To,xic&gy Letters, 19 (1983) X7-271 Elsevier

241

EFFECT OF SACCHARIN ON THE INDUCTION OF CHROMOSOMAL TRANSLOCATfONS IN MALE MICE AND THEIR Ft OFFSPRING

JUGUSLAVA PEt?EVSKI, LJILJANA VUKS~~~OYI~, NADA SAVKOVIe, DRAGAN ALAVAN- TIC and DLJSANKA RADIVOJEVI(?

Institute of Nuclear Sciences ‘Boris KidriC ‘, Laboratory of Radiobiology, Belgrade (Yugoslavia)

(Received April 20th, 1983) (Revision received July 7th, 1983) (Accepted July l&h, 1983)

SUMMARY

The dose-dependent effects of sodium saccharin on the fertility and the induction of chromosomaf transIocations in treated mice and their FI males were examined. C3N x 101 mice were treated with sac- charin for 2 successive weeks at doses of 200 and 500 mg/kg of body wt. Data from the fertility study showed that saccharin did not induce any significant effect on fertility. Cytological analysis of meiotic cells showed no chromosomal rearrang~ents. Semisterility and sterility were noted in F, males of saccharin-treated animats. In semi-sterile and sterile FI males of saccharin-treated animals chromosomal translocations were not found.

INTRODUCTION

The artificial sweetener saccharin has been tested for mutagenicity in bacterial systems [3, 4, 15, 201, drosophila 19, 171, dominant Iethal test in the mouse [I, 10, 12, 13, 17, 191 and in somatic cells in the mouse [6, 111. Kramer has published a review of mutagenic action of saccharin on many different test systems [8] and has shown that the contradictory results may be due to differences in the route of ad- ministration or the presence of varying amount of impurities. Very often the mutagenicity of saccharin is ascribed to its contaminants and for this reason the standards for saccharin purity are very high. The purpose of this study was to in- vestigate the dose-dependent effect of saccharin on the induction of chromosomal translocation in directly treated male mice and their transmission to F1 male off- spring.

0378-4274/83/s 03.00 0 Elsevier Science Publishers B.V.

268

MATERIAL AND METHODS

2.5-month-old C3H x 101 strain male mice were treated with sodium saccharin {Pharmaceutical company ‘Galenika’, Belgrade) for 2 successive weeks (5 days per week). Sodium saccharin was dissolved in tap water and solutions were prepared freshly each day. Sodium saccharin was administered by gavage at dosage of 200 and 500 mglkg per day. Controls received equal amounts of tap water. At each dose level 15 males were studies, and 15 males served as controls. Immediately after the termination of treatment, each male was placed in a cage with 3 virgin females. The females were removed after 7 days. All treated and control males were killed after 8 weeks of treatment. The meiotic chromosome preparations were prepared by an air-drying method [5] and the spermatocytes in the diakinesis-first metaphase were analysed. From each male 100 cells were scored. Fr males chromosome preparations were made on maturation (2.5-3 months).

From each litter 3 Fr males, 2.5 months old, were randomly selected and each male was mated with 3 virgin females. Fertility of Fr males was evaluated in the following manner: Males were classified as sterile if none of 3 females delivered a litter. A male was considered semi-sterile if any of the following three conditions held: (a) if all 3 females produced litters, and all 3 had litters less than 3: (b) if only 2 females produced litters and both had litters less than 6, with one less than 3: (c) if 1 female was pregnant and litter size was less than 3. Males not satisfying the above criteria were considered fertile. Thus, a male could be considered fertile on the basis of singe litter, providing the size of the litter was greater than 3 [15].

RESULTS

Saccharin-treated animals and controls as well as their Fr offspring did not show any statistically significant changes relating to body and testis weight. Observations of growth and development of offspring did not reveal any changes.

Effect of saccharin on the gentility

As seen in Table 1 no statistical difference was noted between males receiving dif- ferent doses of saccharin (200 and 500 mg/kg) and controls.

