Nuclear Morphometric Analysis of Leydig Cells of Male Pubertal Rats Exposed In Utero to Di(n-butyl) Phthalate.

We recently reported that prenatal rat exposure to di(n-butyl) phthalate (DBP) induced Leydig cell (LC) hyperplasia after nine weeks (wks) of age, yet the number of LCs was similar to that of the vehicle group until seven weeks. Nuclear pleomorphism of hyperplastic LCs is common and is considered to be continuous progressive degeneration. Thus, computer-assisted image cell nuclear analysis of LCs was performed on 5- and 7-wk-old Sprague-Dawley (SD) rats whose dams had been administered DBP (i.g.) at 100 mg/kg/day or vehicle (corn oil) on gestation day 12 to 21. The results of the 5-wk-old DBP group were similar to those of the vehicle group; LC nuclei of the 7-wk-old DBP group showed normal ploidy and similar amounts of DNA. However, the size, elongation and peripheral chromatin aggregation parameters were significantly higher, and the reticular chromatin distribution and isolated chromatin aggregation parameters were significantly lower compared with the vehicle group. The present study quantitatively demonstrated nuclear morphological alterations in rat LCs at 7 wks old (puberty) due to the prenatal DBP administration before apparent LC hyperplasia developed.

Dysplastic cells are distinguished from normal cells by alterations in nuclear structures, and morphological changes of the nuclei are considered characteristic features of genomic alteration[1],[2]. Several morphometric nuclear analysis studies have been performed to evaluate pathological changes in the human prostate[3],[4],[5],[6],[7],[8],[9],[10], breast[11],[12],[13],[14], adrenal gland[15], cervix[16],[17],[18] and experimental chemically-induced rodent carcinogenesis[19],[20],[21].

Phthalates are chemicals used as plasticizers in polyvinyl chloride to impart flexibility and durability, and comprise up to 40% of the plastic volume. The phthalate esters, including di(n-butyl) phthalate (DBP), have an estrogenic or anti-androgenic effect on the development of the male reproductive system, and the specific primary cellular target of DBP has been considered to be testicular Leydig cells (LCs)[22],[23],[24],[25],[26]. Recently we reported that prenatal administration of DBP induced atypical Leydig cell (LC) hyperplasia at nine weeks and older, although the numbers and proliferative activities of LCs were similar to those of the vehicle group until seven weeks[27],[28]. The progression from normal structure to hyperplasia has been considered a continuous event[29]. The degree of abnormal morphological aberration of individual cell nuclei is one of the important features in assigning a grade to pathological changes, and nuclear aberrations are always analyzed by a subjective assessment of chromatin pattern, size, and shape of the nuclei[18],[19],[20],[21],[30],[31]. Progressive degeneration of LC nuclei before suffering hyperplasia is difficult to recognize by routine light microscopy, because the qualitative morphological alterations of LCs nuclei after prenatal DBP exposure are unclear (Fig. 1)[27],[28].

Fig. 1.

Representative features of Leydig cells nuclei of the 7-wk-old vehicle group (A) and 7-wk-old DBP group (B). DNA Feulgen stain without counterstain; bar = 25 μm.

The present study used a computer-assisted image analysis system that provided morphometric measurements based on optical density as well as a multitude of parameter measurements: DNA ploidy, nuclear morphology, and nuclear chromatin parameters[18],[19],[20],[21],[30],[31]. The aim of the present study was to demonstrate the potential utility of computer-assisted morphometric analysis of nuclear features by several parameters for use in routine toxicologic pathological examinations. Although there were some studies concerning the alteration of quantitative nuclear chromatin in chemical-induced carcinogenesis[19],[20],[21], more detailed studies using several chemicals are required to establish the usefulness of this system.

