Variability in Weight and Histological Appearance of the Prostate of Beagle Dogs Used in Toxicology Studies

Toxicologic Pathology, 36: 917-925, 2008 Copyright 2008 by Society of Toxicologic Pathology ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1177/0192623308324958 Variability in Weight and Histological Appearance of the Prostate of Beagle Dogs Used in Toxicology Studies LAËTITIA DORSO, FRANCK CHANUT, PAUL HOWROYD, AND ROGER BURNETT MDS Pharma Services, Drug Safety Assessment, Les Oncins, 69210 Saint-Germain-sur-L Arbresle, France ABSTRACT This review was performed to assess variations in weight and histologic appearance of the prostate of untreated male beagle dogs between 23 and 108 weeks of age, from two breeding centers. Data from 125 control beagle dogs from twenty-seven regulatory toxicology studies were used. Age, terminal body weight, and prostate weight were analyzed. Prostate sections were examined microscopically, and histological changes such as development of acini, amount of secretion, and patterns of dilation and inflammation were recorded and graded when appropriate. The influence of age, terminal body weight, and source on the weight and histological appearance of the prostate, and the degree of interanimal variation were evaluated Keywords: prostate; beagle dog; histology; body weight, maturation, preclinical safety evaluation. INTRODUCTION Nonrodent safety evaluation studies are frequently performed with purpose-bred beagle dogs. Incomplete sexual maturity of dogs can interfere with an accurate identification of compound-related changes by introducing variation in the weight and histological appearance of reproductive tissues. Knowledge of anatomical variations and development will permit accurate interpretation of changes observed in preclinical drug safety studies and help in extrapolating this interpretation to the risk in human reproduction (Foley 2001). Although histological changes in the aging dog prostate are well reported, there are no published surveys of the variation in the weight and histological appearance of the prostate in young dogs of the ages that are used in routine safety evaluation studies. This paper reports the findings of such a study. These data allow an understanding of the range of histomorphology of the normal prostate in dogs and therefore a more accurate interpretation of potential treatment-related changes in the prostate. The prostate is the major accessory sex gland present in the male dog. According to James and Heywood (1979), sexual maturity is reached between thirty-five and forty weeks of age in male beagle dogs, whereas Taha et al. (1981) reported that dogs do not achieve adult sexual function, based on seminal characteristics, until forty-five weeks of age. The size and weight of the prostate vary depending on the age, breed, and body weight of the dog. The first histological signs of activity appear at about four months of age, and prostatic acinar cells become periodic acid-schiff (PAS)-positive at twenty-four weeks of age, suggesting the beginning of secretory activity Address correspondence to: Franck Chanut, MDS Pharma Services, Drug Safety Assessment, Les Oncins, 69210 Saint-Germain-Sur-L Arbresle, France; e-mail: Franck.Chanut@mdsinc.com. (Kawakami et al. 1991). Cell proliferation and differentiation of the prostatic epithelium are controlled primarily by androgens, the most potent of which is testosterone. The canine prostate is broadly similar to the human prostate, although there are subtle differences in the vascular supply and musculature around the urinary outflow tract (Stefanov et al. 2000; Steiner 1993). Prostatic hyperplasia also occurs in aging dogs and humans, and for this reason, the dog has been widely used as a model for benign prostatic hyperplasia of man (Waters et al. 1996). MATERIALS AND METHODS Animals These dogs were provided either by Harlan France (Gannat, France) or by Marshall Farms, Inc. (North Rose, NY, USA). Marshall Farms USA dogs were in the range of twenty-five to eighty-nine weeks of age, whereas dogs from Harlan France were from 23 to 108 weeks of age. No data were available for Harlan dogs from fifty-one to ninety weeks of age. For statistical analysis, groups of ten-week intervals were arbitrarily formed. Histological data from 125 male beagle dogs, from 23 to 108 weeks of age (birth date provided by supplier) from the control groups of twenty-seven acute and chronic toxicity studies were examined. In addition, terminal body weight and prostate weight were also available from another thirty-two male beagle dogs from eight other studies. The studies were conducted in an Association for Accreditation and Assessment of Laboratory Animal Care (AAALAC)-accredited facility, in compliance with European regulations governing the housing and use of animals. During the course of all studies, there were no differences of diet, housing, and exercise between the dogs from different commercial sources. 917

