Fd Cosmet. ToxicoL Vol. 8, pp. 617-623. Pergamon Press 1970. Printed in Great Britain

Research Section In vitro Cytogenetic Investigation of Calcium Cyclamate, Cyclohexylamine

and Triflupromazine

S. GREEN, K. A. PALMER and M. S. LEGATOR

Division of Toxicology, Food and Drug Administration, Department of Health, Education, and Welfare, Washington, D.C. 20204, USA

(Received 1 June 1970)

Abstract--Cytogenetic effects after varied times of exposure to calcium cyclamate, cyclo- hexylamine (CHA) or triflupromazine HCI (TFP) were determined in a cell line derived from the kidney of the rat kangaroo (Potorous tridactylis apicalis). A preliminary study, using afla- toxin and mitomycin C, which are known to produce abnormalities in cell systems, established the suitability of this cell line for the detection of chromosomal aberrations. In the present study, mitomycin C produced frequent chromatid gaps and breaks in the arms of the chromo- somes. Aberrations of the chromosome-type as well as exchanges were noted with aflatoxin. The X-chromosome was extremely sensitive to these agents. CHA produced single chromatid breaks and TFP produced single chromatid breaks and exchange figures. The X-chromosome was not exceptionally sensitive to either of these agents. Calcium cyclamate at concentrations up to 200 p.g/ml produced no chromosomal aberrations.

INTRODUCTION

Several in vitro cytogenetic analyses have been developed for testing agents for possible mutagenic activity (Legator, Kelly, Green & Oswald, 1969; Green, Legator & Jacobsen, 1967; Auerbach, 1967; Price, Buck & Lein, 1964). Such systems, in addition to yielding basic information about mutagenicity, also serve as valuable corollaries to in rive studies. This paper reports results obtained from the in vitro analysis of chromosomes from a cell line derived from the rat kangaroo, Potorous tridactylis apiealis, after treatment with calcium cyclamate, cyclohexylamine (CHA) or triflupromazine (TFP) hydrochloride. The selection of these agents was based on their extensive use by the general population including those of child-bearing age.

In recent years calcium cyclamate has been widely used as a non-nutritive sweetener, and its metabolism is therefore of interest. Previous studies have shown that the rat (R. C. Senders, unpublished report 1968), dog (Kojima & Ichibagase, 1966) and man (Leahy, Wakefield & Taylor, 1967) can metabolize cyclamate to CHA. However, recent studies have indicated that the intestinal flora are actually responsible for this conversion (Golberg, Parek_h, Patti & Soike, 1969). Therefore, an examination of the potential mutagenicity of calcium cyclamate and its metabolite seemed warranted.

The phenothiazine tranquillizers represent a class of drugs widely used in the therapy of neurological disorders, such as excited psychoses and psychoneuroses. They are also used as antiemetics. Cohen, Hirschhorn & Frosch (1969) reported negative results with chlorpro- mazine, thioridazine and fluphenazine in cytogenetic studies with human leucocytes.

617

618 S. GREEN, K. A. PALMER and M. S. LEGATOR

Because of its widespread use, TFP, another phenothiazine derivative, was investigated in our laboratory for cytogenetic effects.

Mitomycin C and aflatoxin are known to induce chromosomal aberrations in numerous cell systems. Shaw & Cohen (1965) reported chromosomal abnormalities in human leucocytes treated with mitomycin C. Crude aflatoxin was previously reported to produce alterations in the chromosomes of Viciafaba (Lilly, 1965) and in human peripheral leucocytes (Dolimpio, Jacobson & Legator, 1968). Therefore these two agents were tested in a preliminary study to establish the suitability of the rat-kangaroo cell line for these cytogenetic analyses.

EXPERIMENTAL

Cell culture. Monolayers of a rat-kangaroo cell line grown in milk dilution bottles were incubated at 37°C in an atmosphere of 3 ~ CO2. A typical karyotype is shown in Fig. 1. The growth medium consisted of Eagle's Minimum Essential Medium with the following additives: I0~o foetal calf serum, 1 ~ glutamine, 1 ~ non-essential amino acids, and peni- cillin and streptomycin (I0 IU of each/ml). The medium and additives were obtained from Flow Laboratories, Rockville, Md.

