In vivo cytogenetic effects of trimethylphosphate and of tepa on bone marrow cells of male rats

Mutation Research Elsevier Publishing Company, Amsterdam Printed in The Netherlands

263

I N V I V O C Y T O G E N E T I C E F F E C T S OF T R I M E T H Y L P H O S P H A T E AND OF

T E P A ON BONE MARROW CELLS OF MALE RATS

I. D. ADLER, G. RAMARAO AND S. S. E P S T E I N * Laboratories of Environmental Toxicology and Carcinogenesis, Children's Cancer Research Foundation, and Department of Pathology, Harvard Medical School, Boston, Mass. ( U . S . A . ) (Received June 7th, 1971) (Revision received August Ist, 1971)

SUMMARY

The cytogenetic effects of trimethylphosphate (TMP) and Tris[2-methyl-iaziridinyllphosphine oxide (TEPA) on rat bone marrow cells in vivo were examined at intervals following single and repeated intraperitoneal administration of each drug. Dose-related chromatid aberrations, including open breaks and reunion figures, were maximal 24 and 4 8 h following administration of T E P A and TMP, respectively. Cytogenetic damage induced by the highest dose of TMP, 2000 mg/kg, was less severe than that induced by the highest dose of T E P A tested, IO mg/kg; additionally, the proportion of surviving cells was greater with TMP than with TEPA. On the basis of these data it appears likely that TMP poses a greater potential genetic hazard than TEPA.

INTRODUCTION

Chromosome analysis of bone marrow cells in vivo from mammals, including mice 7,1~, rats~, 37, and Chinese hamsters4,29, ai has become a standard method for testing for the potential mutagenic effects of viruses a°, radiation 1, drugs and chemical pollutants 35. This paper presents data on in vivo cytogenetic effects of TMP and T E P A on bone marrow cells of male rats. TMP is extensively used industrially as a methylating agent2, .1, a solvent for paints and polymers, a catalyst in polymer and resin synthesis% and a chemical intermediate in the production of polymethyl polyphosphates48; until recently TMP was also used as a gasoline additive 11. The acute toxicity of TMP in rodents is lowS; long-term administration of high doses induces weight loss and paralysis. TMP acts as an alkylating agent both in vivo ~4 and in vitro 2~, induces reverse mutations in Neurospora crassaaL and reduces litter sizes sired from treated male rats ~4. Recent dominant lethal assays in mice have demonstrated mutagenic effects of TMP in postmeiotic stages of spermatogenesis 11. * Present address: Case Western Reserve University, School of Medicine, Cleveland, Ohio 441o6 (U.S.A.). Abbreviations: TEPA, Tris[2-methyl-i-aziridinyl]phosphine oxide; TMP, trimethylphosphate. llgutation Res., 13 (1971) 263-273

264

I. I). AI)LER, G. RAMAI,~AO, S. S. EPSTI'.'IN

T E P A has a wide range of industrial applications",m,~", 4r'. More restrictedlv it i~ used as an insect chemosterilanta,~8,~% ~8,aa.a4,~2,4<~1,and in tile therapy of neoplastic~", ~' and non-neoplastic ~7,21 diseases; its use has also been recommended for rodent 4a and plant 20 control. T E P A induces dominant lethal mutations in mice~a; abnormal development of zygotes recovered from female mice mated with TEPA-treated males has also been demonstrated~", av. The mutagenic activity of T E P A in male mice has been further proven by the induction of sterility and semisterility, due to reciprocal translocations, in their F~ and F2 male progeny~4, 4". T E P A also induces chromosome aberrations in human leucocyte cultures ~. MATERIALS AND METHODS