918 DORSO ET AL. TOXICOLOGIC PATHOLOGY Necropsy At the end of the experimental period, the dogs were euthanized with an overdose of barbiturates, and a full macroscopic postmortem examination was performed. Animals were weighed, and all major organs including prostate, testes, and epididymides were dissected free of fat and the weights recorded. Prostatic sections were prepared according to MDS standard operating procedures and were standard across studies. Prostate samples were fixed in 10% buffered formalin, whereas testis and epididymides samples were fixed in modified Davidson s fixative. They were then processed into paraffin wax, sectioned at 3 4 µm, and routinely stained with hematoxylin-eosin (HE). Microscopic Examination The prostatic sections were re-examined and the histological appearance recorded in a consistent fashion, using the following criteria: 1. Epithelial maturation of acini. Grade 1 corresponded to totally immature prostatic epithelium, characterized by a cuboidal to flattened basophilic epithelium with a high nucleocytoplasmic ratio. Grade 2 corresponded to partially immature prostatic epithelium, with some areas showing an immature prostatic epithelium and other areas showing a mature prostatic epithelium. Grade 3 corresponded to totally mature prostatic epithelium, characterized by a columnar epithelium with basal nuclei and an abundant eosinophilic apical cytoplasm. 2. Absence or presence of prostatic secretion. 3. Presence and pattern of acinar dilation. 4. Absence or presence of inflammation. Testis and epididymide maturity was also evaluated to allow an animal-by-animal correlation with prostate morphology. As described by Foley (2001), the immature testis was characterized by small-diameter, immature tubules, often without a lumen, lined by Sertoli cells and immature germ cells; the presence of multinucleated giant cells; and the absence of spermatids and spermatozoa. Immature animals may have few or no spermatozoa within the epididymal duct. By contrast, the mature testis was characterized by visible lumina in seminiferous tubules, the presence of spermatids and spermatozoa, and infrequent multinucleated giant cells. Statistical analysis was performed using R Development Core Team (R Foundation for Statistical Computing, Vienna, Austria, 2007) A Student test was used to compare changes in terminal body weight and prostate weight between animals originating from the two different suppliers. Correlations between different parameters (age, terminal body weight, and absolute prostate weight) were determined by the linear regression method. RESULTS The age, numbers, and final body weight of the dogs are presented in Table 1. Terminal Body Weight and Prostate Weight Terminal body weight and absolute and relative prostate weight were tabulated by week of age and supplier. The mean terminal body weight was higher and statistically significant in dogs from Harlan France than in dogs from Marshall Farms USA (Figure 1 and Table 1). The difference in mean relative prostate weight was not statistically significant between the two suppliers for the period between twenty-three and fifty weeks of age (Figure 2 and Table 2). There was a positive correlation between terminal body weight and age in dogs from Marshall Farms USA and from Harlan France (p <.01) (Figure 3). In dogs from both suppliers, there was a positive correlation (p <.01) between absolute prostate weight and age (Figure 4). The correlation between terminal body weight and absolute prostate weight was positive in dogs from the two suppliers (p <.01). Relative prostate weight also increased with age between twenty-three and fifty weeks, however, implying that the prostate increased in weight relatively more than the rest of the body during this period (Figure 5). Microscopic Findings Owing to the absence of animals from Harlan France of fifty-one to ninety weeks