UDC 612.61.014.46:[613.298:666.293.5]-087

Yu. V. Ivanov, T. L. Dobroslavskaya

Cytological Studies of the Gonadotoxic Effects of a Mixture of Substances Migrating from Enameled Articles Intended for Contact with Foodstuffs

Moscow F. F. Erisman Research Institute of Hygiene

As the range of chemical compounds with which humans come into contact continues to expand, some of which may potentially affect the gonads, there is an urgent need for clear quantitative and qualitative…

…sufficiently rapid assessments of the effects of these substances on the gonads. At the same time, quantitative morphological and cytological characterization of the gonadotropic effects of these substances on the body remains inadequate in many respects. To date, studies of spermatogenic abnormalities have relied primarily on various modifications of two quantitative morphological methods. The first method, proposed by Fogg and Cowing, involves determining the proportion of tubules in a transverse section of the testis that contain particular types of germ cells. The second consists of counting individual types of spermatogenic epithelial cells per transverse section of a seminiferous tubule, on average (L. Ya. Zhorno and K. K. Surikova). Given the identified stages of spermatogenesis and the propagation of a “wave” of different spermatogenic epithelial cell associations along the seminiferous tubule, it is clear that counting cells per transverse section of a seminiferous tubule requires prior identification of the stage of spermatogenesis in each selected tubule section. This can be accomplished in specially prepared and stained sections of the testes (Leblond and Clermont).

The present study was undertaken to assess the possible gonadotoxic effects of a mixture of substances migrating into extracts from enameled products manufactured using new grades of titanium enamel. Its primary objective was to develop an appropriate quantitative method for characterizing abnormalities of spermatogenesis.

Drawing on established methods for preparing cellular homogenates of organs and counting the total number of cells in an organ using a Goryaev counting chamber (E. N. Shcherbova et al.), we applied this method to testicular tissue, additionally staining the homogenate to identify the different types of spermatogenic epithelial cells. The testicular cell homogenate was prepared as follows. The tunica albuginea was opened and the testicular tissue was dissected free of it. The tissue was then homogenized first with scissors. After adding up to 10 ml of a 1% solution of gentian violet in 5% acetic acid to the homogenate, gentle homogenization was continued using a syringe fitted with a needle 2.5 mm in diameter until a uniform cell suspension was obtained. The testicular cell suspension was drawn into a leukocyte pipette to the 1 mark, and the pipette was filled to its full volume with gentian violet solution. Cells were counted in a Goryaev chamber as in an erythrocyte count, with adjustment for dilution in the pipette and the volume of the entire homogenate, using the formula \(П=\frac{a}{2}\cdot10^7\), where \(П\) is the total number of cells and \(a\) is the number of cells counted in the chamber. Spermatogonia were counted first, followed by spermatocytes, spermatids, spermatozoa, and Sertoli cells. Thus, counting cells in a single chamber yielded separate data on the absolute numbers of the principal spermatogenic epithelial cell types in the testis.

The following additional methods were used to assess spermatogenic abnormalities: determination of testicular weight coefficients; preparation of smears from homogenized pieces of testis, stained with periodic acid–Schiff reagent and counterstained with hematoxylin, and stained for RNA and DNA by Brachet’s method; and preparation of paraffin sections of testes from material fixed in formalin and Carnoy’s solution, stained with periodic acid–Schiff reagent and hematoxylin, or with hematoxylin and eosin.

In smears stained for DNA and RNA, the level of synthetic activity in the testicular tissue was assessed visually. In smears of testicular homogenate stained with periodic acid–Schiff reagent and hematoxylin, individual types of spermatogenic epithelial cells were counted in the microscope’s field of view; the results were expressed as percentages. In sections of the testes…

The individual types of germ cells were counted per cross-section of a seminiferous tubule, on average, and the stage of the cells in each tubule section was identified. As with the cell counts in smears, the results were expressed as percentages. The toxicological experiment was conducted on outbred male albino rats weighing 120–140 g.

Considering the formulation of titanium enamels and the toxicity of their individual components, it may be concluded that the principal limiting indicators in the hygienic assessment of enameled articles intended for contact with food are the amounts of boron, fluorine, cobalt, and nickel compounds migrating from these articles into extracts. In selecting tests for the toxicological studies, we took into account published reports indicating that boron has a specific gonadotropic effect.

Taking into account the results of sanitary-chemical analyses and the conditions under which the articles are used, the experiment was conducted in 7 groups of animals (175 rats). Group 1 received boiled water (control); group 2, an aqueous extract obtained by treating the same specimen daily (repeated treatment); group 3, an aqueous extract obtained by treating a new specimen daily; group 4, an aqueous extract obtained by treating a new specimen daily, with the addition of 0.8 mg/mL of boron; group 5, an aqueous extract with boron added at the maximum amount detected in the 1% acetic acid extract of the articles during sanitary-chemical testing of this batch of specimens, namely 1.6 mg/L; group 6, an aqueous extract obtained by treating a new specimen daily, with boron added at the maximum amount detected in the 4% acetic acid extract, namely 3.2 mg/L; and group 7, an aqueous extract obtained by treating a new specimen daily, with the addition of 16 mg/L of boron.

In assessing the total numbers of individual spermatogenic epithelial cell types in stained testicular homogenate, we identified, as already noted, 5 principal cell types: spermatogonia, spermatocytes, spermatids, spermatozoa, and Sertoli cells.

To verify the accuracy of cell counting and identification in stained testicular homogenate in a Goryaev counting chamber, the same cell types were counted in smears of testicular homogenate stained with Schiff–periodic acid and hematoxylin, which made it possible to identify the principal types of spermatogenic cells in rats clearly. The percentage data on cell content in the homogenate and smears were then compared. The results of this comparative count of spermatogenic epithelial cells were identical. In addition, the mean numbers of the above-mentioned testicular epithelial cell types were determined in cross-sections of seminiferous tubules, after first identifying the stage of spermatogenesis represented by each tubule section.

