It has been proposed that the hepatocellular proliferation induced by peroxisome proliferators may occur through an indirect mechanism involving cytokine release as opposed to direct regulation of cell growth genes by PPARα. We compared the induction of peroxisome proliferation and cell proliferation in C57Bl/6 mice treated with 100 mg/kg/day WY14,643 in the presence or absence of increasing doses of dexamethasone (DEX), an inhibitor of the release of proinflammatory cytokines. Biochemical markers of peroxisome proliferation, including fatty acyl-CoA oxidase activity, CYP4A content, and liver-to-body-weight ratios were markedly increased in the WY14,643-treated mice. DEX coadministration, up to a maximum dose of 50 mg/kg/day, did not prevent the induction of these parameters. Acyl-CoA oxidase mRNA levels increased 5-fold with WY14,643 treatment and 15-fold with DEX coadministration at 5 mg/kg/day. ApoCIII mRNA levels were decreased by 50% in WY14,643-treated mice. DEX alone at 5 mg/kg/day increased the ApoCIII mRNA 4-fold, but WY14,643 coadministration also inhibited this induction by greater than 50%. In addition, immunohistochemical detection of peroxisomes with anti-PMP-70 antibody demonstrated marked increase in hepatocellular peroxisomes in WY14,643-treated mice regardless of DEX treatment. In contrast, coadministration of DEX at 2 mg/kg/day partially inhibited the hepatocyte proliferation response (measured by BrdU incorporation or Ki-67 immunohistochemical detection). Moreover, DEX at doses of 5 mg/kg/day or higher completely inhibited the induction of cell proliferation and, at these higher doses, reduced the cell proliferation rate to levels below the vehicle-treated control mice. Our studies clearly demonstrate that the hepatocellular proliferation induced by a peroxisome proliferator can be modulated independently of the other pleiotropic effects usually induced by these agents, suggesting an indirect mechanism of hyperplasia.

Levels of apoptosis induction, nuclear p53 expression, and cytoplasmic Bax protein expression were evaluated in various tissues before and after exposure of 2 neonatal and 6 adult rats to gamma irradiation. These studies served to provide positive control material for an evaluation of the effects of aflatoxin B1 administered to adult rats at several doses followed by sacrifice at several timepoints. The irradiation work also was run to determine any differences between neonate and adult expression of apoptosis, p53 and Fax. For the aflatoxin B1 study, only dUTP-biotin nick end labeling (TUNEL) staining to demonstrate apoptotic bodies and immunohistochemical staining to localize p53 nuclear protein were performed. In neonates, untreated control liver showed slight apoptosis. Following irradiation, apoptosis increased markedly in liver; other neonate tissues showed no apoptosis before or after irradiation. Results in untreated adult rats revealed slight apoptosis in some tissues with high cell turnover rates (thymus, spleen, intestine, and lymph node). Little apoptosis was evident in liver or kidney. Following irradiation, apoptosis increased in all tissues examined, especially at the 6 hr timepoint. In aflatoxin B1-treated adults, apoptosis evaluated by TUNEL appeared to increase slightly 24 hr post-dose, although morphological assessment showed increases at 3, 6, and 24 hr post dose sacrifices. The degree of apoptosis was directly proportional to increasing aflatoxin B1 dose. In neonates, p53 showed a significant increase only in thymus following irradiation, especially at the 4 hr timepoint. In adults, irradiation produced significant increases of p53 expression in all tissues except kidney. Following aflatoxin administration, p53 was present only in rat liver at 24 hr post-dose; and 1, 3, and 10 mg/kg doses showed slight dose-related increases in the number of positive cells. Bax protein staining was ubiquitous in adults and was present in all tissues; its detection was not affected by irradiation.

A method for preparing skin biopsies for cryosectioning was developed to accurately obtain samples from specific areas of the dermis, while minimizing contamination with epidermal tissue. Routine preparation of 6mm punch biopsies from freshly excised, full-thickness skin produced contraction and folding of the edges of the biopsy prior to mounting for snap-freezing and cryosectioning. Sample orientation was ruined, and cryosections were heterogeneous with respect to dermal structures and/or to dermal and epidermal layers. Biopsy artifacts were prevented by prefreezing skin over dry ice prior to taking biopsies. The biopsies were held frozen on dry ice until they were mounted on cryostat pegs with flattened, frozen OCT surfaces; then they were snap-frozen in chilled OCT in an isopentane bath cooled with liquid nitrogen. The method for determining skin level homogeneity of cryosections consisted of taking 10 mu m cryosections for histology between sections sampled for drug level analysis. The histological sections were fixed in 5% acetic acid in methanol and stained with hematoxylin and eosin to define the skin layers and structures associated with each sample for analysis. Histological sections from prefrozen skin had fewer processing artifacts, and dermal cryosections free of epidermal contamination were dramatically increased compared to the routine procedure.

Effective methods exist for separating epidermis from dermis for many species; however, a simple and effective skin separation method for non-human primates is not available. This investigation describes an easy and reliable method for separating epidermis from dermis in Rhesus monkeys. Skin was shaved and washed prior to necropsy. Skin samples were placed on cardboard and then in Whirl-Pak bags, frozen on dry ice and stored at -70 degrees C. Just prior to the separation procedure, Whirl-Pak bags were returned to dry ice storage. Immediately after removal from dry ice, each closed Whirl-Pak bag was placed into a waterbath maintained between 60 and 67 degrees C. After 2 minutes, the Whirl-Pak bag was removed from the waterbath, opened and the skin surface of the application site was gently scraped with a scalpel blade to remove the epidermis. Effectiveness of removal was verified by histologic examination of the remaining dermal samples.

Clofibrate, a peroxisome proliferator, is hepatocarcinogenic in rats in a dose-dependent fashion. While there is a relationship between peroxisome proliferation and rodent liver carcinogenesis, recent evidence also suggests an association between the tumorigenicity of peroxisome proliferators and sustained cell proliferation. To investigate the role of early cell proliferation in clofibrate-induced carcinogenesis and the predictive potential of this end-point, in a 3-month study, rats were fed clofibrate doses equivalent to those used in the chronic bioassay, and cell proliferation was determined after 1 week and 3 months, using a 1-week continuous bromodeoxyuridine (BrdU)-labeling technique. Adult Sprague-Dawley rats were fed clofibrate at 1500, 4500, or 9000 ppm. Six rats/sex/group were killed after 1 or 13 weeks of treatment. Osmotic minipumps containing BrdU were implanted into rats 7 days prior to necropsy to determine the cumulative 7-day hepatocyte labeling index immunohistochemically. A dose-related increase in hepatocyte labeling index was seen after 1 week of treatment. However, at 13 weeks, sustained increases in hepatocyte proliferation were not seen; but a dose-related decrease in the hepatocyte labeling index was observed. Liver stereology at 13 weeks demonstrated a dose-related increase in liver weight and volume, but a decrease in hepatocyte nuclei per unit volume, a minimal increase or no change in the total number of hepatocyte nuclei per liver, and an absolute decline in the total number of BrdU-labeled hepatocyte nuclei per liver. These data suggest that in rats, clofibrate may influence hepatocarcinogenicity by decreases in normal hepatocyte proliferation over time and this effect may influence the pathogenesis of tumors at time points beyond 13 weeks of treatment.