Random bred male CD-rats (Charles River Laboratories), 3-4 weeks old and weighing 7 ° 14o g, were used in these experiments. Commercial grade TMP and T E P A were obtained from K & K Chemical Company (Plainview, New York). Both drugs were dissolved in water. Experiments were designed to investigate cytogenetic effects at varying times after administration of single doses of each drug, and also the dose-response relationships at a fixed time following administration of each drug. Additionally, both drugs were tested for possible cumulative effects. In initial experiments marrow samples were obtained from 2 test and from 2 solvent control animals at each dose and/or time interval; experiments were subsequently replicated with samples from i control and from 3 test rats. Time response. Male rats were injected intraperitoneally with single doses of 20oo mg/kg TMP, or of io mg/kg TEPA. Bone marrow samples were prepared at subsequent intervals of 6, 12, 24, 48, 72 and 96 h. These doses of both drugs had been shown to be subtoxic in previous experiments n,13. Dose response. Bone marrow samples were prepared 24 h following single intraperitoneal injections of TMP at arithmetically increasing doses of 5oo, 75o, IOOO, 125o, 15oo and 175o mg/kg, or of T E P A at logarithmically increasing doses of o.65, 1.25, 2.5, 5.o and IO mg/kg. Cumulative effects. Each drug was injected on four consecutive daily occasions at doses of 5oo mg/kg of TMP, or 5 mg/kg of TEPA. Marrow samples were prepared 6 and 24 h following the termination of treatment. Marrow samples were prepared on the basis of previously described procedures3L Preparations were evaluated, using a Leitz or Nikon microscope at iooo × magnification, for structural chromatid aberrations such as gaps (Fig. I), chromatid breaks (Fig. 2), isochromatid breaks (Fig. I) and reunion figures (Fig. 3)- Each affected cell was classified, depending on the progressive severity of their chromosome damage, as belonging to one of the following four categories: cells with gaps only, cells with chromatid breaks, cells with reunion figures, and cells with more than IO aberrations (Fig. 4). In Tables I - I I I the latter three categories are classified as "Cells with aberrations"; cells with gaps only are listed separately. Chromosome aberrations, such as deletions or translocations, are not reported quantitatively as chromosomes were not karyotyped. Total numbers of breaks per cell, based on the number of open breaks and the number of breaks involved in reunion figures, were not scored because more than two chromosomes were often involved in a complex reunion figure or because Mutation Res., I3 (~97 t) 263-273

]~ viT)o CYTOGENETIC EFFECTS ON BONE MARROW CELLS

265

2

1

%

+ w

,.

.".;',_,,

~

air

~R\.

m

M

I

.

B t,

~ %i4 B

•

~,e

-

. * l to

b

¢a b

I m

• q m

m

3

4

Figs. i - 4. Examples of chromatid aberrations caused by TEPA and TMP. I-3: G, gap; B, break; IB, isochromatid break; R, reunion figure. 4: Cell with more than IO aberrations. the n u m b e r of total aberrations was not countable. Tile percentage mitotic index was based on counts of 50o-6oo cells per animal. RESULTS No acute toxic effects, as evidenced b y m o r t a l i t y or weight loss, were observed in a n y groups of animals t r e a t e d with either T M P or T E P A at the specific doses. Time response. Chromatid aberrations induced b y T E P A were m a x i m a l at 24 h (Table I), when most affected cells exhibited more t h a n IO aberrations (Fig. 5). At 96 h these effects were still in slight excess of control levels. Mitotic a c t i v i t y was m a r k e d l y reduced u p to 72 h, a n d approached control values b y 96 h following treatment.

Mutation Res., z3 (I97z) 263-273

266

I. I). A])LER, G. RAMARAO, ~. S. I'I'KTI'~IN

***%

ro

*%

l

.°I 85 70

I

l

l

I

I

1

I

®

65

60

55

~o

~0

25

20

15

~li

CELLS WITH MORE THAN I0 ABERRATIONS

ID~

CELL S WITH REUNION FIGURES

f'"l

CELLS WITH GAJ~SONLY

JO

CELLS WITH BREAKS

0

HOURS AFTER

TREATMENT

Fig. 5. Mitotic index curve and distribution of c h r o m a t i d aberrations a m o n g the affected bone m a r r o w cells of male rats t r e a t e d with T E P A (IO.O mg/kg).

Following TMP treatment the incidence of chromatid aberrations was maximal at 48 h, although the 24-h value approached similar levels (Table I). Cells with gaps and breaks predominated up to I2 h, while cells with reunion figures and cells with more than IO aberrations were equally represented at later stages (Fig. 6). The chromosome-damaging effect of 2000 mg/kg of TMP was markedly lower than that of IO mg/kg of TEPA. Mitotic activity was only slightly decreased and approached control levels b y 48 h (Table I and Fig. 6). Analysis of variance for the frequency of cells with chromatid aberrations in TEPA-treated animals gave a pooled error standard deviation of 5.2 (I3%), with 16 degrees of freedom between animals. As can be seen (Table I) variability was maximal in the most highly affected groups. The pooled error standard deviation between animals for TMP was 2.3 (5.75%) with 18 degrees of freedom. The corresponding deviation between slides was 2 (5.0%) with 17 degrees of freedom based on Mutation Res., 13 (1971) 263-273

267

I n vivo C Y T O G E N E T I C E F F E C T S ON B O N E M A R R O W C E L L S TABLE

[

EVALUATION OF BONE MARROW CELLS OF MALE RATS TREATED WITH T E P A OR T M P SAMPLED AFTER DIFFERENT TIMES POSTTREATMENT (single i.p. i n j e c t i o n )

Drug

Dose (mg/hg)

Time (h)

Number Mitotic of index" animals (%)a

Total Cells with number gaps only of cells Number %

Cells with chromatid aberrations Number ~ ~c S.D.