of age, most histological data were derived from Marshall Farms dogs. There were no differences in histological appearance between dogs from the two sources at the same stage of maturity. The microscopic findings were consistent with what was described by Evans (1993). Prostates from sexually immature animals were characterized by a branching ductular system. In contrast to the normal mature gland, the acinar portions of the gland were not developed and had little lumen, and there was no evidence of secretory activity (eosinophilic material) in the acinar cell cytoplasm or acinar lumen. Immature prostatic epithelium was characterized by a cuboidal to flattened basophilic epithelium with a high nucleocytoplasmic ratio (Figure 6). The amount of connective tissue was relatively higher in the immature gland than in the fully developed gland. This stroma was composed predominantly of prominent fibrous connective tissue, and smooth muscle was seen in the outer capsule and in major trabeculae radiating into the gland (Evans 1993). The percentage of grade 1 or 2 acini (totally or partially immature) decreased with age, and the percentage of grade 3 acini (mature) increased with age (Figure 7). By contrast, mature prostates in these studies were characterized by compound tubuloalveolar glands, which radiated from their duct openings into the urethra. The acinar portion of the gland was dilated to form an alveolar structure and contained primary and secondary infoldings of secretory epithelium,

Vol. 36, No. 7, 2008 WEIGHT AND APPEARANCE OF PROSTATE IN BEAGLE DOGS 919 TABLE 1. Mean terminal body weight. Data shown reflect a statistically significant higher mean terminal body weight in dogs from Harlan France than in dogs from Marshall Farms USA between twenty-three and fifty weeks of age. Marshall Farms USA Harlan France Age (weeks) Number of animals Mean terminal body weight (g) ± SD Number of animals Mean terminal body weight (g) ± SD 23 30 16 7665 ± 722 13 8647 ± 901* 31 40 49 8582 ± 883 11 10242 ± 1300* 41 50 17 8734 ±1132 3 11259 ± 1017* 51 60 11 9713 ± 867 0 NA 61 70 6 9168 ± 1893 0 NA 71 80 17 9806 ± 1358 0 NA 81 90 8 9383 ± 1702 0 NA 91 100 0 NA 2 12386 ± 952 101 110 0 NA 4 12440 ± 2014 *p.01 Abbreviations: NA, not available; SD, standard deviation. Harlan France, Gannat, France Marshall Farms, Inc., North Rose, NY, USA FIGURE 1. Comparison of mean terminal body weight between the two suppliers: there is a statistically significant higher mean terminal body weight in dogs from Harlan France (n = 27) than in dogs from Marshall Farms USA (n = 82) between twenty-three and fifty weeks of age (*p <.05, **p <.01). FIGURE 2. Comparison of mean relative prostate weight between the two suppliers, between twenty-three and fifty weeks of age. There is no statistically significant difference between the two suppliers (Harlan France [n = 27], Marshall Farms USA [n = 82]). TABLE 2. Mean absolute prostate weight. Data shown reflect a statistically significant higher mean absolute prostate weight in dogs from Harlan France than in dogs from Marshall Farms USA between thirty-one and forty weeks of age. Marshall Farms USA Harlan France Age (weeks) Number of animals Mean absolute prostate weight (g) ± SD Number of animals Mean absolute prostate weight (g) ± SD 23 30 16 1.708 ± 1.020 13 2.217 ± 1.194 31 40 49 3.428 ± 1.852 11 4.875 ± 2.512* 41 50 17 5.036 ± 2.127 3 4.687 ± 2.085 51 60 11 5.847 ± 1.936 0 NA 61 70 6 6.702 ± 1.705 0 NA 71 80 17 7.072 ± 2.089 0 NA 81 90 8 7.719 ± 1.169 0 NA 91 100 0 NA 2 8.705 ± 2.638 101 110 0 NA 4 9.090 ± 1.926 *p.05 Abbreviations: NA, not available; SD, standard deviation. Harlan France, Gannat, France Marshall Farms, Inc., North Rose, NY, USA

920 DORSO ET AL. TOXICOLOGIC PATHOLOGY FIGURE 3. Correlation between terminal body weight and age. There is a correlation between terminal body weight and age in dogs from Marshall Farms USA (n = 124) and in dogs from Harlan France (n = 33) (p <.01). FIGURE 4. Correlation between absolute prostate weight and age. There is a correlation between absolute prostate weight and age in dogs from the two suppliers (Marshall Farms USA [n = 124], Harlan France [n = 33]) (p <.01). FIGURE 5. Correlation between terminal body weight and absolute prostate weight. There is a correlation between terminal body weight and absolute prostate weight in dogs from Harlan France (n = 33) and in dogs from Marshall Farms USA (n = 124) (p <.01).