TABLE I

EFFECT OF SACCHARIN ON FERTILITY

Treatment Dose Number of animals Time of application 70

(mdkg) Q/Q (weeks) fertility (r

Saccharin 200 15145 2 88.8

Saccharin 500 I5/45 2 77.7

Controls 15145 2 86.6

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TABLE II

EFFECT OF SACCHARIN ON CHROMOSOMAL TRANSLOCATIONS

Treatment Dose No. of

(mg/kgI males

Time

of killing

(weeks1

Number of

cells

analvsed

X/Y Cells with

trans-

locations

Saccharin 2ocl 15 8 1500 142 0

Saccharin 500 15 8 1500 90 0

Controls - 15 8 1500 20 0

TABLE III

EFFECT OF SACCHARIN ON THE FERTILITY OF THE F, GENERATION

Treatment Dose

(mg/kgl

Males of FI generation

Total Sterile

number of number %

males

Semi-sterile

number %

Fertile

number %

Saccharin 200 -II 3 3.9 9 11.6” 65 84.4”

Saccharin 500 12 9 12.5 14 19.4b 49 68.1b

Controls - 66 0 0 2 3.0 64 96.9

a0.05>P>0.001

bP>O.OOl

Effect of saccharin on the spermatogenesis

The cells analysed in diakinesis-first metaphase were affected on the sperm-

atogonial stage. No chromosomal translocations were observed in 3000 cells of

treated males, and in 1500 cells of 15 control animals (Table II).

Effect of saccharin on the fertility in FL males

The results (Table III) showed that saccharin induced statistically significant

reduction of fertility of Fi males. At the dose of 500 mg/kg the percentage of fertile

males was significantly reduced.

Cytological analysis of the F1 males

In the experiment 144 Fi males originated from treated males and 66 controls Fr

males were analysed for the presence of chromosomal translocations. FI males

classified as fertile, semi-fertile and sterile were cytogenetically analysed. Neither in

Fi males derived from saccharin treated males nor in controls were chromosome

translocations detected.

270

TABLE IV

CYTOLOGICAL ANALYSIS OF THE OFFSPRING

FI males Dose Number of Number of males % of animals

(mg/k) males analysed with translocations

Fertile 200 65

500 49

Semi-sterile 200

500

9

14

Sterile 200

500 9

Fertile

Semi-sterile

Sterile

controls

64

2

0

62 0

47 0

9 0

14 0

3 0

9 0

64 0

2 0

0 0

DISCUSSION

The results obtained with saccharin-induced chromosomal translocations con-

flict. &am et al. [19] observed a markedly significant increase of chromosome aber-

rations in spermatocytes 12 weeks after i.p. injections with 5 x 200 mg/kg male

mice. In the saccharin group 1.6% of cells carrying translocations were detected.

Whereas Leonard et al. [lo] did not find chromosomal translocations in dividing

spermatocytes of male mice treated with saccharin i.p. at a dose of 2 g/kg or after

chronic oral treatment. Also, the results obtained with saccharin-induced

chromosomal translocations in somatic cells are not in agreement. Newell et al. [14]

scored metaphase in rat bone marrow cells after administration of 30, 2500 and 5000

mg/kg saccharin and found a significant increase of chromosome aberrations 6 h

after i.p. injections of 2500 mg/kg saccharin. Other authors did not find any

chromosomal damage [2, lo].

Our results show that sodium saccharin given in doses of 200 and 500 mg/kg in-

duced no chromosomal translocations in diakinesis -first metaphase in directly

treated animals as in their offspring. In Fi generation the fertility reduction was

significant after a dose of 500 mg/kg. The results presented here confirm the fin-

dings of Leonard et al. [lo] that saccharin is not capable of inducing reciprocal

translocations in the mouse. Our results were the first obtained after treatment of

saccharin on the heritable translocations in Fr sons. Presented data also confirm the

findings of others [7, 121 that saccharin does not induce any effects on fertility in

directly treated animals. These results support other investigations of this sweetener,

widely used in the diet and therefore these results may be important from a medical

aspect.

271

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