Toxicologic Pathology

Toxicology of the male reproductive system has received increased interest in recent years partly fuelled by the growing reports of falling sperm counts and rising reproductive disorders in the human population. Recently revised regulatory guidelines for the safety assessment of pharmaceuticals and chemicals on reproduction and fertility have emphasized the importance of detailed histopathological examination of the testes as a sensitive method for detecting disturbances in spermatogenesis. Unfortunately this has been accompanied by a general confusion regarding a practical approach to undertaking such a detailed examination, particularly in respect to the use of spermatogenic or tubular staging to identify subtle disturbances in spermatogenesis. The ability to identify tubular stages of the spermatogenic cycle in sections of testis plus a good understanding of the spermatogenic process and its dynamics are essential in order to carry out a sensitive examination of testicular histopathology and to interpret the changes seen. A rational approach is required initially to detect and subsequently to characterize toxic effects to the male reproductive system. It is important that a distinction is made between these two objectives since different study designs are required and different methodology may be employed to produce the type of information or data required.

Hydroquinone exposure has been reported by the National Toxicology Program (NTP) to produce renal tubule adenomas and to exacerbate spontaneous chronic progressive nephropathy (CPN) in male F344 rats. A mechanism for hydroquinone-related tumorigenesis has not been established, but CPN is known to involve, and hydroquinone produces, enhanced renal tubule cell proliferation. Through an independent review of the renal histopathology from the NTP study, the grade of CPN and the presence of atypical tubule hyperplasia and adenomas was evaluated. Hydroquinone exposure in males at 50 mg/kg produced a statistically significant increase in the grade of CPN. At 0, 25, and 50 mg/kg, 0/44, 4/49, and 15/51 male rats had either atypical tubule hyperplasias or adenomas; all were within areas of severe or end-stage CPN and were statistically significantly associated with CPN grade. Additionally, there was a dose-related increase in profiles believed to represent new tubule proliferation within areas of advanced CPN, as well as an apparent expansion of these into unusual complex tubule profiles in end-stage kidneys of the high-dose male group. In summary, this histopathological review suggests a mechanism for hydroquinone-related adenoma formation that includes enhancement of the severity of CPN coupled with stimulation of tubule proliferation.

We examined the bone changes in recombinant human granulocyte colony-stimulating factor (rhG-CSF)-treated young and young adult rats in order to investigate the effect of age-related conditions of bone growth on the bone changes induced by rhG-CSF. Recombinant human G-CSF (100 and 1,000 μg/kg/day) was given to rats by daily intravenous injection for 28 days starting at the age of either 6 or 14 wk, and the hindlimb bones were evaluated histopathologically. In the young rats, bone lesions were observed in the 100- and 1,000-μg/kg groups. In the young adult rats, lesions were found only in the 1,000-μg/kg group. The lesions involved accelerated osteoclastic bone resorption and osteogenesis due to intramembranous ossification, and there was no age-related difference in these histopathological findings. However, both the incidence of bone involvement and the severity of lesions were greater in the young rats than in the dose-matched young adult rats. The results suggest that the higher dose of rhG-CSF may intrinsically induce bone lesions of a particular histopathological nature in rats regardless of their age, and the action of rhG-CSF on bone may be stronger in young growing rats than in young adults.

Rats showing an ataxic gait induced by 20 wk of treatment with 0, 30, or 60 mg/kg of difluorobenzhydrylpiperadine (DFBP), a detriazinyl metabolite of almitrine, were examined by light microscopy and transmission electron microscopy. Vacuolar degeneration associated with lamellar inclusions was observed in musculus soleus and m. interossei of the hindlimbs in DFBP-treated rats. The inclusions were also produced within sensory neurons, satellite and Schwann cells, and vascular endothelial cells of thoracic and lumbar dorsal root ganglia as well as muscle spindles of affected muscles. Membrane-bound vacuoles containing electron-dense granules were seen in the peripheral nerves. This study demonstrated neuronal and muscular toxicity of DFBP in rats.

