1994, Dietrich, Daniel R., Candrian, Regula, Marsman, Daniel S., Popp, James A., Kaufmann, William K., Swenberg, James A.

Cell proliferation (S phase response) in archival liver tissues of partially hepatectomized rats was determined via proliferating cell nuclear antigen (PCNA) immunohistochemistry. These results were compared with the S phase response assessed previously in the same tissues via tritiated thymidine (Tdr) autoradiography. The effect of prolonged tissue fixation on PCNA immunohistochemistry was compared in two studies: study A, the liver was fixed for a maximum of 7 days and then embedded in paraffin and stored for not, vert, similar 18 months, while in study B, the liver was fixed in formalin for 7 years and then embedded in paraffin and stored for not, vert, similar 18 months until sectioning and immunostaining. PCNA immunostaining was successful in the liver sections of both studies, irrespective of the length of formalin fixation. Furthermore, the S phase labeling indices (LI) determined via PCNA and Tdr were comparable, although not identical, in the two studies. Therefore, use of PCNA immunohistochemistry should allow retrospective staining of rodent tissues for the assessment of cell proliferative activity in formalin-fixed organs from previously conducted toxicity and carcinogenicity studies.

1991, Dietrich, Daniel R., Swenberg, James A.

The NCI-Black-Reiter (NBR) rat is the only strain of male rat known not to synthesize the hepatic form of the low molecular weight protein, α2u-globulin. In previous studies, NBR rats were shown not to develop renal disease when exposed to decalin, a compound known to induce α2u-globulin nephropathy in other rat strains. The objective of this study was to show that the presence of α2u-globulin (α2u) is essential for the development of this syndrome in rats exposed to 2,2,4-trimethylpentane (TMP), 1,4-dichlorobenzene (DCB), isophorone (IP), PS-6 unleaded gasoline (UG), and d-limonene (d-L). The induction of α2u-nephropathy in F344 male rats with lindane was used as a positive control and this response was contrasted to male NBR and female F344 rats treated with lindane. Five to seven 11-week-old male NBR rats were exposed to TMP (500 mg/kg/day), DCB (500 mg/kg/day), IP (1000 mg/kg/day), UG (500 mg/kg/day), d-L (1650 mg/kg/day), or lindane (10 mg/kg/day) and five 11-week-old male and female F344 rats were exposed to lindane (10 mg/kg/day) by oral gavage on 4 consecutive days. NBR male and F344 male and female rats gavaged with corn oil were incorporated in the study as vehicle controls. The presence of hyaline droplets was assessed in perfusion-fixed kidneys by staining paraffin sections with Mallory-Heidenhein stain and in GMA sections with Lee's methylene basic blue fuchsin stain. Paraffin sections were also analyzed immunohistochemically for the presence of α2u. Under exposure conditions that clearly induce α2u-nephropathy in male F344 rats, no lesions, hyaline droplets, or α2u were detectable in treated or control male NBR and female F344 rats. It is thus concluded that the presence of α2u is causal to the development of renal disease in rats exposed to TMP, DCB, IP, UG, d-L, and lindane.

1990, Dietrich, Daniel R., Swenberg, James A.

1992, Swenberg, James A., Dietrich, Daniel R., McClain, R. Michael, Cohen, Samuel M.

1991, Dietrich, Daniel R., Swenberg, James A.