Vol. 36, No. 7, 2008 WEIGHT AND APPEARANCE OF PROSTATE IN BEAGLE DOGS 921 FIGURE 6. Immature prostatic epithelium characterized by a cuboidal (a) to flattened (b) basophilic epithelium with a high nucleocytoplasmic ratio. Bar = 200 µm. FIGURE 7. Evolution of epithelial development of acini with age: the percentage of grade 1 or 2 acini decreased and the percentage of grade 3 acini increased with age of dogs from the two suppliers (Marshall Farms USA [n = 97], Harlan France [n = 28]). FIGURE 8. Mature prostatic epithelium characterized by a columnar epithelium with basal nuclei, an abundant eosinophilic apical cytoplasm, forming infoldings into alveolar lumen. Bar = 50 µm. which projected into the alveolar lumina. Mature prostatic epithelium was characterized by columnar epithelial cells with basal nuclei and abundant eosinophilic apical cytoplasm (Figure 8). The lumina were slightly dilated, although in the majority of cases there was no secretory material. When present, prostatic secretion was characterized by a deeply eosinophilic secretion dilating the acini but without compression of adjacent acini (Figures 9 and 10). Secretion increased with age between twenty-three and eighty-nine weeks of age. Focal areas of acini with eosinophilic intraluminal content were sometimes found in prostates showing features of immaturity. In mature prostates, acinar dilation presented two patterns. Simple dilation was defined as many dilated acini with or without luminal eosinophilic secretion, which did not compress the adjacent acini. Acini were lined by a flattened basophilic epithelium with a high nucleocytoplasmic ratio (Figure 11a). Focal glandular ectasia was defined as focal dilation of a few acini with eosinophilic content, sharply demarcated, with

922 DORSO ET AL. TOXICOLOGIC PATHOLOGY FIGURE 9. Evolution of prostatic secretion with age: prostatic secretion increased with the age according to prostatic development in dogs from both suppliers (Marshall Farms USA [n = 97], Harlan France [n = 28]). FIGURE 10. Prostatic secretion characterized by dilated acini filled with a deeply eosinophilic secretion with no compression of adjacent prostatic acini. Bar = 400 µm. compression of the adjacent prostatic parenchyma. Acini were lined by a columnar epithelium with basal nuclei and abundant eosinophilic apical cytoplasm (Figure 11b). The alveoli were separated by a dense stroma that also contained smooth muscle cells. These smooth muscle septa were continuous with the smooth muscle and fibrous tissue located in the periurethral area (Evans 1993). The patterns of inflammation were classified as: minimal lymphoid cell infiltration in the connective tissue without involvement of acinar epithelium; periurethral lymphoid cell infiltration; and subacute inflammatory cell foci, with inflammatory cells (mainly lymphocytes, plasma cells and macrophages) with involvement of the acinar epithelium (atrophy, exocytosis, basophilia or fibrosis) (Figure 12). FIGURE 11. Simple dilation (11a) and focal glandular ectasia (11b). Bar = 1000 µm. The incidences of dilation and inflammation are presented in Table 3. No relation could be established between inflammatory lesions and dilation pattern. With the available data, there is no

Vol. 36, No. 7, 2008 WEIGHT AND APPEARANCE OF PROSTATE IN BEAGLE DOGS 923 FIGURE 12. Lymphoid cell inflammation with atrophy of adjacent acini. Bar = 300 µm. clear relation between the incidence of these changes (dilation and inflammation) and the age of animals. Testis Maturity The percentage of immature and mature testes according to age is summarized in Figure 13. There was a strong correlation between maturity of prostate and maturity of testes and epididymides in each dog. Moreover, 90% of thirty-one- to fortyweek-old dogs from Harlan France were mature, whereas only 10% of dogs from Marshall Farms USA were mature at the same age. Sexual maturity appeared to occur earlier in dogs from