Mutation Research, 224 (1989) 405-408 405 Elsevier

MUTGEN 01480

Effect of D N O C , Ferbam and Imidan exposure on mouse sperm morphology

I. Quinto, E. De Marinis 1, M. Mallardo, A. Arcucci, R. Della Morte and N. Staiano Dipartimento di Biochimica e Biotecnologie Mediche, I1 Facolt~ di Medicina e Chirurgia, Universit3 di Napoli, 80131 Naples (Italy)

and ~ T~xicology Department, Ente Farmacologico ltaliano, 80131 Naples (Italy)

(Received 15 November 1988) (Revision received 17 May 1989)

(Accepted 22 May 1989)

Keywords: DNOC; Imidan; Ferbam; Sperm morphology assay

Summary

DNOC, Ferbam and Imidan were tested in (C3H × C57BL/6) F 1 mice to assess their potential testicular toxicity. Chemicals were administered i.p. and per os at different doses for 5 consecutive days. After 35 days the testicular toxicity was evaluated by measuring the testicular weights, the sperm counts and the percentage of abnormal sperm. DNOC and Imidan failed to induce teratospermia in mice treated by both routes of administration. Conversely Ferbam induced a statistically significant increase in teratospermia only following per os administration to mice at a dose of 1000 mg/kg b.w./day. These data indicate that per os administration of Ferbam succeeded in producing active metabolites able to interfere with the differentiation process of spermatogenic cells.

The genetic risk from human exposure to pesticides has become of universal concern since many of them have been shown to be tumorigenic in rodents and active in short-term mutagenicity assays.

In our previous study on the mutagenicity of 13 pesticides (Remondelli et al., 1986), DNOC, Ferbam and Imidan were shown to be direct mutagens in S. typhimurium. DNOC was also shown to induce recessive lethals in Drosophila melanogaster (Muller and Haberzettl, 1980) while Imidan increased the incidence of chromosomal aberrations in human lymphocyte cultures (Kurin-

Correspondence: Ileana Quinto, Dipartimento di Biochimica e Biotecnologie Mediche, Naples University Medical School, Via S. Pansini 5, 80131 Naples (Italy).

nyi and Pilinskaia, 1977). Due to the reported mutagenic activity of these pesticides and their widespread agricultural use, it seemed of interest to us to evaluate the effects of DNOC, Imidan and Ferbam on germ cells by means of the mouse sperm morphology assay (Wyrobek and Bruce, 1975).

The mouse sperm morphology assay has been widely utilized to detect those toxins that are able to interfere with the differentiation processes of spermatogenic cells (Wyrobek and Bruce, 1975; Topham, 1979). Although the mechanisms for production of abnormally shaped sperm are un- known, the involvement of a genetic component is suggested by the observations that: (a) the sperm shape is genetically determined and highly herita- ble (Wyrobek and Bruce, 1978; Topham, 1980) and (b) most mutagens induce teratospermia in

0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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mice (Wyrobek and Bruce, 1975; Topham, 1979; Bruce and Heddle, 1979).

In this study we report the effects of DNOC, Ferbam and Imidan on the sperm morphology, sperm counts and testicular weights of mice treated by 2 different routes of administration (per os and i.p.).

Materials and methods

Chemicals Ferbam and DNOC (technical grade) were

purchased from Pennwalt; Imidan (purity: 99.8%) was purchased from Stauffer. Mitomycin C (Mit C) was purchased from Kyowa Hakko Kogyo Co.

Method 11-15-week-old (C3H × C57BL/6) F1 male

mice were used. The parental strains (C3H and C57BL/6) were obtained from Charles River, Calco, Como (Italy). The mice were given food and water ad libitum. Room temperature (22- 24 ° C), humidity (60%), air changes (10 cycles/h) and lighting (12 h on and 12 h off) were con- trolled. Chemicals were dissolved in distilled water and administered i.p. or per os to groups of 6 mice once a day for 5 consecutive days. The negative control groups received only distilled water. As

positive control, a group of animals was injected i.p. with Mit C (0.4 mg/kg b.w./day). The animals were killed by cervical dislocation 35 days after the first administration. The testes weights and the sperm counts of each mouse were recorded. Sperm suspensions, smears, slides and evaluation of sperm abnormalities were made according to Wyrobek and Bruce (1975).

Five hundred spermatozoa of each animal were counted at 400 × under blue-green filters. Vari- ance analysis and Dunnett's multiple comparison test were performed on testes weight values. The mean percentages of sperm abnormalities were analyzed by the Mann-Whitney U-test (Sach, 1974).

Results

The testicular weights, epididymal sperm counts and percentage of sperm abnormalities in mice following DNOC, Ferbam and Imidan treatments by 2 different routes of administration are shown in Tables 1 and 2.