DBP (99.8% pure) was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Eight-week-old time-mated female Sprague-Dawley rats (n=10) were procured from SRL Co. (Shizuoka, Japan) on gestation day 0; the day of copulation was confirmed. Upon arrival, animals were distributed into dose groups using body weight randomization. Animals were housed individually in polycarbonate cages containing wood chip bedding in a high-efficiency particulate air (HEPA)-filtered, mass-air-displacement room maintained on a 12-h light-dark cycle at approximately 22 ± 2 °C with a relative humidity of 55 ± 5%. Animals were fed a conventional diet and had free access to food and water (MF, Oriental Yeast, Osaka, Japan). All experimental procedures were conducted under the approval of the Animal Care and Use Committee at Azabu University School of Veterinary Medicine; medical guidelines established by the National Institutes of Health and Public Health Service Policy on the Humane Use and Care of Laboratory Animals were followed. Two groups of pregnant rats (n=5 per group) were intragastrically (i.g.) administered DBP in 0.5 ml corn oil (Nacalai Tesque Inc., Osaka, Japan)/animal at 0 (vehicle group) or 100 mg/kg/day on gestation days 12 to 21. Dose solutions were prepared fresh every morning and administered at 9:00 am. The regimen was based upon previous reports that the lowest-observed-adverse-effect (LOAEL) dose of DBP in fetal male rats was 100 mg/kg/day[22],[23],[24],[25],[26],[27],[28]. Offspring were weighed and sexed at birth. Litters were reduced to 10 offspring, five males and five females per dam. Weaning was carried out at 21 days postpartum, and pups were then removed from the mothers. Offspring were housed in polycarbonate cages (n=5 per cage; single sex) with wood chip bedding that was replaced every 5 days. All animals were weighed at birth and again at 5 and 7 wks of age. At each time point, ten males (five males from the DBP or the vehicle group) were randomly selected, weighed, anesthetized, and euthanized by a CO2 overdose. Animals did not used for the present studies were utilized in other investigations (data not shown). The testes were removed and weighed, and representative samples were fixed in 10% formaldehyde in 0.1 M phosphate buffer (pH 7.4). The formalin-fixed testes were cut and embedded in paraffin blocks so that a 5-μm-thick histological section showed a cross-section of the entire seminiferous tubule. For each rat, five histological slides of the testis were prepared and stained with a Blue Feulgen DNA Ploidy Analysis Staining Kit (Scytek Laboratories Inc., Logan, UT, USA) without a counterstain (Fig. 1). A CAS 200 image analyzer (Bacus Laboratories Inc., Lombard, IL, USA) was used to establish quantitative DNA via pixel optical density data generated from Feulgen stained slides. However, the Blue Feulgen optical density results from DNA Ploidy Analysis Staining Kit (Scytek Laboratories Inc., Logan, UT, USA) were not uniform and thus indiscernible differences were always observed. Consequently, prior to analyzing target LCs in the present study we would always calibrate the CAS 200 image analyzer as follows: the optical density of at least 50 normal rat lymphocytes, as 2C control diploid cells with migrating adjacent LCs in each Feulgen-stained sample, were analyzed[29],[30]. Thereafter, for every section from each group, a minimum of 800 cells were randomly selected and analyzed using the CAS 200 image analyzer. The measurements were transformed into a QDA v3.0 Image List Mode file (Bacus Laboratories Inc.), analyzed by the Cell Sheet v.2.0 software program (Bacus Laboratories Inc.) and statistically compared between the DBP group and the vehicle group as follows. For each data set, the mean value and standard deviation were compared by Mann-Whitney U test using the Stat View-J 5.0 statistical analysis software (Abacus Concepts, Piscataway, NJ, USA). A P value of less than 0.01 was regarded as statistically significant[30].

The morphological features measured in this study to characterize each nucleus parameter are listed in Table 1. These parameters included four basic categories following the description of Bacus et al. (1996)[30]: [1] DNA description parameters, measurements of the DNA content of a nucleus; [2] general nuclear mophometry parameters, measurements of nuclear dimensions such as nuclear area, nuclear shape (circularity), maximum nuclear diameter and others; [3] general chromatin morphometry parameters, counts per nucleus of defined individual point texture measurements used to assess alterations in fine chromatin parameters; and [4] Markovian measurements of texture that summarize differences in absorbance between a reference pixel and other pixels at defined distances from the reference pixel for the entire nucleus[30]. Because it was difficult to understand what biological characters of the nucleus account for the Markovian texture measurements, the present study applied the first three parameters, discriminating the degree of chromatin granularity, peripheral chromatin aggregation, and symmetrical chromatin distribution (Table 1), following the previous studies by Pressman (1979)[32] and Dawson et al. (1993)[33].

Table 1.

Summary of Morphometrical Parameters Obtained by Extracting with a CAS 200TM Image Analyzer, and Explanation of the Meaning for Each Parameter According to the Descriptions of J.W. Bacus[24], J.P. Pressman[28] and Dawson et al.[29]

Pregnant dams were orally dosed with DBP (100 mg/kg/day) from gestation days 12 to 21; body weights were similar in control and DBP-treated dams both at the beginning and end of the experiments. Additionally, no decrease in litter size or pup survival, alteration of sex ratio or difference in body weights of male pups compared with controls on any day examined were found and the relative testicular weights of DBP groups were similar to those of the vehicle group (data not shown)[27],[28]. However, by conducting routine toxicological pathological observation, the LCs observed in the 5- and 7-wk-old DBP groups did not display nuclear alterations or any other apparent toxicity (Fig.1).