TEPA

zo

6 12 24

4 4 5

4.5 1.9 2.0

200 200 200

9 15 2

4.5 7-5 I.O

17 12o

I75

48 72 96

5 2 2

4.9 3.8 9.8

200 ioo IOO

3 I I

1. 5 I.O I.O

12 6 3

6.0 _~ 1. 3 6.0 :[- 5.0 3.0 2~ 0.6

6 12 24

5 4 5

7 -1 5 .0 5.9

360 360 360

II 19 15

3.1 5.3 4 .2

7 3° 73

1.9 ~ 2.5 8-3 i 5 .o 20.3 ± 5.75

48 72 96

5 3 2

8.9 8.1 6.2

36o 200 ioo

8 2 I

2.2 I.O I.O

81 12 3

22.5 i 9.45 6.0 ~ 1.8 3 .0 :L o.o

17

11. 7

lO9 °

20

1.83

13

TMP

2000

Composite control

8.5 i 2.05 60.0 =E 26.0 87. 5 ~ 10. 9

1.19

a B a s e d o n 5 o o - 6 o o cells per a n i m a l .

3 slides per animal. Thus variability could not be reduced by increasing the number of slides examined per animal. One chromosome translocation was found in both TEPA- and TMP-treated groups 96 h after treatment. Dose response. Both T E P A and TMP induced a dose-related increased incidence of cells with chromatid aberrations and a concomitant dose-related decrease in the mitotic index (Table II). The dose scale was arithmetic for TMP and logarithmic for

®

# O/

I

i

I

i

i

i

I

25

of 15

~ m

I0

<[ ~

[]

Con~o!

6

12

24

HOURS A F T E R

48

72

--

CELLS WITH MORE THAN tO ABERRATIONS CELLS WITH REUNION FIGURES

m

CELLS WITH BREAKS

~

CELLS WITH GAPS ONLY

96

TREATMENT

F i g . 6. M i t o t i c i n d e x c u r v e a n d d i s t r i b u t i o n of c h r o m a t i d a b e r r a t i o n s a m o n g t h e a f f e c t e d b o n e m a r r o w cells of m a l e r a t s t r e a t e d w i t h T M P (2ooo m g / k g ) .

Mutation Res., 13 (1971) 2 6 3 - 2 7 3

268

1. 1). A 1 ) L E R ,

(1. t~AM.\I{A(), S. S. I,;I'STI(IN

' I ' A t31A'; 11 EVALUATION

OR T M I '

Drug

()[: B O N E MARRO~V (H-LLS ()I" MALl'2 R.\'I'S TREATI61) \V1TI| 1)II,'I;I';RI,IN F IIONE,'-; OI; "1"['[1~,\

(single i.p. injections}

Dose (nzg/kg)

TEPA

.\:umber oj cuTimals

Mitotic index (°i',)

Total nttmbcr orcells

Cells with gaps only Number °c,

0.65 1.25 2.5

4 4 5

~'.4 7.2 0. l

2oo 200 200

5 .0

5

5.2

200

IO.O

5

2.0

200

2

Control T31I )

,XNO SAM[>LIi:I) 24 h POSTTREATMt.'.NT

o 0 (> I2

(hI[s with chromatid aberrations A'~tmbcr o

S.1L

~ () 23

0 . 5 :i: ~.25 3 .o : 4 . 7 i [ - 5 1 8.-t

[3.0

78

J O -O ~

I.o

175

o 3.0 3-0

87.5

9"45

i: 1 o . 0

S

9.5

32o

I

o. 3

o

500 750 IOOO

5 5 2

8-4 Io.9 7.8

24o 200 20o

4 4 5

1,7 2.o 2. 5

l 0 ~2

I25 °

5

7.8

2oo

2

I.O

io

5.o

15oo

4

7.5

240

2

0.8

2()