The spontaneous occurrence of abnormal sperm in (C3H × C57BL/6) F1 mice ranged between 1.6 and 2.4%. These values are equivalent to those previously reported for the same strain (Wyrobek and Bruce, 1975; Quinto and De Marinis, 1983;

TABLE 1

EFFECT OF ORAL ADMINISTRATION OF FERBAM, IMIDAN AND DNOC ON TESTES WEIGHT, SPERM CELL COUNT AND SPERM MORPHOLOGY IN MICE

5 per os administrations Testes weight Sperm count Sperm (mg/10 ml/kg b.w./day) (rag/10 g b.w.) ( x 106/epididymis) abnormalities

(~)

Control

Ferbam

Imidan

DNOC

81.6± 7.8 2.3±0.9 1.6~0.2

250 86.0± 8.0 1.5±0.5 1.4~0.6 5 ~ 87.0±10.0 2.0±0.5 2.4~0.7

1000 91.5±12.7 1.1±0.4 6 .3~2 .6"

12.5 76.4± 9.5 1.3±0.7 2,0±0.4 25 75.6± 4.8 2.7±1.4 1,8~0.5 50 86.2± 9.3 2.8±1.4 1,4±0.4

3 85.6±10.4 2.2±1.7 1.6±0.4 6 80.2± 9.7 2.0±0.7 1.9~0.7

12 86.05:9.2 3.7±1.6 1.7~0.5

Each value is the mean 5: SE. * P < 0.05 (Mann-Whitney U-test).

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TABLE 2

EFFECT OF i.p. INJECTIONS OF FERBAM, IMIDAN AND DNOC ON TESTES WEIGHT, SPERM CELL COUNT AND SPERM MORPHOLOGY IN MICE

5 i.p. administrations Testes weight Sperm count Sperm (mg/10 ml/kg b.w./day) (mg/10 g b.w.) ( × 106/epididymis) abnormalities

(~)

Control

Ferbam

I~dan

DNOC

71.8i3.3 9.1±2.5 2.4±0.6

125 63.0±5.3 10.7±6.9 2.6±0.4 250 61.8±2.9 8.4±4.9 2.4±1.1 5~ 66.3±4.0 13.5±8.2 1.5±0.5

12.5 65.0±3.6 8.9±4.4 1.5±0o3 25 54.3±3.0 13.1±1.7 2.0±1.1 50 52.8±6.3 14.9±5.2 2.5±0.4

3 68.0±3.1 10.1±3.2 2.0±0.4 6 65.6±4.7 11.6±3.3 1.4±0.3

12 61.6±6.0 11.2±4.8 3.0±0.6

Each value is the mean + SE.

Quinto et al., 1988). Mit C-i.p. injected groups of mice showed 18.7% sperm abnormalities at a dose of 0.4 mg /kg b .w. /day without reduction in sperm counts and testes weight (data not shown).

The testicular weights and the sperm counts were not affected by either i.p. or per os treatment with any of the 3 chemicals. A statistically signifi- cant increase in sperm abnormalities (6.3%) was observed following per os administration of Ferbam at a dose of 1000 mg/kg b.w./day. I.p. injections of Ferbam and both i.p. and per os administrations of DNOC and Imidan did not induce teratospermia.

Discussion

The mouse sperm morphology assay was ini- tially proposed as an in vivo assay for mutagenic activity of chemical and physical agents at the testicular level (Wyrobek and Bruce, 1975). In- deed, teratospermia induced by testicular toxins could arise either by genetic events (i.e., mutations of genes coding for sperm shape) or by epigenetic events (interaction with enzymes, hormonal or ischemic factors).

Under our experimental conditions Ferbam in- creased the percentage of teratospermia in treated mice while DNOC and Imidan did not. The posi- tive effect of Ferbam was observed only following

per os administration of the chemical, thus indi- cating that this route of administration was espe- cially effective in providing testicular toxins (parental compound or its metabolites).

DNOC, Ferbam and Imidan have all been shown to be direct mutagens in S. typhimurium (Remondelli et al., 1986). In the present study the lack of testicular toxicity of D N O C and Imidan might indicate the failure of the drugs to reach the germ cells. DNOC also failed to induce chro- mosomal abnormalities in mouse germ cells (Nehez et al., 1982). On the other hand, the increased percentage of abnormal sperm produced by Ferbam indicates the ability of this dithiocarba- mate compound to interfere with the differentia- tion process of spermatogenic cells. A similar ef- fect on sperm morphology was previously re- ported for Thiram, another dithiocarbamate pesticide (Zdzienicka et al., 1982). Moreover, oral administration of Ferbam and Thiram produced squamous metaplasia in the thyroid of male rats (Lee et al., 1978). As our investigation did not extend to sperm morphology in the offspring of treated mice, we cannot provide evidence for in- duced transmissible mutations in germ cells. Re- gardless of the genetic or epigenetic mechanism possibly involved in the production of terato- spermia, our results indicate that exposure to Ferbam induces sperm abnormalities in mice and

408

thus it represen ts a r isk for diseases such as steri l- i ty a n d abor t ions .

Acknowledgements

This work was s u p p o r t e d b y a g r an t f r o m the

Min i s t e ro del la Sani th , I ta ly . W e t h a n k Mrs . R.

Ba ldon i for t y p i n g the m a n u s c r i p t .

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