Age-Dependent Response of the Rat Testes to Di(2-ethylhexyl) Phthalate

Spermatogenesis starts soon after birth in the Sprague-Dawley rat but is not fully established until about 56 days of age. When high oral doses of the plasticizer di(2-ethylhexyl) phthalate (DEHP) ...


Introduction
Di(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer which has become widely distributed in the environment (1)(2)(3). Furthermore, blood and blood products may leach DEHP out of plasticized medical devices leading to inadvertent patient exposure during, e.g., hemodialysis and blood transfusions (4,5). As yet, exposure in humans is not known to be associated with any adverse effects. Administration of high oral doses of DEHP to rats have resulted in liver abnormalities and testicular damage (6,7). The effect on testes has been observed to vary with the age of the animal, younger rats being more sensitive than older ones (8). In this communication, possible mechanisms involved in this age dependency will be discussed.

Aspects of the Development of the Testis
Before going into age-related effects of DEHP it is necessary briefly to discuss some of the factors involved in the normal development of the testis and to define testicular development.
Testicular development and function are dependent on many factors (9). It is known that gonadotrophic hormones from the pituitary act on the Sertoli cells, either directly (FSH = follicle stimulating hormone) or via the Leydig cells (LH = luteinizing hormone); it has also been shown that the Sertoli cells are involved in the regulation *Department of Anatomy and Histology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala, Sweden. tDepartment of Drugs, National Board of Health and Welfare, Uppsala, Sweden. of germ cell differentiation (10,11). The complicated cellcell interactions within the testis are not fully understood (12).
The development of the testis in the Sprague-Dawley rat is morphologically well described. In the newborn rat the seminiferous cords contain only undifferentiated Sertoli cells and gonocytes. At about 10 days of age most gonocytes have differentiated into spermatogonia (13). The first primary spermatocytes appear at day 12 or 13, and formation of the occluding inter-Sertoli cell junctions takes place between 15 and 19 days of age (14,15). At 24 days the first meiotic divisions are observed ( Fig. 1), and spermatids in the Golgi phase have been demonstrated at day 25 (16). At 44 days the first mature spermatids ("testicular spermatozoa") are observed, but not until about 56 days of age is the frequency of such late spermatids the same as in the adult testis (17). Up to 48 days of age a considerable number of exfoliated, more or less degenerated, testicular cells are found in the epididymis.
From day 20 to day 60 there is a 17-fold increase in testicular weight but only a 7-fold increase in body weight. Tubular diameter increases about 2.5 times during this period and total tubular length increases about 3 times (17).
Both testicular and plasma testosterone concentrations are low until about 50 days of age (16,18), except for a short postnatal period when testosterone levels are high, due to the influence of maternal gonadotrophins (19). The rise in testosterone is preceded by an increase in serum LH, which starts at about 30 days of age and reaches a maximum at about 50 days (20). The number of LH receptors shows a similar increase (19). FSH levels rise somewhat earlier (about day 20) and from 30 days of age FSH levels are fairly constant (20). In the 56-day-old rat mature spermatids are common. From this brief description of the development of the testis it can be concluded that it is of utmost importance to consider the age of the animals when using them in toxicity studies. If the exact age of the rat is not known proper evaluation of the testicular effects will be difficult. Moreover, when extrapolating data from rats to man it must be borne in mind that germ cell proliferation in the rat starts very early; the first meiotic cells appear within 2 weeks after birth. In humans, spermatogenesis starts at puberty (12-14 years of age). The term puberty, which is well defined in humans (21), should not be used in the rat, since there is no general agreement on the definition. In fact, if the situation in humans is translated to rats regarding testicular development, the rats are almost born in puberty Consequently when data obtained in the rat are used for evaluation of the risk oftesticular damage in childhood these fundamental differences should be considered.
Oral Administration of DEHP and Influence of Age Gray and Butterworth (8) reported that oral administration of DEHP (2.8 g/kg/day for 10 days) to 4-, 10-, and 15-weeks-old Wistar rats produced testicular damage in the two younger groups but not in the older group.
In an attempt to further elucidate this age-dependent difference in response, we treated groups of Sprague-Dawley rats (25,40, and 60 days old at the beginning of the treatment) with DEHP DEHP was administered in the diet and the dose was adjusted to give a daily intake of 1.0 or 1.7 g/kg body weight for 14 days. In this study we found that body weight gain was retarded in all groups and that testicular weight was markedly reduced in 25-and 40-day-old rats given 1.7 g/kg (Table 1). In these animals there was severe testicular damage, all tubules being affected in the 25-day-old rats (Fig. 2). When the mean daily intake was 1.0 g/kg, only a few tubules (1-10%) in each animal were affected. No clear changes were observed in the testes of mature rats at either dose of DEHP (Fig. 2). This mode of administration affected daily food intake, and therefore possible effects of the low food consumption were studied in pairfed rats. Only minor testicular changes were observed in the pair-fed animals, excluding low food consumption as the only factor responsible for the testicular damage.
There may be several causes for the age-dependent variation in testicular response to oral administration of DEHP Gray and Beamand (22) investigated the possibility of a difference in tissue sensitivity using primary cultures of testicular cells. They found that germ cell detachment from the Sertoli cells was significantly higher in cultures from younger animals than in those from older animals when incubated with equal amounts of mono(2ethylhexyl) phthalate (MEHP), the primary metabolite of DEHP! DEHP itself had no effect. It is at present difficult to relate these findings to the in vivo situation but they seem to suggest that there is an age-dependent difference in tissue sensitivity However, spermatids, which in mature rats constitute most of the epithelium, are known to be lost during culture. Therefore, the testicular cell culture system may not adequately reflect the testicular epithelium of mature animals. Another possible cause of the differing response between younger (immature) and older (mature) rats could involve differences in absorption, distribution, metabolism, and/or excretion. To study this possibility and to overcome the problem of the reduced food intake, another series of experiments were carried out (23). Groups of rats (25,40, and 60 days old) were given DEHP by gavage for 14 days at a dose of 1.0 g/kg/day. Animals that were 25 days old at the onset of the treatment showed severe testicular damage (Fig. 3), whereas the older animals were unaffected. In other animals, repeated blood samples were collected in order to estimate the exposure to DEHP and its primary metabolite MEHP DEHP was not found to any significant extent in peripheral plasma whereas there were high levels of MEHP The mean area under the plasma concentration-time curve of MEHP in the youngest age group was twice as high as in the two older groups. Finally, 25and 60-day-old rats were given carbonyl-1 C-DEHP (1.0 g/kg) to measure the amount of DEHP-derived metabolites excreted in urine. Twice as much radioactivity was found in the 25-day-old group.
The results indicated that the extent of absorption and hence total exposure to MEHP and its metabolites was higher in the younger rats. Thus it seems probable that the age-related difference in testicular response after oral administration may, at least in part, be explained by age differences in the pharmacokinetics of DEHP