Materials. Crude aflatoxin was obtained from Dr. A. D. Campbell, Food and Drug Administration, and mitomycin C from Dr. A. E. Osterberg, Cancer Chemotherapy National Service Center, National Institutes of Health. Calcium cyclamate and CHA were obtained from City Chemical Corp., New York, and TFP.HC1 came from E. R. Squibb and Sons, Inc., New York. The aflatoxin was a crude preparation consisting of 15 ~o aflatoxin B1 and lower concentrations of the other three fractions, B2, G1 and G2. The sample of CHA used was tested for purity by gas chromatography. No peaks were observed other than the peak from CHA. The purity of calcium cyclamate was tested by extracting CHA according to procedures outlined by Howard, Fazio, Klimeck & White (1969). The salt was then prepared and chromatographed on thin-layer plates. For the identification and semi-quantitative determination of the amount of CHA, the spots were compared visually with appropriate standards after spraying with ninhydrin. By this procedure, 20-40 ppm CHA was found in the calcium cyclamate.

Experimental procedures. In the experiments with aflatoxin and mitomycin, the growth medium was removed 1 or 2 days after planting and the cells were incubated for 8 hr in medium containing 12-5 or 25/zg/ml aflatoxin or 10/zg/ml mitomycin C. Cells treated on day 1 or day 2 were examined 64 and 40 hr, respectively, after removal of the agents. Similarly CHA (adjusted to pH 7.3 with 1 N-HC1) was added on day 1 for 24 hr at concentrations of l, 10, 50, 100 or 500/zg/ml. The cells were examined at 1, 24 and 48 hr after the removal of CHA. Calcium cyclamate was added on day 1 for 24 hr at concentrations of 100 or 200 /~g/ml, and cells were examined 48 hr after removal of the agent. TFP was added on day 3 for 8 hr at concentrations of 5, 10 or 20/zg/ml and cells were examined 16 hr after removal of TFP. The treatment times indicate the minimum length of exposure of the ceils to the agent before abnormalities could be detected. These agents had dissimilar effects on the cells and therefore the length of exposure and consequently the recovery time differed.

After exposure to the various chemicals the cells were washed with Hank's Balanced Salt Solution, refed with fresh medium and allowed to grow until they were harvested. Colcemid (Ciba Pharmaceutical Co., Summit, N.J.) was added at a concentration of 0.33/~g/ml 16 hr before harvesting. The cells were processed by the procedure of Moorhead, Nowell, Mellman, Battips & Hungerford (1960), stained with Giemsa stain and magnified

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CYTOGENETIC STUDY OF CYCLAMATE 619

by I000 for examination. At least 100 metaphases were analysed for each concentration of treated cells, except in the case of aflatoxin, and at least 300 were analysed for non-treated controls. The mitotic index, defined as the percentage of cells in mitosis, was determined on the basis of at least 1000 cells. In the cytogenetic analysis, breaks in the chrornatid and the centromere of chromosomes were determined and scored according to the chromosome group. A break was defined as a deletion greater than one chromatid width. A distinction should be made between the centromere and heterochromatic regions. The former is defined as the region immediately adjacent to the centromere, whereas the latter in reference to the X-chromosome is a secondary constriction in the long arm proximal to the centromere. Breaks in this area of the X-chromosome are listed under the centromere region later in this paper.

Confidence intervals were computed to determine whether there were significant differ- ences between treated and control sample populations for the aflatoxin and mitomycin data and for the data on chromosome breaks and the mitotic index in CHA- and TFP-treated ceils. A chi-square analysis of the distribution of breaks among chromosome groups was performed for each type of treatment; in this analysis, the breaks in the centromere region and in the arms were combined.

RESULTS

Concentrations of 12.5 and 25 p.g aflatoxin/ml added on day I resulted in 51/150 and 31/75 cells with breaks, respectively; the same concentrations added on day 2 resulted in 30/128 and 33/78 cells with breaks, respectively. Mitomycin at I0 p.g/ml added on day 2 resulted in 43/100 cells with breaks. The number of breaks in control cells was 2/300. All concentrations of aflatoxin and mitomycin significantly inhibited mitosis and significantly increased the incidence of chromosomal aberrations (P < 0.05). In contrast to the negligible effect of mitomycin C and aflatoxin on regions adjacent to the centromere of the autosomes, the X-chromosome showed a disproportionate sensitivity to these chemicals (Table I). This sensitivity was attributed to the heterochromatic region proximal to the centromere in the long arm.