The time course of contrast media (CM)-induced renal proximal tubular vacuolation was investigated in rats by light microscopy, transmission electron microscopy (TEM), and ultrastructural histochemistry for acid phosphatase activity. Young adult male rats were treated with a single dose of 3.0 g I/kg Iotrolan (Isovist® 300 mg I/ml) and sacrificed at 0 min, 5-min, 15-min, 2-hr, and 24-hr intervals. Light microscopy of vibratome sections of freshly excised tissue and of cryostat and paraffin sections was also performed to allow comparison of the appearance of the vacuoles in the fresh state with light and electron microscopy. The sequence of events seen to occur can be summarized as follows.

CM-induced vacuolation occurred at a low level as soon as 5 min after compound administration. The vacuolation was observed by TEM but could not be detected by light microscopy. This was followed by an increase in size and numbers of vacuoles up to the 24-hr timepoint with a sequential increase in the staining for acid phosphatase activity of the vacuoles, most marked at the 24-hr timepoint. At timepoints less than 24 hr there appeared to be no marked increase in the normal complement of lysosomes or in the components of the Golgi-endoplasmic reticulum-lysosome pathway. At 24 hr, the vast majority, but not all, of the CM-induced vacuoles were positive for acid phosphatase activity. The intensity of staining varied, and there was evidence of fusion of small lysosomes with CM-induced vacuoles. These results suggest that formation of CM-induced vacuoles is a 2-stage process, following a normal pathway for the handling of endogenous and exogenous substances.

Peroxisome proliferators are believed to induce liver tumors in rodents due to sustained increase in cell proliferation and oxidative stress resulting from the induction of peroxisomal enzymes. The objective of this study was to conduct a sequential analysis of the early changes in cell-cycle kinetics and the dynamics of rat liver DNA synthesis after treatment with a peroxisome proliferator. Immunofluorescent detection of proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) incorporation into DNA during S phase were used to assess rat hepatocyte proliferation in vivo during dietary administration of Wy-14,643, a known peroxisome proliferator and hepatocarcinogen in rodents. Rats were placed on a diet containing 0.1% Wy-14,643 and implanted subcutaneously with 5-bromo-2'-deoxyuridine containing osmotic pumps 4 days prior to being sacrificed on days 4, 11, and 25 of treatment. Isolated liver nuclei labeled with fluorscein isothiocyanate (FITC)-anti-BrdU/PI and FITC-anti-PCNA/PI were analyzed for S-phase kinetics using flow cytometry. Morphometric analysis was performed to evaluate nuclear and cell size and enumeration of BrdU labeled cells, binucleated hepatocytes, and mitotic index. The BrdU labeling index increased 2-fold in livers of Wy-14,643-treated rats at day 4, but distribution of cells in G₁, S phase, and G ₂-M did not differ significantly from controls. PCNA-positive cells decreased from 36% on day 4 to 17% on day 25, whereas the percentage of PCNA-positive cells in controls increased 2-fold from day 4 to day 11 and remained unchanged up to day 25. The differences in the number of PCNA-positive nuclei between control and Wy-14,643-treated groups were statistically significant only on day 4. Binucleated hepatocytes, determined by morphometric analysis, increased slightly on day 25 in treated rats parallel to an increase in the percentage of cells in G₂-M phase. Significant shifts were noted in nuclear diameter and nuclear area after 11 and 25 days of treatment with Wy-14,643. Hepatic cell populations with nuclei >9 μm diameter and nuclear area >64 μm² increased in Wy-14,643-fed rats during the treatment period compared with the control, indicating hepatic karyomegaly and hyperploidy, whereas percentage of distribution of nuclei based on diameter and area remained consistently unchanged in control animals from 4 through 25 days of sham treatment. The flow cytometric and morphometric analysis indicated an initial wave of DNA synthesis in response to Wy-14,643. The hepatomegaly was sustained over the treatment period accompanied by increase in ploidy with a significant shift toward hyperploidic hepatocytes. The increase in DNA content was almost entirely accounted for by the overall polyploidy increase rather than by an absolute increase in cells.