Harlan France than in dogs from Marshall Farms USA. In summary, the sources of variation in prostate weight between individual animals were (1) the sexual maturity of the animals (sexual maturity being reached between forty-one and fifty weeks in Marshall Farm dogs but at thirty-one to forty weeks in Harlan France dogs); (2) the source of the animals was very important in this result, as Harlan France dogs were heavier at a given age and sexually mature earlier than Marshall s dogs, but there were no differences in histological appearance between dogs from the two sources at the same age of maturity; and (3) individual variation between dogs of the same sexual maturity. DISCUSSION These data allowed a better understanding of the interindividual variability in prostate weight and appearance of beagle dogs (Figure 6, Figure 9, and Table 3). The mean terminal body weight of dogs of the same age was statistically significantly higher in dogs from Harlan France than in dogs from Marshall Farms USA between twenty-three and fifty weeks of age. Because each supplier bred its own dog colony, these differences in mean terminal body weight could be attributable to a different genetic background. Moreover, the mean relative weight was not statistically significantly different between the two suppliers. The testicular histology results suggested that sexual maturity occurred earlier in dogs from Harlan France than in dogs from Marshall Farms USA. However the number of animals was higher for Marshall Farms USA (n = 82) than for Harlan France (n = 27) between twentythree and fifty weeks of age. The analysis of data concerning terminal body weight and age suggested that there is a positive correlation between terminal body weight and age. The positive correlation between absolute prostate weight and age in dogs from the two suppliers showed that prostatic development occurred according to age. The positive correlation between terminal body weight and absolute prostate weight suggested that prostatic development also occurs according to the conformational growth. Regarding prostatic epithelial development, the percentage of grade 1 or 2 acini (totally or partially immature) decreased with age, and the percentage of grade 3 acini (mature) increased with age in dogs from Marshall Farms USA. We considered that maturity of epithelial acini is reached at forty-three weeks of age for the two suppliers. Histologically, prostatic secretion increased with age, in the same way as prostatic development. Acinar dilation and prostatic inflammation were events that were seen regardless of age, period of study, and method of urine collection. The etiology of inflammatory foci in prostate parenchyma was uncertain but was probably caused by lowgrade bacterial infection (James and Heywood 1979). Bacterial infection, endocrine influence, immunological dysfunction, and stress are reported to be factors in development of prostatic inflammation in rat prostate (Suwa et al. 2001). Histologic findings in the prostate could not be interpreted without histologic evaluation of testes because of the close hormonal link between these two organs. The combined histologic examinations of the prostates and testes in those dogs allowed the determination of age of sexual maturity. In Harlan France s dogs, for this data set, the oldest reported immature dog was thirty-one weeks old, whereas in Marshall s dogs, the oldest reported immature dog was forty-one weeks old. This result confirmed that Harlan s dogs were sexually mature earlier than Marshall s dogs. These data were consistent with those obtained by James and Heywood (1979), who showed that male beagle dogs reached full sexual maturity between thirty-five and forty weeks of age (Figure 13). Moreover, in thirty-one- to forty-week-old dogs, there was a greater percentage of mature dogs from Harlan France than from Marshall Farms USA. This survey showed the spontaneous variations