Calcium cyclamate added at concentrations up to 200/zg/ml for 24 hr on day 1 did not inhibit mitosis or cause chromosome breaks.

With CHA, mitotic inhibition was found after 1 or 24 hr but not after 48 hr recovery. At 48 hr, however, the maximum number of chromosomal abnormalities was found. Table 2 illustrates the percentage of cells showing breaks at the various concentrations of CHA. The number of breaks per chromosome in the CHA-treated cells, shown in Table 1, indicated a significant difference between the expected and observed breaks based on chromosome length. The no. 3 autosome accounted for almost half of the total breaks.

TFP produced a significant decrease in the mitotic index on day 3 at 20 p.g/ml; below this level, inhibition was insignificant. Table 1 gives the location of breaks and Table 2 sum- marizes the percentage of cells with breaks. Rearrangements as well as chromatid breaks occurred with this agent.

Figure 2 (A-D) illustrates the typical chromosomal abnormalities observed with each agent.

DISCUSSION

The difference in the behaviour of the X-chromosome and the autosomes to aflatoxin may be explained by the probable origin of the X-chromosome from an X-autosomal transloca-

0

Tab

le 1

. Loc

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g ch

rom

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llow

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ith

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tro

mer

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t w

ith

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A

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A

TF

P

Ch

rom

oso

me

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p A

flat

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M

ito

my

cin

C

T

C

T

A

flat

oxin

M

ito

my

cin

C

T

C

T

Con

trib

utio

n to

x 2

CH

A

TF

P

Afl

atox

in

Mit

om

yci

n

C

T

C

T

rrl z

1 30

27

5

25

3 17

6

4 3

3 4

12

1-03

20

.70

1.01

0-

05

0.49

4.

29

2 19

17

3

19

5 11

7

6 0

2 2

6 3.

99

6-97

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94

0.63

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07

0.06

3

9 2

4 36

3

20

5 1

3 12

8

12

8.04

7-

86

2.44

38

-59

6.74

23

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4 5

2 2

3 0

0 3

2 0

3 I

I 4.

55

1-21

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06

6.23

1.

26

7.14

5

1 0

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2 0

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5.06

2-

30

1.54

7.

37

1.88

5.

96

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15

8 2

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6 10

6.

10

6.09

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28

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Tot

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28.7

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45.1

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T

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01 (

5 d.

f.).

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the

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ated

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all

ch

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CYTOGENETIC STUDY OF CYCLAMATE 621

Table 2. Percentage o f cells with breaks and the mitotic index o f cells treated with CHA for 24 hr or with TFP for 8 hr

Concentration Proportion of cells of agent Mitotic indext with breakst (/~g/ml) (~ ) (with %)

CHA 0 (control) 12.3 23/301 = 7.64 1 12.4 15/182 = 8-24 10 12.4 27/232 = 11.64 50 12.25 14/100 = 14.00" 100 12.0 28/165 = 16.97' 500 12.0 34/168 = 20.24*

TFP 0 (control) 9.7 28/327 = 8'56 5 9.5 8/102 = 7.84 10 8.2 14/108 = 12-96" 20 6.7* 31[100 = 31.00"

tDetermined 48 hr after removal of CHA and 16 hr after removal of TFP. Cells were treated with CHA and TFP 24 and 72 hr, respectively, after planting.

Values marked with an asterisk differ significantly from those of controls: *P < 0'05.

t ion, as p roposed by Moore (1965). The he te rochromat ic segment in the long a rm may mark the point of fusion of the two chromosomes . This could change the sensit ivity of the X- ch romosome and also explain the difference in local iza t ion of breaks as compared to the autosomes. W h y this area was not affected by the other three agents is not known. The act ion of af latoxin in this m a m m a l i a n system may be s imilar to that o f maleic hydraz ide in plants (Kih lman, 1966); that is, the ch romosome-break ing effect may increase with the presence of he terochromat in .