io.S

175o 2000

5 5

7.1 5.9

280 360

3 I5

r.1 4 .2

33 73

I i . 8 ~: 7 . 0 2 o . 3 :~ 5 . 7 5

9

8,9

36o

2

o.56

Control

~

o 0.4 i 4.5 6.0 ~

I.I 3.7 t.8

{: 3 . o 5 [ 4.3

o,28

T E P A ; thus, examination of the dose-response slopes shows that for both compounds the percent of cells with chromatid aberrations increases 2- to 3-fold when the dose is doubled (Figs. 7 and 8). The dose of 2000 mg/kg TMP produced an incidence of aberrations comparable to that induced by doses of T E P A in the range of 2.5-5.0 mg/kg. At these dose levels not only is the overall percentage of affected cells similar but the distribution of these cells among the categories is approximately equal. However, when T E P A is given at IO mg/kg the cells with extensive aberrations predominate, whereas the dose of 2ooo mg/kg TMP causes less severe damage. The pooled error standard deviation between animals was 3.3 (8.25%) with 18 degrees of freedom for TEPA. The corresponding deviation for TMP was 1.6 ( 4 ° ) with 24 degrees of freedom. Cumulative effects. Repeated T E P A treatment resulted in a relatively low number of cells with aberrations (Table I I I ) as compared with single administration of the individual dose of 5 mg/kg (Table II). This is probably due to cell death as shown in tile drastically reduced mitotic index. The affected cells generally exhibit gaps, open breaks and reunion figures (Fig. 9). The chromosome-damaging effect of repeated doses of TMP was more marked (Table I I I ) than that induced by single administration of the individual dose of 5oo mg/kg (Table II). There was a substantial number of cells exhibiting more than IO aberrations in the sample taken 6 h after the termination of treatment (Fig. 9). Cells with gaps only were less frequent in the TMP group than in the controls. In contrast to T E P A the mitotic index was not significantly decreased as compared with controls. DISCUSSION

Various in vivo cytogenetic tests on bone marrow of mice have been reported %1~. The use of mice in these studies in part derives from their laboratory convenience, M u t a t i o n R e s . , 13 ( 1 9 7 1 ) 2 6 3 - 2 7 3

I n vivo

I0

269

C Y T O G E N E T I C E F F E C T S ON B O N E M A R R O W C E L L S

o°%

# I

1

I

I

I

l

45

40

:55

30

25

20 I

15

CELLS WITH MORE THAN IO ABERRATIONS CELLS WITH REUNION FIGURES

IC I~

CELLS WITH BREAKS CELLS WITH GAPS ONLY

0 Control

0.65

1.25

2.5

5.0

I0.0

TEPA (=~lk~)

Fig. 7. Mitotic index curve and distribution of c h r o m a t i d aberrations a m o n g the affected bone m a r r o w cells of male rats t r e a t e d with logarithmically increasing doses of T E P A .

their well established genetic background, and their common use in other mutagenicity tests such as the specific locus and dominant lethal assays. Unfortunately, the chromosome complement of mice is uniform ; all chromosomes are telocentric and thus poorly appropriate for cytogenetic analysis of mitosis. A more suitable species, in regard to its low chromosome number and differentiation in chromosome morphology, is the Chinese hamster which has been used in different laboratories4,31,41, ~°. However, hamsters require special breeding facilities and their use for routine studies is thus limited. Rats have certain advantages as experimental animals including a welldefined karyotype and a chromosome number (2n = 42) that approximatesthehuman. Rats have been successfully used in several laboratories to studycytogenetic effects in bone marrow6,aT, 39. The presumptive human relevance of in vivo cytogenetic data, as an indicator of genetic hazards, has been repeatedly emphasized recentlya~,38, 4°. Persisting cell lines with an abnormal chromosome complement in the bone marrow M*~tation Res., 13 (1971) 263-273

270

I. l). AI)I,I,;t,~, (;. I~,A3IAI{A(), S. S. E I ' S T I ~ I N

u)

o.......

0 /

I

I

i

I

I

I

i

~

(~)

25

A

15

1

I'-

l

CELLS WITH MORE THAN I0 ABERRATIONS

~

CELLS WiTH REUNION RGURES

I0 <[

1

1

Comlrol

500

I

750

I000

1250

1500

1750

~

CELLS WITH BREAKS

~

CELLS WiTH GAPSONLY

2000

TMP (mo/k~) F i g . 8. M i t o t i c i n d e x c u r v e a n d d i s t r i b u t i o n of c h r o m a t i d a b e r r a t i o n s a m o n g t h e a f f e c t e d b o n e m a r r o w cells of m a l e r a t s t r e a t e d w i t h a r i t h m e t i c a l l y i n c r e a s i n g d o s e s of T M P .