In a 2-year carcinogenesis bioassay, d-limonene (dL) induced kidney tumors in male F344 rats, but not in female F344 rats or either sex of mice. d-Limonene-1,2-oxide, a metabolite of dL, has been shown to bind reversibly to the male rat-specific urinary protein, α2u-globulin (α2u-G), resulting in an α2u-G-chemical complex that is more resistant to lysosomal degradation than α2u-G alone. This reduced degradation of α2u-G-chemical complex leads to an accumulation of this protein in the proximal convoluted tubules of the male rat kidney and to the morphological changes characteristic for α2u-globulin nephropathy. The only male rat strain known to be resistant to this renal disease is the α2u-G deficient NCI-Black-Reiter (NBR) rat. The objectives of this study were to determine whether or not dL causes sustained increases in cell proliferation and has promoting activity for renal adenomas in male rats and if the male rat-specific urinary protein, α2u-G, is required. In a 32-week initiation-promotion assay, male F344 and NBR rats were treated with either 0 or 500 ppm N-ethyl-N-hydroxyethylnitrosamine (EHEN) in the drinking water for 2 weeks. Experimental groups of 31 to 38 rats then received 0 or 150 mg d-limonene/kg/day in corn oil for 30 weeks by p.o. gavage 5 days/week. Cell proliferation in the proximal tubules was assessed via 5-bromo-2'-deoxyuridine-filled osmotic mini-pumps and immunohistochemistry after 7 weeks (2 weeks EHEN + 5 weeks dL) and at the end of the study (2 weeks EHEN + 30 weeks dL). Preneoplastic and neoplastic lesions were quantified in perfusion-fixed kidneys.
A 5-fold increase in the labeling index of P2-cells was found after 5 weeks and 30 weeks of promotion in all dL-treated F344 rats, whereas no difference between treatment groups was detected in NBR rats. No increase in tumors or preneoplastic lesions was detected in dL-treated NBR rats, whereas a 10-fold increase in renal adenomas and atypical hyperplasias was found in the EHEN-dL-treated F344 rats compared with F344 rats treated with EHEN-corn oil. d-Limonene treatment alone caused a significant increase in the number of atypical tubules and atypical hyperplasias in F344 rats when compared with the F344 vehicle control. On the other hand, a significantly lower incidence of liver tumors was found in EHEN-dL-treated F344 rats compared with F344 rats treated with EHEN-corn oil, suggesting a chemopreventative effect of dL on EHEN-induced liver carcinogenesis in F344 rats. It is thus concluded that dL promotes preneoplastic lesions and renal tumors only in the presence of the male rat-specific urinary protein, α2u-G. Since α2u-G is a species- and sex-specific protein that is causal for both the cytotoxic and carcinogenic response in the male rat, extrapolation of dL carcinogenicity data from rat studies to other species, including humans, is probably not warranted.

1991, Dietrich, Daniel R., Swenberg, James A.

The current literature on non-genotoxic renal carcinogens and the associated neoplastic and preneoplastic lesions has been reviewed in order to determine their occurrence and predictive nature with regard to tumor formation. In addition the mechanisms involved in the genesis of renal tumors are discussed. A more generalized classification of preneoplastic and neoplastic renal lesions was introduced, based on studies conducted with genotoxic and non-genotoxic renal carcinogens. Reports on preneoplastic lesions were found in the literature for control animals as well as animals treated with non-genotoxic carcinogens. Due to the paucity of data regarding preneoplastic lesions in control animals and animals treated with non-genotoxic carcinogens, new data were also generated by rereading kidney slides of control animals of a randomly selected NTP study and kidney slides of male rats treated with the highest dose of ochratoxin A, one of the most potent non-genotoxic renal carcinogens known. The control slides and the slides from the ochratoxin A study indicated that the cytologic and morphologic types of preneoplastic lesions characteristically observed in bioassays using genotoxic carcinogens are also present in control animals and animals treated with non-genotoxic carcinogens. The incidence of preneoplastic lesions was low in control animals and higher in animals treted with non-genotoxic carcinogens. The diversed classifications used in the literature did not allow a direct comparison of lesions and corresponding incidences with those of the newly generated data. However, three major tendencies were observed: (a) whenever a high incidence of preneoplastic lesions was reported, renal neoplasms were also found, (b) the larger the size and the further a lesion had progressed, the higher was the probability of tumor formation, and (c) not all preneoplastic lesions are irreversible, but reversibility seemed to decrease with increasing lesion size and progression. It must be emphasized that the data available for these conclusions are limited. This is not due to the lack of adquate numbers of bioassays with non-genotoxic carcinogens, but rather to the lack of consistent reporting of data. A generalized and more widely used classification which incorporates early lesions would certainly improve the curret data base on renal lesions and provide future improvements in the predictive nature of these lesions.

1991, Foley, Julie F., Dietrich, Daniel R., Swenberg, James A., Maronpot, Robert R.

Proliferating cell nuclear antigen (PCNA), an endogenous cell replication marker, was detected by an improved immunohistochemical procedure (super sensitive biotin-streptavidin), in formalin fixed, paraffin embedded tissue. Small intestine, liver, kidney, testis, and esophagus from rats were stained for the detection and evaluation of PCNA as asuitable marker for cell proliferation. Different patterns of staining that are believed to correlate with the individual phases of the cell cycle (G1, S, G2, M, G0) were recognized. PCNA offers an alternative to established cell labeling techniques for investigating cell proliferation.