Table 1 also shows that C H A and T F P affect the no. 3 au tosome d i spropor t iona te ly . This is also shown by the high chi-square values for this ch romosome for C H A and T F P . The cont ro l chi-square value in bo th instances was not significant, a l though the no. 3 au tosome did indicate a higher lability.

This s tudy also shows that the two major agents, C H A and T F P , cart be separa ted on the basis o f the t ime at which ch romat id aber ra t ions begin to appear in metaphase after t rea t - ment. C H A had a delayed effect; no aber ra t ions could be detected un t i l 40-48 hr after t rea tment . The effect of T F P , in contrast , was not delayed and aber ra t ions could be found 16 hr after chemical t reatment . K ih lman (1966) suggested that the t ime at which aber ra t ions occur may give useful in format ion abou t an agent and also serve as a me thod o f drug classification.

This study confirms the in vivo work o f Legator , Palmer , Green & Petersert (1969). The in format ion ob ta ined f rom this cell line, e.g. the specific ch romosome affected and the locat ion of the aberra t ion , may help in character iz ing the mutagenic act ivi ty o f C H A derivatives, such as N-hydroxy C H A . Agents act ing on the same ch romosome and at the same locat ion on that ch romosome are thought to have similar mutagenic ac t ion; but whether or not C H A derivatives would have the same act ion as C H A is not yet known.

622 S. GREEN, K. A. PALMER a n d M. S. LEGATOR

E v a l u a t i o n s o f th i s type c a n n o t be t a k e n as conc lus ive e v i d e n c e t h a t a n a g e n t is m u t a g e n i c

in m a n . H o w e v e r , in vitro cy togene t i c s s h o u l d be c o n s i d e r e d as v a l u a b l e i n d i c a t o r sy s t ems ,

p o i n t i n g t o w a r d s the n e e d fo r a d d i t i o n a l , a n d p a r t i c u l a r l y in vivo, s tud ies .

Acknowledgement--The authors acknowledge the assistance of Dr. Leonard Friedman, Lillian Yin, Holdine Roginski and William Pankey.

R E F E R E N C E S

Auerbach, C. (1967). The chemical production of mutations. The effect of chemical mutagens on cells and their genetic material is discussed. Science, N. Y. 158, 1141.

Cohen, M. M., Hirschhorn, K. & Frosch, W. A. (1969). Cytogenetic effects of tranquilizing drugs in vivo and in vitro. J. Am. reed. Ass. 207, 2425.

Dolimpio, D. A., Jacobson, C. & Legator, M. (1968). Effect of aflatoxin on human leukocytes. Proc. Soe. exp. BioL Med. 127, 559.

Golberg, L., Parekh, C., Patti, A. & Soike, K. (1969). Cyclamate degradation in mammals and in vitro. Toxic. appl. Pharmac. 14, 654.

Green, S., Legator, M. & Jacobson, C. (1967). Utilization of a cell line derived from the rat-kangaroo for cytogenetic studies. Mature. Chrom. 8, 36.

Howard, J. W., Fazio, T., Klimeck, Barbara A. & White, R. H. (1969). Determination of cyclohexylamine in various artificially sweetened foods and artificial sweeteners. J. Ass. off. analyt. Chem. 52, 492.

Kihlman, B. A. (1966). Actions of Chemicals on Dividing Cells. 1st ed. p. 121. Prentice-Hall, Inc., Englewood Cliffs, New Jersey.

Kojima, S. & Ichibagase, H. (1966). Studies on synthetic sweetening agents. VIII. Cyclohexylamine, a metabolite of sodium cyclamate. Chem. pharm. Bull., Tokyo 14, 971.

Leahy, J. S., Wakefield, M. & Taylor, T. (1967). Urinary excretion of cyclohexylamine following oral administration of sodium cyclamate to man. Fd Cosmet. Toxicol. 5, 447.

Legator, M. S., Kelly, F. J., Green, S. & Oswald, E. J. (1969). Mutagenic effects of captan. Ann. N. Y. Acad. Sci. 160, 344.