in the weight and morphology of the beagle prostate that may influence the assessment of toxicological data. Harlan s dogs were heavier at a given age and sexually mature earlier than Marshall s dogs. The histologic appearance of the prostate was the same regardless of the supplier. There were spontaneous variations in histologic appearance of dogs prostates and a difference of age of maturity according to the commercial sources. This finding raises the importance of the pathologist s awareness of such spontaneous lesions in the dog strain used and knowledge of the particular supplier s dogs. Immature acini could lead to a misdiagnosis of treatment-related effect of acinar atrophy. To

924 DORSO ET AL. TOXICOLOGIC PATHOLOGY TABLE 3. Incidence (%) of histological changes of dilation and inflammation in dogs from Marshall Farms USA and Harlan France. The incidence of these changes is not clearly influenced by the age of the animal. Age (weeks) 23 30 31 40 41 50 51 60 61 70 71 80 81 90 91 100 101 110 No dilation Marshall 25 18 23 0 17 22 17 NA NA Harlan 27 25 33 NA NA NA NA 0 0 Grade 1 dilation Marshall 44 39 18 20 33 11 50 NA NA Harlan 19 37.5 33 NA NA NA NA 0 0 Grade 2 dilation Marshall 12.5 10 18 20 50 11 17 NA NA Harlan 36 25 34 NA NA NA NA 0 0 Grade 3 dilation Marshall 12.5 13 18 40 0 0 0 NA NA Harlan 18 12.5 0 NA NA NA NA 50 0 Focal glandular ectasia Marshall 6 26 41 40 33 78 17 NA NA Harlan 0 37.5 0 NA NA NA NA 50 100 No inflammation Marshall 75 63 53 80 50 33 33 NA NA Harlan 91 25 100 NA NA NA NA 0 25 Minimal lymphoid cell infiltration Marshall 19 26 23 20 33 33 0 NA NA Harlan 0 50 0 NA NA NA NA 0 0 Periurethral lymphoid cell infiltration Marshall 6 5 6 0 0 0 17 NA NA Harlan 9 25 0 NA NA NA NA 100 75 Subacute inflammatory cell foci Marshall 0 8 18 0 17 33 17 NA NA Harlan 19 37.5 33 NA NA NA NA 0 0 Abbreviation: NA, not available. FIGURE 13. Percentage of immature and mature testes according to age: in Harlan France s dogs, the oldest reported immature dog was thirtyone weeks old, whereas in Marshall s dogs, the oldest reported immature dog was forty-one weeks old. This result confirmed that Harlan s dogs were sexually mature earlier than Marshall s dogs (Marshall Farms USA [n = 97], Harlan France [n = 28]). reduce interanimal variability, we consider that mature animals should be used wherever possible in studies unless there is a specific requirement to study juvenile development. ACKNOWLEDGMENTS The authors thank all their pathologist colleagues and the archivists who helped in the compilation of these data. REFERENCES Evans, H. E. (1993). Miller s Anatomy of the Dog, 3 rd ed. W. B. Saunders Company, 514 15. Foley, G. L. (2001). Overview of Male Reproductive Pathology. Toxicol Pathol 29, 49 63. James, R. W., and Heywood, R. (1979). Age-related variations in the testes and prostate of Beagle dogs. Toxicology 12, 273 79.

Vol. 36, No. 7, 2008 WEIGHT AND APPEARANCE OF PROSTATE IN BEAGLE DOGS 925 Kawakami, E., Tsutsui, T., and Ogasa, A. (1991). Histological observations of the reproductive organs of the male dog from birth to sexual maturity. J Vet Med Sci 53, 241 48. Stefanov, M., Martín-Alguacil, N., and Martín-Orti, R. (2000). Distinct vascular zones in the canine prostate. Microsc Res Tech 50, 169 75. Steiner, M. S. (1993). Role of peptide growth factors in the prostate: a review. Urology 42, 99 110. Suwa, T., Nyska, A., Peckham, J. C., Hailey, J. R., Mahler, J. F., Haseman, J. K., and Maronpot, R. R. (2001). A retrospective analysis of background lesions and tissue accountability for male accessory sex organs in Fischer-344 rats. Toxicol Pathol 29, 467 78. Taha, M. A., Noakes, D. E., and Allen, W. E. (1981). Some aspects of reproductive function in the male beagle at puberty. J Small Anim Pract 22, 663 67. Waters, D. J., Patronek, G. J., Bostwick, D. G., and Glickman, L. T. (1996). Comparing the age of prostate cancer diagnosis in humans and dogs. J Natl Cancer Inst 88, 1686 87.