TABLE

III

EVALUATION OF BONE MARROW CELLS OF MALE RATS TREATED WITH T E P A OR T M P SUBACUTELY (4 d a i l y i.p. i n j e c t i o n s ) SAMPLED AT TWO TIMES AFTER THE LAST TREATMENT

Drug

Dose (mg/kg)

Time Number ]klitotic (h) of index (%)a animals

Total

Cells with

Cells with

number gaps only

q[cells

Number %

chromatid aberrations

Xrumber % 4~-S.DI TEPA

4"5 4" 5

6 24

Control

TMP Control

4"500 4-500

6 24

4 5

2.1 o-96

2

IO 12

6. 3 ~ 6.6 6.0 ~- 4.9

o

o

2.0 1.5

14 9

7.0 d2: 1. 5 4.5 4 1.8

2.5

o

o

16o 2oo

5 9

3 -1 4.5

8.8

8o

o

o

2 2

5-7 6.5

200 200

4 3

4

6-7

20o

5

a B a s e d o n 500 cells per a n i m a l .

or in other somatic cells m a y undergo malignant neoplastic change; germinal cells m a y be affected with the induction of heritable chromosome aberrations. In the present studies TMP and TEPA both induced chromosome damage, further confirming the results of other mutagenicity testsn-", zT. The sensitivities of the cytogenetic response to TEPA and TMP and their dose-response relationships appear similar to such parameters previously reported in the dominant lethal assay n, ia. The cytogenetic effects of these two drugs was, however, markedly distinct. 24 h after treatment the highest dose of TEPA produced extensive chromosomal damage and marked inhibition of cell replication; only a minimal number of cells with chromosomal abnormalities, however, were detected after 48 h. It thus appears that the chromo3¢utation Res., 13 (1971) 2 6 3 - 2 7 3

[tl vivo

CYTOGENETIC EFFECTS ON BONE MARROW CELLS

TEPA

TMP

...... ~

~

o

O/

I

! Control

I

271

I

I

I

6

24

I

I

®

I

m

CELLS WITH MORETHAN I0 ABERRATIONS

B

CELLS WiTH REUNION FIGURES

f nMm

HOURS A F T E R

I Control

I 6

CELLS WITH BREAKS CELLS WITH GAPSONLY

I 24

TREATMENT

Fig. 9. M i t o t i c i n d e x c u r v e s a n d d i s t r i b u t i o n of c h r o m a t i d a b e r r a t i o n s a m o n g t h e affected bone m a r r o w cells of m a l e r a t s s u b a c u t e l y t r e a t e d w i t h T E P A or TMP.

somal damage induced cell death. The highest dose of TMP tested produced a less severe overall effect. With io mg/kg T E P A about 88% of the cells showed aberrations. If an approximate linear regression is drawn to the TMP dose-response pattern and projected, a dose of 64000 mg/kg TMP would be required to induce aberrations in 88% of the cells. The cumulative treatment still further illustrates the different effects of these drugs. Repeated administration of 500 mg/kg of TMP caused an accumulation of cells with aberrations to 4.5~o in cells examined 24 h after the termination of treatment in contrast with the 0.4% level detected after a single dose. However, the cumulative treatment with 5 mg/kg of T E P A resulted in a 6~o incidence of cells with aberrations, as opposed to 39% following treatment with a single dose; this difference again probably reflects the elimination of severely damaged cells. This conclusion is confirmed b y the extremely low mitotic index. Those dose-response curves do not reflect the actual maximal effects of all doses since the duration of the cell cycle is affected differently by lower and higher doses of alkylating agents. The m a x i m u m number of mitoses with chromatid aberrations might occur earlier than 24 h posttreatment with lower doses because mitotic delay is less severe. Detailed time responses for all doses and autoradiographic estimation of the duration of the different stages of cell cycle depending on kind and dose of test compound exceed the feasibility of a screening test like the present. Both drugs induced increased incidences of chromatid aberrations. No dicentric or ring chromosomes were observed. One chromosome translocation was found in both the T E P A - and TMP-treated groups 96 h after treatment; detailed karyotyping might have revealed more aberrations of the chromosome type. Since the cytogenetic effect of the high doses of TMP is less severe than that induced by the lower doses of TEPA, cells affected by TMP would be more likely to survive, yielding cell lines with persisting and transmissible chromosome aberrations. TMP m a y thus pose greater potential genetic hazards than TEPA. Mutation Res., 13 (1971) 263-273