Legator, M. S., Palmer, K. A., Green, S. & Petersen, K. W. (1969). Cytogenetic studies in rats of cyclo- hexylamine, a metabolite of cyclamate. Science, N. Y. 165, 1139.

Lilly, Lorna J. (1965). Induction of chromosome aberrations by aflatoxin. Nature, Lond. 207, 433. Moore, R. (1965). A biometric analysis of the chromosomes of the marsupials--Macropus major, Macropus

rufus and Potorous tridactylis. Cytogenetics 4, 145. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M. & Hungerford, D. A. (1960). Chromosome

preparations of leukocytes cultured from human peripheral blood. Expl Cell Res. 20, 613. Price, K. E., Buck, R. E. & Lein, J. (1964). Incidence of antineoplastic activity among antibiotics found to be

inducers oflysogenic bacteria. In Antimicrobial Agents and Chemotherapy. p. 505. Edited by J. C. Sylvester. American Society for Microbiology, Ann Arbor, Michigan.

Shaw, Margery & Cohen, M. M. (I 965). Chromosome exchanges in human leukocytes induced by mitomycin C. Genetics, N.Y. 51, 181.

l~tude cytog~n~tique in vitro du cyclamate de calcium, de la cyclohexylamine et de la triflupromazine

R6sum6----Les effets cytog6n6tiques cons~cutifs ~ des dur6es d'exposition variables au cyclamate de calcium, a la cyclohexylamine (CHA) e t a la triflupromazine-HCl (TFP) ont 6t6 d6termin6s sur une lign6e de cellules provenant du rein du kangourou-rat (Potorous tridactylis apicalis). Des reoherches pr61iminaires utilisant l'aflatoxine et la mitomycine C, substances connues pour provoquer des anomalies des syst~mes cellulaires, avaient 6tabli que cette lign6e de cellules convenait pour d6pister des aberrations chromosomiques. La mitomycine C a provoqu6 de nombreuses lacunes de chromatides et de cassures de bras des chromosomes au cours des recherches ddcrites ici. L'aflatoxine a donn6 lieu ~. des aberrations du type chromosomique ainsi qu'a des 6changes. Le chromosome X 6tait ex'tr~mement sensible ~t ces deux agents. La CHA a provoqu6 des cassures isol6es de chromatides et la TFP des cassures isol6es de chroma- tides et des figures d'6change. Le chromosome X n'6tait pas e×ceptionnellement sensible h l 'un ou b. l'autre de ces agents. Le cyclamate de calcium b. des concentrations allant jusqu'h. 200 t~g/ml n 'a pas provoqu6 d'aberrations chromosomiques.

CYTOGENETIC STUDY OF CYCLAMATE 623

Cytogenetische in-vitro-Untersuchung von Calciumcyclamat, Cyclohexylamin wad Triflupromazin

Zusammenfassung--Cytogenetische Wirkungen nach unterschiedlichen Einwirkungszeiten von Calciumcyclamat, Cyclohe×ylamin (CHA) und Triflupromazin-HCl (TFP) wurden an einem aus der Niere der K/inguruhratte (Potorous tridactylis apicalis) gewonnenen Zellstamm untersucht. Eine vorangehende Untersuchung mit Aflatoxin und Mitomycin C, von denen bekannt ist, dass sie in Zellensystemen Anomalit/iten hervorrufen, ergab die Eignung dieses Zellstamms for die Entdeckung von Chromosomenaberrationen. Bei der gegenw~irtigen Unter- suchung verursachte Mitomycin C h~ufige Chromatidlficken und Br/.iche in den Ausl~ufern der Chromosomen. Abweichungen vom Chromosomentyp und Austauscherscheinungen wurden bei Aflatoxin beobachtet. Das X-Chromosom war gegen diese Verbindungen ~usserst empfmd- lich. CHA rief einzelne Chromatidbr~iche und TFP einzelne Chromatidbr/iche und Austausch- erscheinungen hervor. Das X-Chromosom war gegen keine dieser Verbindungen ungew6hn- lich empfindlich. Calciumcyclamat in Konzentrationen bis zu 200/~g/ml rief keine Chromoso- menaberrationen hervor.