To clarify relationships between serum thyroid-stimulating hormone (TSH) levels and ultrastructural changes in thyrotrophs caused by intermittent or continuous treatments with antithyroid compound, male Fischer-344 rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN) were given water containing 0.1% sulfadimethoxine (SM) for 8 wk and then sacrificed (Group 1). Additional groups were examined 2 wk after withdrawal (Group 2), after 4 wk retreatment following a 2-wk withdrawal period (Group 3), and after 14 wk continuous exposure (Group 4). Control rats (Group 5) remained untreated for 8 wk after the DHPN initiation. Microscopic examination revealed hypertrophy of thyrotrophs and depletion of TSH-positive material in Groups 1, 3, and 4 but a return to normal in Group 2. Electron microscopic examination of thyrotrophs in the anterior pituitary in Groups 1, 3, and 4 revealed dilated rough endoplasmic reticulum (ER) cisternae with intracisternal dense granules as well as diminished numbers of intracytoplasmic secretory granules, these changes being most marked in Group 1 and least pronounced in Group 4. The number of intracytoplasmic secretory granules in Group 3 was much lower than in Group 4, as demonstrated by morphometric analysis. In Group 2, thyrotrophs showed dilated rough ER cisternae without intracisternal dense granules and essentially the same component of intracytoplasmic secretory granules as Group 5. The present study suggests that while prolonged continuous treatment with SM results in gradual acclimation to an increased demand for TSH, intermittent treatment elicits a persistent state of reduced TSH storage in thyrotrophs due to a continued strong feedback through the hypothalamus-pituitary-thyroid axis.

The herbicide dichlobenil (2,6-dichlorobenzonitrile) is a tissue-specific toxicant inducing necrosis in the olfactory mucosa. Transmission electron microscopy showed vacuolations (1 hr) and necrosis (4 hr) in the Bowman's glands following intraperitoneal injection of dichlobenil (25 mg/kg) in C57B1 mice, whereas no lesions were noted in the olfactory neuroepithelium at these timepoints. Twenty-four hr following injection (25 mg/kg), most of the lamina propria and the olfactory epithelium were severely damaged; the basal lamina, nerves, and blood vessels in the lamina propria, however, remained intact. Following a lower dose (12 mg/kg), the lesions were not as pronounced. The effects of dichlobenil (6, 12, and 25 mg/kg) on cell replication in the olfactory mucosa, as determined by incorporation of 3H-thymidine 3 days later, were more pronounced in the lamina propria than in the neuroepithelium and occurred at a lower dose in the lamina propria than in the neuroepithelium. Together these studies support the previous proposal that dichlobenil induces a primary damage in the Bowman's glands.

Male and female Fischer-344 (F-344) and male NCI-Black-Reiter (NBR) rats were dosed with 0, 35.5, or 71 mg 1,3,5-trinitrobenzene (TNB)/kg/day for 10 days. Male F-344 rats were dosed with TNB (0 and 35.5 mg/kg) for 20 and 30 days. Hematoxylin and eosin and Mallory-Heidenhain stains and alpha-2u-globulin and proliferating cell nuclear antigen immunohistochemical stains were performed on kidney sections. All treated male F-344 rats exhibited dose-related accumulation of hyaline droplets containing alpha-2u-globulin in proximal tubules. The kidney weights were significantly increased in male and female rats treated with TNB. Significant increases in cell proliferation in proximal tubules were observed in male F-344 rats. Renal changes observed in TNB-treated rats appeared identical to those from other chemicals that induce alpha-2u-globulin nephropathy in male rats. No hyaline droplet accumulation was found in female F-344 and male NBR rats at any doses. We can conclude that TNB induces dose-related exacerbation of hyaline droplets containing alpha-2u-globulin in male rat kidney and subsequent cell proliferation.