272

1. 1). AI)Lt._'I-,', (;. RAMAI{A{), >. 5. EPSI'I,.IN

ACKNOWLEDGMENTS

We thank Dr. Y. BISHOP and Mrs. E. ARNOLD for l~elpful discussions and statistical assistance. This work was supported by NIH Grants C-6516 and Kr-o5526, and by the Environmental Protection Agency Contract CPA-7o-I7. REFEtlENCES I BARNES, C. A., AND 9. L. T. ILBERY, C y t o g e n e t i c t e c h n i q u e s in radiobiological procedures. Australasian Radiol., 12 (I968) 395-402. 2 BILLMAN, F. H., A. RADIKE AND B. W. MUNDY, A l k y l a t i o n of anlines, I, J. Am. Chem. Soc., 64 (1942 ) 2977-2978. 3 BORKOVEC, A. B., Insect Chemosterilants, W i l e y (Interscience), N e w York, r966. 4 BROOKS, A. L., AND R. O. ~/[cCLEELAN, C h r o m o s o m e a b e r r a t i o n s a n d o t h e r effects p r o d u c e d by 90Sr_ 90y in Chinese h a m s t e r s , Intern. J. Radiation Biol., 16 11969 ) 545--56I. 5 CHANG, T. H., AND VVr. CLASSEN, C o m p a r a t i v e effects of t e t r a m i n e , T E P A , a p h o l a t e a n d their s t r u c t u r a l a n a l o g s on h u m a n c h r o m o s o m e s in vitro, Chromosoma, 24 11968) 314-323 . 6 CHAIJDHURI, V. P., AND H. LANGENDORFF, Chemical r a d i o p r o t e c t i o n of m a m m a l i a n c h r o m o seines in rive; R a d i o p r o t e c t i o n of r a t bone m a r r o w c h r o m o s o m e s with a single p r o p h y l a c t i c dose of A E T , Intern. J. Radiation Biol., 14 11968) 463-467 . 7 DATTA, P. K., AND E. SCHLEIERMACHER, T h e effects of c y t o x a n on t h e chromosomes of m o u s e bone m a r r o w , Mutation Res., 8 11969) 623-628. 8 DEICHMANN, W. B., AND S. WITERUP, O b s e r v a t i o n s on t h e effects of t r i m e t h y l p h o s p h a t e u p o n e x p e r i m e n t a l animals, J. Pharmacol. Exptl. Therap., 88 (1946) 338-342. 9 DRAKE, G. L., F l a m e r e t a r d a n t s a n d t h e i r a p p l i c a t i o n to textiles, Bull. N. Y. Aead. Med., 43 11959) 656-662. io DRAKE, G. L., AND J. D. GUTHRIE, I m p a r t i n g crease r e s i s t a n c e a n d crease r e t e n t i o n to c o t t o n with A P e , Textile Res. J., 29 11959) 155-164. i I EPSTEIN, S. S., W. BASS, E. ARNOLD AND Y. BISHOP, M u t a g e n i c i t y of t r i u l e t h y l p h o s p h a t e in mice, Science, 168 (197 ° ) 584-586. i2 EPSTEIN, S. S., S. R. JOSHI, E. ARNOLD, E. C. PAGE AND Y. t~ISHOP, A b n o r m a l z y g o t e d e v c l o p m e n t in mice after p a t e r n a l e x p o s u r e to a chemical m u t a g e n , Nature, 225 (I97 o} 126o-126i. 13 EPSTEIN, S. S., E. ARNOLD, 1~. STEINEERG, D. MACKINTOSH, H. SHAPNER AND Y. BISHOP, M u t a g e n i c i t y a n d a n t i f e r t i l i t y effects of T E P A a n d M E T E P A in mice, Toxicol. Appl. Pharmacol., 17 1197o ) 23-40. 14 EPSTEIN, S, S., W. BASS, E.. ARNOLD, Y. BISHOP, S. JosHI AND I. D. ADL~:R, Sterility a n d s e m i s t e r i l i t y in m a l e p r o g e n y of male mice t r e a t e d w i t h t h e chemical m u t a g e n T E P A , To~'icoA Appl. Pharmacol., 19 (I97I) I 3 4 - I 4 6 , 15 ERGOZCUE, J., Effect of LSD-25 on m i t o t i c a n d meiotic chronlosonles of mice a n d m o n k e y s , Humangenetik, 8 (1969) 86 9316 EARBER, S., t{. APPLETON, V. DOW'NING, I:. HEALD, J. KING AND R. TOClI, Clinical s t u d i e s of t h e carcinolvtic action of t r i e t h y l e n e p h o s p h a m i d e s , Cancer, 6 11953 ) 135-14 t. 17 GRoss, D., "Chemical s y n o v e c t o m y w i t h m u s t a r d gas in p r i n l a r y chronic polyarthritis, Z. Rheumaforsch., 22 (1963) 456 459. 18 HATHAWAY, D. O., Sterilizing of codling m o t h s b y aerosol t r e a t n l e n t with T E P A , ,/. Eco,z. Entomol., 6t 11968) 322-323 . 19 HEDIN, P. A., Sterility i n d u c e d b y T E P A in t h e boll weevil, Effective dose a n d p e r m a n e n c y , g o n a d a l c h a n g e s , a n d biological t u r n o v e r of labeled c o n l p o u n d s , J. Econ. Entomok, 60 0967) 2o9-214. 20 HOLMESEN, T. W., AND J. E . LEASURE, G r o w t h - i n h i b i t i n g action of tris-(-i-aziridinyl}-phosp h i n e oxide in grasses, Science, 153 11966) 1659. 21 HOWLS, R. G., I. C. ISDALE, B. S. ROSE AND P. ~T. CONLON, E x p e r i e n c e s w i t h intra-artictflar t h i o - T E P A in r h e u m a t o i d arthritis, New Zealand Med..1., 66 11967) 667-669. 22 HUDSON, P. S., AND C. C. BraE, Use of A P e a n d like c o m p o u n d s as i n g r e d i e n t s of solid rocket p r o p e l l a n t c o m p o s i t i o n s , U.S. P a t e n t 3 o87 844 (to ]Phillips P e t r o l e u m Co.). 23 HUNGERFORD, D. A., AND P. C. NOWELL, Sex c h r o m o s o m e p o l y m o r p h i s m a n d t h e n o r m a l k a r y o t y p e in t h r e e s t r a i n s of t h e l a b o r a t o r y rat, J . zllorphd., I t 3 (I963) 275--28524 JACKSON, H., AND A. R. JONES, Antifertility action a n d m e t a b o l i s m of t r i m e t h y l p h o s p h a t e in rodents, Nature, 220 (1968) 591-592. 25 JONES, A. R., AND H. JACKSON, C h e m o s t e r i l a n t action of t r i m e t h y l p h o s p h a t e in rodents, Brit. J. Pharmacol., 37 11969) 531 532.

