division and no new DNA issynthesized until after fertilization (Sirlin and

Edwards, 1959; Rudkin and Griech, 1962).

Male offspring of 6-MP treated mothersappeared to exhibit an “¿�all-or-none―responseto treatment. The untreated females with whichthey were paired either were not pregnant orwere pregnant and had normal litter sizes.Mortality rate of the G2 fetuses produced by

the G1 males was not increased. Male germ cells

divide mitotically during embryonat and fetal

development and do not enter meiosis until

puberty is attained. The drug appeared to have

interfered with mitosis of the male germ cells.

If spermatogonia incurred genetic damage,they were eliminated or the damage was laterrepaired because the G2 fetuses from resulting

spermatozoa survived normally.

Thousands of patients have received azathio

prine, 6-mercaptopurine or other potentially

mutagenic drugs to treat many rheumatological,dermatological , hematologicat , gastrointestinal,

hepatic, ophth atmological, osteological, neuro

logical, pulmonary or renal disorders (Gerber

and Steinberg, 1976a,b). Many children have

been born to mothers treated with azathioprineduring pregnancy (Board et al., 1967; Kaufman

et al., 1967; Caplan et at., 1970; Leb et at.,1971 ; Lower et at., 1971 ; Merkatz et al., 1971;Penn et al., 1971; Merrill et al., 1973; Cot@ et

at., 1974; Jacob et al., 1974; Sharon et at.,

1974; Price et al., 1976). Although one must becautious in extrapolating from animal experi

ments to human clinical situations, these

children should be observed carefully to deter

mine if treatment adversely affected their

reproductive function.

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