The cell nuclear analysis parameters of LCs of the 5-wk-old DBP group shown in Table 1 were not significantly different from those of the vehicle group (Table 2). Although the DNA ploidy of LCs of the 7-wk-old DBP group and that of the vehicle group were the diploid type (Fig. 2) and the DNA amount of the DBP group was similar to that of the vehicle group (Fig. 3, Tables 1 and 3), the general nuclear morphometric parameters of LCs of the DBP group were significantly different from those of the vehicle group. The area, perimeter, shape, maximum diameter and elongation values of LCs of the 7-wk-old DBP group were significantly higher than those of the vehicle group (Fig. 3, Tables 1, 3), but the minimum diameter values of the DBP group were similar to those of the vehicle group (Fig. 3, Tables 1, 3). Moreover, the general morphometric parameters of LC chromatin of the 7-wk-old DBP group were significantly different from those of the vehicle group. The values of parameters configurable (Cfg) run length, valley and peak of the DBP group were significantly lower than those of the vehicle group (Fig. 3, Tables 1, 3), and the slope value of the DBP group was significantly higher than that of the vehicle group (Fig. 3, Tables 1, 3). Besides the Markovian analysis, several parameters of LCs of the 7-wk-old DBP group were significantly different from those of the vehicle group. The chromatin granularity values of the DBP group were significantly lower than those of the vehicle group (Fig. 3, Tables 1, 3), the chromatin peripheral aggregation parameters values of the DBP group were significantly higher than those of the vehicle group, and the symmetrical chromatin distribution parameters values were similar to those of the vehicle group (Fig. 3, Table 3).

Table 2.

Summary of Morphometrical Parameters Obtained by Extracting with a CAS 200TM Image Analyzer and Analysis by Cell-SheetTM of Leydig Cell Nuclei in 5-wk-old Rats

Fig. 2.

Representative DNA histograms: distribution of DNA mass generated by CAS 200 computer-assisted cytometry of the 7-wk-old vehicle group (A) and 7-wk-old DBP group (B). Cells that contain normal amounts of DNA [2C]; cells in the G2/M-phase area of the cell cycle [4C].

Fig. 3.

Box plots of the nuclear morphological parameters listed in Table 1. Values were analyzed using at least 800 nuclei; Mann-Whitney U test; ** P<0.001.

Table 3.

Summary of Morphometrical Parameters Obtained by Extracting with a CAS 200TM Image Analyzer and Analysis by Cell-SheetTM of Leydig Cell Nuclei in 7-wk-old Rats

In general, the normal cell nucleus tends to be round or at least smoothly curved, and the chromatin tends to be evenly distributed, but this is not true for dysplastic cells, which tend to have irregularly shaped nuclei and chromatin distributed in apparently clumped and disordered patterns[3],[4],[6]; it has been proposed these variances might be phenotypic characters of the genomic alterations[1],[2]. Normal LC nuclei show a discriminative chromatin distribution with many distinct isolated large- and/or small-sized chromatin aggregations continuously distributed and thin chromatin aggregation on the nuclear membrane (Fig. 1)[29].

Hyperplastic LCs, in general, show nuclear pleomorphism, which is considered evidence of continuous progressive degeneration[29]. Until puberty, rats do not show LC hyperplasia despite prenatal DBP exposure[27],[28]. Based on the present analysis of the DNA morphological parameters, LCs of the 5- and 7-wk-old DBP groups showed DNA diploidy similar to that of the vehicle groups. Other morphometric parameters indicated that the nuclear structures and chromatin distribution patterns of LCs of the 5-wk-old DBP group were similar to those of the vehicle group, but the nuclear structures and chromatin distribution patterns of LCs of the 7-week-old DBP group were significantly different from those of the vehicle group.

The present quantitative study demonstrated that, compared with the vehicle group, LC nuclei of 7-wk-old rats exposed to prenatal DBP were significantly larger with an oval shape according to analysis using general nuclear morphometry parameters (Fig. 3, Tables 1 and 3), significantly decreased chromatin granulation clumps, and coarse clumping of the nuclear chromatin, while the nuclear borders were significantly thickened with focal aggregations of chromatin at the inner nuclear border according to analysis of general chromatin morphometry parameters and Markovian analysis including chromatin granularity and peripheral aggregation parameters (Fig. 3, Tables 1 and 3). Variances in nuclear area, perimeter and diameter are considered frequent events in progressive degeneration, but not in the degenerative process[34]. Nuclear shrinkage and hyperchromatism were described in LCs of cadmium-exposed rats[35], and these morphological changes due to the effect of cadmium on LCs may be the last step before the appearance of tumor lesions[36],[37]. The present study revealed that the effects of prenatal DBP exposure on rat LC nuclear structures were significant at seven weeks of age without increased cellular proliferation, and these morphological variances suggested that the genomic alterations of LCs following prenatal DBP exposure might be induced before hyperplastic LC formation. Further study is required to elucidate the detailed genomic alterations including mutation, deletion, amplification, and/or epigenomic modification.

Although pathologists have traditionally described changes in nuclear chromatin as “increased chromatin clumps” and “irregular thickening and sharp margination of nuclear borders,” it was impossible to quantify these characteristics[5],[6],[7],[20]. The present nuclear morphometric analysis study provided quantitative data that confirmed the description indicating the pathological status of cells. It is clear that the differences in quantitative chromatin parameters observed in the present study are important morphologic criteria that might be used in the diagnosis of toxicological pathology.