Mutation Res., 13 (1971) 263-273

In vivo CYTOGENETIC EFFECTS ON BONE MARROW CELLS

273

26 JONES, F. W., G. O. OSBORNE, G. J. SUTHERLAND, R. D. TOPSOM AND J. VAUGHAN, T h e n u clear a l k y l a t i o n of a r y l a m i n e s b y trialkyl p h o s p h a t e s , Chem. Commun., I (1966) 18-19. 27 JOSHI, S. R., E. C. PAGE, E. ARNOLD, Y. BISHOP AND S. S. EPSTEIN, Fertilization a n d e a r l y e m b r y o n i c d e v e l o p m e n t s u b s e q u e n t to m a t i n g w i t h T E P A - t r e a t e d male mice, Genetics, 65 (197 ° ) 483-494 . 28 KALOOSTI&N, G. H., C h e m o s t e r i l i z a t i o n of m a l e p e a r p s y l l a w i t h T E P A , J. Econ. Entomol,, 61 (1968) 573-57429 KATO, M. B., AND Y. TEGNER, C h o m o s o m e b r e a k a g e i n d u c e d in vivo b y a carcinogenic h y d r o c a r b o n in bone m a r r o w ceils of t h e Chinese h a m s t e r , Hereditas, 61 (1969) 1-8. 3 ° ]~ATO, R., Localization of s p o n t a n e o u s a n d r o u s s a r c o m a v i r u s - i n d u c e d b r e a k a g e in specific regions of t h e c h r o m o s o m e s of t h e Chinese h a m s t e r , Hereditas, 58 (1967) 221-247. 31 KATO, R., C h r o m o s o m e b r e a k a g e i n d u c e d b y a carcinogenic h y d r o c a r b o n in Chinese h a m s t e r cells a n d h u m a n l e u k o c y t e s in vitro, Hereditas, 59 (1968) 12o-141. 32 KOLMARK, G., M u t a g e n i c properties of certain esters of inorganic acids i n v e s t i g a t e d b y t h e N e u r o s p o r a b a c k - m u t a t i o n test, Compt. Rend. Tray. Lab. Carlsberg, 26 (1956) 205-220. 33 LABRECQUE, G. G., AND C. N. SMITH, Principles of insect chemosterilization, A p p l e t o n - C e n t u r y Crofts, N e w York, 1968. 34 LADD, 5 R, T. L., M a s s sterilization of J a p a n e s e beetles with T E P A a n d d e t e r m i n a t i o n of residues, J, Econ. Entomol., 61 (1968) 942-944 . 35 LEGATOR, M. S., AND C. JACOBSON, Chemical m u t a g e n s as a genetic h a z a r d , Clin. Proc. Children's Hosp., 14 (1968) 184-189. 36 LEGATOR, M. S., AND C. JACOBSON, D e t e c t i o n of c y t o g e n e t i c effects of toxic a g e n t s in m a m m a lian s y s t e m s , in F. W. SUNDERMAN (Ed.), Manual of Procedures for Laboratory Diagnosis of Diseases Caused by Toxic Agents, Assoc. Clin. Sci., Section 20, 1968, pp. i - I O . 37 LEGATOR, M. S., K. A. PALMER, S. GREEN AND K. W. PETERSEN, C y t o g e n e t i c s t u d i e s in r a t s b y c y c l o h e x y l a m i n e , a m e t a b o l i t e of c y c l a m a t e , Science, 165 (1969) 1139-114o. 38 NICHOLS, W. W., P. MOORHEAD, G. BREWEN, I. D. ADLER, A. D. CONGER, J. GERMAN, 5" A. HEDDLE, D. A. HUNGEREORD, P. A. JACOBS, M. S. LEGATOR, P. C. NOWELL, A. H. SPARROW AND W. W. ZUELZER, C h r o m o s o m e nlethodologies in m u t a t i o n testing, E M S Newsletter, in t h e press. 