The results of these counts, expressed as percentages, were comparable to the results of determining cell content in testicular homogenate only at stages 5–6 of spermatogenic epithelial development. It should be noted that 14 stages of distinct germ-cell associations have been identified in rats, 12 in guinea pigs, and 6 in humans.

At the first 4 stages, the spermatogonia data used to assess spermatogenesis differed from the cell counts in the homogenate; at stages IX through XIV, the spermatozoa data differed (mature spermatozoa are not detected at these stages at all).

Thus, certain shortcomings of the method of counting spermatogenic cells in sections of seminiferous tubules become apparent in comparison with counts in organ homogenate. The principal drawback is the need to relate data on changes in spermatogenesis either to one arbitrarily selected stage of spermatogenesis or to all stages, for example,

Table 1

Results of spermatogenic epithelial cell counts in experimental animals of the main generation, in millions (9 months of the experiment; \(M\pm m\))

* Here and in Table 2, differences are significant at \(P\leq 0.05\).

across all 14 in the rat, as noted above. The absolute content of particular spermatogenic epithelial cell types in the testis is a more informative measure than their relative numbers expressed with reference to one arbitrarily selected stage of spermatogenesis. Having established the validity of the method for counting spermatogenic epithelial cells in testicular homogenate, we used it as the basis for assessing the possible gonadotoxic effects of a mixture of substances migrating from products made with new titanium enamels intended for contact with food. The results are summarized in Tables 1 and 2. As Table 1 shows, no changes in the total number of spermatogenic cells relative to the control were found in animals that received aqueous extracts of the products, or aqueous extracts supplemented with 0.8 mg/L boron, for 9 months. Nor were changes found in cell types highly sensitive to any exposure, such as spermatogonia and spermatocytes. In animals in groups 5–7, which were given aqueous extracts supplemented with boron at concentrations of 1.6, 3.2, and 16 mg/L, respectively, daily for 9 months, significant differences from the control were found both in the total content of spermatogenic cells in the testis and in individual germ cell types.

Examination of the individual spermatogenic cell types revealed particularly marked shifts under the influence of extracts from the enameled products in the populations of spermatogonia, spermatocytes, and spermatids, especially the latter. The results indicate that the population of mature spermatozoa—

…is fairly resistant to the action of boron in extracts from enameled products. If a dose–response curve is constructed for the effect of boron on the testes in terms of the reduction in the total cellularity of the organ, a boron concentration of 1 mg/L should be regarded as subthreshold for the testes. This agrees with published data (M. T. Abashidze).

As the results summarized in Table 2 show, after a 3-month recovery period all spermatogenesis parameters had returned to normal in rats from all experimental groups except group 7.

In discussing the gonadotoxic effect of extracts from products made with titanium enamels and intended for contact with food, we consider it necessary to address two further methodological points. Although studies of testis-to-body-weight ratios in the experimental animals revealed a tendency for these ratios to decrease as the boron concentration in the extracts increased, they did not permit clear and confident conclusions about either threshold doses or the specificity of the substance’s action on the gonads. This is because natural age-related testicular atrophy may occur during a prolonged exposure period. Moreover, true testicular atrophy may be masked by connective tissue proliferation, edema, and other changes. Biochemical studies of DNA and RNA content in testicular tissue and histochemical examination of smears prepared from organ homogenates also showed that the two approaches differed in their informativeness. In histochemical examination using Brachet staining for DNA and RNA, the pronounced changes produced by extracts supplemented with increasing boron concentrations from 0.8 to 16 mg/L did not always correlate with the results of biochemical studies, in which the differences proved to be…

Table 2

Results of counts of spermatogenic epithelial cells in the testes of experimental animals of the parental generation, in millions, after a 3-month recovery period (\(M\pm m\))

[[unclear: beginning of word]] significant only at the dose of 16 mg/L. In this case, calculations per 1 g of testicular tissue introduced errors whose origin has already been discussed above.

Thus, studies of the gonadotoxic effects in animals of a mixture of substances migrating from articles manufactured with titanium enamels and intended for contact with food demonstrated the promise of the proposed rapid quantitative cytological method for assessing disturbances of spermatogenesis.

References

Abashidze M. T. — Gig. i san., 1973, No. 4, pp. 10–14. — Zhorno L. Ya., Surikova K. K. — Dokl. AN SSSR, 1963, vol. 150, pp. 170–173. — Shcherbova E. N., Rozhdestvenskii A. M., Bezin G. I. — Pat. fiziol., 1974, No. 4, pp. 85–87. — Fogg G. C., Cowing R. F. — Cancer Res., 1951, vol. 11, pp. 23–30. — Leblond C., Clermont G. — Am. J. Anat., 1952, vol. 90, pp. 167–170.

Received July 12, 1978.

Cytologic Study of the Gonadotoxic Action of a Group of Substances Migrating from Enamel Utensils Intended for Contact with Food Products

Yu. V. Ivanov, T. L. Dobroslavskaya

A developed cytological method of counting various types of cells from the spermogenic epithelium of the whole organ was used for assessment of the action on gonads of a group of substances migrating from enamel utensils, intended for contact with food products. The suggested method of cytological examination of the testis is an express method. It allows to assess quickly the disturbances of the spermatogenesis and present them in absolute numerical values judging by the number of main types of cells of the spermatogenic epithelium. This method may be used for evaluation of disturbances of the spermatogenesis instead of the existing laborious method of counting spermatograms in a series of sections of the testis.