Diagnostic criteria are presented for degenerative, inflammatory, nonneoplastic proliferative, and neoplastic lesions in the liver of medaka (Oryzias latipes), a small fish species frequently used in carcinogenesis studies. The criteria are the consensus of a Pathology Working Group (PWG) convened by the National Toxicology Program. The material examined by the PWG was from medaka exposed to N-nitrosodiethylamine for 28 days, removed to clean water, and sacrificed 4, 6, or 9 mo after initiation of exposure. Degenerative lesions included hepatocellular intracytoplasmic vacuolation, hepatocellular necrosis, spongiosis hepatis, hepatic cysts, and hepatocellular hyalinization. Inflammatory lesions consisted of granulomas, chronic inflammation, macrophage aggregates, and focal lymphocytic infiltration. Nonneoplastic proliferative lesions comprised foci of cellular alteration (basophilic focus, eosinophilic focus, vacuolated focus, and clear cell focus) and bile duct hyperplasia. Neoplastic lesions included hepatocellular adenoma, hepatocellular carcinoma, cholangioma, and cholangiocarcinoma. Two lesions composed mainly of spindle cells were noted, hemangiopericytoma and spindle cell proliferation. Rather than being an exhaustive treatment of medaka liver lesions, this report draws from the published literature on carcinogen-induced liver lesions in medaka and other fish species and attempts to consolidate lesion criteria into a simplified scheme that might be useful to pathologists and other researchers using medaka lesions for risk assessment or regulatory purposes.

To clarify the cell of origin of granular cell tumors in the rat brain, light and/or electron microscopic examinations were performed on 40 cases of spontaneous meningeal tumors in Wistar (Jcl: Wistar) rats. The meningeal tumors were histologically subclassified into 3 types: meningothelial meningiomas (MMs), 3 cases; granular cell tumors (GCTs), 28 cases; and mixed forms (MIXs) of GCTs and MMs, 9 cases. Of these tumors, 2 MMs, 2 GCTs, and 3 MIXs were examined by transmission electron microscopy. Tumor cells of MMs were characterized by cytoplasmic intermediate filaments and prominent interdigitating cell processes often connected with cellular junctions without surrounding basal laminae. GCTs were composed of 2 cell types: granular cells with many dense bodies and filamentous cells with fine intermediate filaments. MIXs consisted of granular cells, filamentous cells, and intermediate cells. The tumor cells in GCTs and MIXs were apposed to each other and connected with cellular junctions. MIXs displayed a spectrum of cellular differentiation in that 2 MIXs had a close morphological resemblance to GCTs and the other one to MMs. These findings indicate there might be a histogenetic sequence among MMs, GCTs, and MIXs. The present study supports the suggestion that GCTs and MIXs may be variants of MMs from the meningeal arachnoid cell.

A high incidence of oral squamous cell carcinoma was present in male and female Brown-Norway rats fed ad libitum or food-restricted dietary formulations. One hundred eighty-nine rats were examined from 4 dietary treatment groups: male ad libitum, male food-restricted, female ad libitum, and female food-restricted. The ad libitum treatment groups for both males and females had significantly more cases of oral squamous cell carcinoma than cohort food-restricted groups. In ad libitum rats, 10 of 47 (21%) males and 15 of 47 (32%) females had oral squamous cell carcinoma, whereas only 4 of 47 (9%) males and 5 of 48 (10%) females in the food-restricted groups were similarly affected. The food-restricted rats lived significantly longer than ad libitum cohorts, so the higher incidence of squamous cell carcinoma was not dependent on extended lifespans. In addition to the dietary influence, a significant difference in oral squamous cell carcinoma incidence occurred between various familial lines. Family lines having representatives in both ad libitum and food-restricted groups had lower oral squamous cell carcinoma incidences in the food-restricted group whether comparing affected litters or individuals. Results suggest that the incidence of oral squamous cell carcinoma in our colony of Brown-Norway rats can be influenced by both the dietary treatment group and genetic predilection within certain pedigrees.

A poorly differentiated spontaneous tumor was found in the parotid gland region of a 6-wk-old female Sprague-Dawley rat. The tumor consisted of undifferentiated cells forming dense clusters, well-defined nests, or compact sheets. In some areas, spindle-shaped cells were arranged in a fascicular fashion. Neither ductular nor acinar structures were seen. No keratinization was observed, either. Mitotic figures were frequent. All neoplastic cells were immunoreactive for pankeratin but negative for vimentin, α-smooth muscle actin and S-100 protein. Ultrastructurally, the tumor cells were characterized by desmosome-like junctional complexes, poorly developed cytoplasmic organelles, and microvilli-like cytoplasmic processes. Based on these findings and its anatomical location, the present tumor was diagnosed as a poorly differentiated carcinoma of the parotid gland.