39 PETERSEN, K. W., R e p e a t a b l e b o n e m a r r o w b i o p s y w i t h n o n - c u l t u r e cell p r e p a r a t i o n for cytogenetic s t u d i e s in rats, Stain Technol., 44 (1969) 3o8-3o9. 4 ° Report of the Advisory Panel on Mutagenicity of Pesticides, Report of the Secretary's Commission on Pesticides and their Relationship to Environmental Health, U.S. Dept. H e a l t h , E d u c a t i o n a n d Welfare, D e c e m b e r 1969, pp. 567-653 . 41 SCHMIDT, W., AND G. R. STAIGER, C h r o m o s o m e s t u d i e s on bone m a r r o w f r o m Chinese h a m s t e r s t r e a t e d w i t h b e n z o d i a z e p i n e t r a n q u i l i z e r s a n d c y c l o p h o s p h a m i d e , Mutation Res., 7 (1969) 99 IO8. 42 SHAW, J. G., N. P. PATTON, R. M. SANCV:EZ, L. M. SPISHAKOEE AND B. C. REED, M e x i c a n f r u i t fly control, Calif. Citrograph, 51 (1966) 2oo-2o 3. 43 SKINNER, W. A., AND H. C. TONG, Effects of a l k y l a t i n g a g e n t s derived f r o m d i a m i n e s on fertility of t h e male mouse, Experientia, 24 {I968) 924-925. 44 SOLOMON, J. D., T E P A for sterilizing male c a r p e n t e r w o r m s , J. Econ. Entomol., 59 (1966) 15281529. 45 SOMMER, H., AND F. LINKE, N e w possibilities in sizing w i t h s y n t h e t i c resins, Tantex, 27 (1962) 283-293. 46 SRAM, R. J., z. ZoDoVA, V. BENES AND K. SYMON, I n d u c t i o n of t r a n s l o c a t i o n in mice b y T E P A , E M S Newsletter, 3 (197 o) 13. 47 T o y , A. D. F., T h e u s e of t r i m e t h y l p h o s p h a t e as a m e t h y l a t i n g agent, J. Am. Chem. Soc., 66 (1944) 499. 48 T o Y , A . D. F., P r e p a r a t i o n of t e t r a m e t h y l p y r o p h o s p h a t e , J. Am.Chem. Sot., 71 (1949) 2268-2269. 49 WRIGHT, G. 5", AND U. K. ROWE, E t h y l e n e i m i n e ; Studies of t h e d i s t r i b u t i o n a n d m e t a b o l i s m in t h e r a t u s i n g c a r b o n - I 4, Toxicol. Appl. Pharmacol., II (1967) 575-584. 5 ° YERGANIAN, C., AND K. S. LAVAPPA, P r o c e d u r e s for c u l t u r i n g diploid cells a n d p r e p a r a t i o n of meiotic c h r o m o s o m e s f r o m d w a r f species of h a m s t e r s , in A. HOLLAENDER (Ed.), Chemical Mutagens: Principles and Methods for their Detection, Vol. 2, P l e n u m , New Yo, k, 1971, p. 387 . 51 YOUNG, J. R., AND 5" W. SNOW, T E P A as a c h e m o s t e r i l a n t for t h e corn earworm, a r m y w o r m a n d g r a n u l a t e d c u t w o r m , J. Econ. Entomol., 60 (1967) 1427-143o.

Mutation Res., 13 (1971) 263-273