Intravenous Administration of DEHP
As most toxicity studies aim at obtaining a better knowledge of possible risks for humans, it is of importance to consider the route of administration. In humans, it is generally believed that the highest exposure to DEHP occurs in connection with hemodialysis or transfusions of blood and blood products where DEHP is introduced directly into the blood stream. Considerable exposure to DEHP has been reported to occur in newborn infants undergoing exchange transfusions (24). To reveal possible age-related effects on the testis of repeated intravenous infusions of DEHP, in a preliminary study we gave 25-and 40-day-old rats infusions of 500 mg/kg body weight, the highest tolerable dose of the DEHP emulsion. The infusions lasted 3 hr and were given every other day for 11 days (in all, six infusions/animal). No changes could be detected in paraffin embedded testes, but in Epon sections for light and electron mi-croscopy some degenerating primary spermatocytes were observed in the 40-day-old group dosed with 500 mg/kg/day (Fig. 4). Some alterations were also observed in the Sertoli cells. No such changes were found in the 25-day-old group.
The results of this preliminary study indicate that intravenous administration of DEHP does not cause agedependent testicular response similar to that observed after oral administration, suggesting that testicular sensitivity to intravenous DEHP of 25-day-old rats is not higher than that of 40-day-old animals. To confirm this suggestion, further studies are needed since it is not known if the disposition of DEHP after intravenous infusion varies with age.

Discussion
It is well established that oral administration of high doses of DEHP to rats causes testicular lesions, and that their effects are more pronounced in young animals where spermatogenesis is not fully established. The Sertoli cells play an important role in the establishment and maintenance of the specific microenvironment of the adluminal compartment of the seminiferous epithelium and this is a prerequisite for normal spermatogenesis. Alterations in this microenvironment will severely affect the spermatocytes and the spermatids that reside in the adluminal compartment (12). Thus, any agent interfering with normal Sertoli cell function may secondarily affect spermatogenesis. In this context it should be emphasized that an absence of morphological alterations in the Sertoli cells does not necessarily imply functional normality There are several reports indicating that the Sertoli cells are the cells primarily affected by treatment with phthalic acid esters (25)(26)(27), and so far there is no evidence of direct effects on the germ cell. Assuming that the Sertoli cells are "target cells" for the toxic metabolite(s) of DEHP, the seemingly contradictory results obtained after different modes of administration might in fact be explained by differences in the exposure of Sertoli cells and/or age differences in the metabolic activity of these cells. As previously pointed out, the result of our study with oral administration of DEHP suggested that the total exposure of MEHP and its metabolites was higher in 25-day-old rats than in more mature animals, FIGURE 4. Electron micrographs from 40-day-old rats in the infusion study. Note dilated endoplasmic reticulum (*) in the Sertoli cells (Sert) of the animal given 500 mg DEHP/kg body weight. Note also the primary spermatocytes (Spc I) with signs of degeneration. Arrows point to seemingly normal inter-Sertoli cell junctions. Spg = spermatogonia. which might account for the age difference in response. susceptible. A higher "target cell" exposure in the more The minor differences found in the infusion study with mature animals could also result from age differences in some lesions in 40-day-old but not in 25-day-old rats, could the disposition of DEHP after intravenous administrabe due to a somewhat higher "target cell" exposure in tion.
the older rats, since in these animals blood flow to the testis is higher but the number of Sertoli cells is the same Conclusions as in the younger rats (28). Moreover, the Sertoli cells of older animals have a higher metabolic activity since they Oral administration of high doses of DEHP to male "support" more germ cells, and thus they may be more rats of different ages produces more pronounced testic-ular damage in young rats. This difference in response seems to be due to a higher absorption of the DEHPderived metabolite, MEHP, in the young animals. After intravenous infusions of DEHP, the younger rats were not more susceptible than the older ones. To assess the risk for testicular damage of DEHP exposure during childhood requires additional studies with species in which testicular development is more similar to that of humans. This study was supported by grants from the National Swedish Environment Protection Board and the Swedish Council for Forestry and Agricultural Research.