Morphological changes in the kidney of dogs chronically exposed to exogenous growth hormone.

Porcine growth hormone was administered subcutaneously to beagle dogs at doses of 0.025, 0.1, and 1 IU/kg/d for 14 weeks, markedly elevating serum growth hormone (GH) and insulin-like growth factor-1 (IGF-1) levels. This was accompanied by a significant increase in body weight gain and kidney weights in both male and female dogs. The increase in kidney weight (6 to 54%) was slightly greater than the increase in body weight (6 to 40%). By light microscopy, glomerular deposits, mesangial thickening, and very slight cellular infiltration in glomeruli were seen in mid- and high-dose groups. Based on morphometric evaluation, there was an increase in the renal glomerular area, which was statistically significant (p < or = 0.05) in the mid- and high-dose males and in the high-dose females. This was associated with a statistically significant (p < or = 0.05) increase in the number of total glomerular cells in the mid- and high-dose males. By transmission electron microscopy, thickening of the glomerular basal lamina and diffuse increase of the mesangial matrix were observed in both male and female dogs in the mid- and high-dose groups. Immunohistochemical reactions were negative for IgG, IgM, and C3. The morphological changes in the kidney of dogs resemble the diffuse glomerulosclerosis described in human diabetic nephropathy.

The relative protective effects of moderate dietary restriction versus dietary modification on spontaneous cardiomyopathy in male Sprague-Dawley rats.

The relative protective effects of modifying dietary protein, fat, fiber, and energy content vs moderate food or dietary restriction (DR) on spontaneous cardiomyopathy of Charles River male Sprague-Dawley (SD) rats was evaluated at 1 and 2 years. For 2 years, SD rats were fed Purina Rodent Chow 5002 (21.4% protein, 5.7% fat, 4.1% fiber, 3.1 kcal/g) or a modified rodent chow 5002-9 (13.6% protein, 4.6% fat, 15.7% crude fiber, 2.4 kcal/g) ad libitum (AL) or by moderate DR at approximately 65% of the caloric intake of the AL group fed the 5002 diet. Serum lipids, carcass composition, and organ weights were evaluated and hearts were qualitatively and quantitatively examined microscopically for male SD rats at 1 and 2 years. Cardiomyopathy was characterized by the colocalization of myocardial degeneration, the development of subepicardial, perivascular, subendocardial, and interstitial fibrosis, and mononuclear inflammatory cell infiltration that increased by incidence and severity in an age-dependent manner from 1 to 2 years. SD rats fed the 5002 diet AL had the greatest heart weights and the most severe cardiomyopathy, with the highest myocardial fibrotic index. These parameters were relatively decreased in the AL 5002-9 diet, the DR 5002 diet, and the DR 5002-9 diet rats at 1 and 2 years. Regardless of the type of diet fed, both AL groups had the most severe cardiomyopathy by 2 years. Moderate DR allowed isocaloric comparisons of the relative effects of modified diets on survival, obesity, and heart disease. Only slight improvements in the severity and progression of spontaneous cardiomyopathy were seen by modification of the protein, fiber, fat, and energy content of the diet if fed AL. However, moderate DR with either diet was more effective than changing the diet composition in preventing and controlling the progression of cardiomyopathy in male SD rats.

The carcinogenic risk of high dose total body irradiation in non-human primates.

PURPOSE: High dose total body irradiation (TBI) in combination with chemotherapy, followed by rescue with bone marrow transplantation (BMT), is increasingly used for the treatment of haematological malignancies. With the increasing success of this treatment and its current introduction for treating refractory autoimmune diseases the risk of radiation carcinogenesis is of growing concern. Studies on tumour induction in non-human primates are of relevance in this context since the response of this species to radiation does not differ much from that in man. MATERIALS AND METHODS: Since the early sixties, studies have been performed on acute effects in Rhesus monkeys and the protective action of bone marrow transplantation after irradiation with X-rays (average total body dose 6.8 Gy) and fission neutrons (average dose 3.4 Gy). Of those monkeys, which were irradiated and reconstituted with autologous bone marrow, 20 animals in the X-irradiated group and nine animals in the neutron group survived more than 3 years. A group of 21 non-irradiated Rhesus monkeys of a comparable age distribution served as controls. All animals were regularly screened for the occurrence of neoplasms. Complete necropsies were performed after natural death or euthanasia. RESULTS: At post-irradiation intervals of 4-21 years an appreciable number of tumours was observed. In the neutron irradiated group eight out of nine animals died with one or more malignant tumours. In the X-irradiated group this fraction was 10 out of 20. The tumours in the control group, in seven out of the 21 animals, appeared at much older age compared with those in the irradiated cohorts. The histogenesis of the tumours was diverse with a preponderance of renal carcinoma, sarcomas among which osteosarcomas, and malignant glomus tumours in the irradiated groups. CONCLUSIONS: When corrected for competing risks, the carcinogenic risk of TBI in the Rhesus monkeys is similar to that derived from the studies of the Japanese atomic bomb survivors. The increase of the risk by a factor of 8, observed in the monkeys, indicates that patients are likely to develop malignancies more frequently and much earlier in life after TBI than non-exposed individuals. This finding underlines the necessity of regular screening of long-term surviving patients subjected to TBI and BMT.

Insulin-like growth factor-1 and growth hormone (GH) levels in canine cerebrospinal fluid are unaffected by GH or GH secretagogue (MK-0677) administration.

Elevation in circulating GH levels results in a dose-related increase in serum insulin-like growth factor-1 (IGF-1) levels in dogs. However, it is not known whether elevations in systemic IGF-1 and GH levels contribute to the cerebrospinal fluid (CSF) levels of these hormones. Therefore, a study was designed in dogs to determine if elevated circulating GH levels was a result of a GH secretagogue (MK-0677) or if exogenous GH administration resulted in increased IGF-1 and GH levels in the CSF of dogs. A total of 12 normal, young adult male dogs were randomized to three treatment groups (4 dogs/group) based on body weight. There were 4 vehicle control dogs. A group of 4 dogs were dosed orally with MK-0677 (5 mg/kg/day) dissolved in deionized water. A third group of 4 dogs received subcutaneous injections of porcine GH (pGH) at a dose of 0.1 IU/kg/day. From all dogs, blood and CSF samples were collected prior to the initiation of treatment and on days 7 and 15 of treatment. All samples were assayed using a validated radioimmunoassay. Administration of MK-0677 or pGH resulted in a statistically significant (P < or = 0.05) increased body weight gain and increased serum IGF-1 and GH levels. In contrast, administration of MK-0677 resulted in no significant (P > 0.05) increase in CSF IGF-1 or GH levels on days 7 or 15 of the study. The CSF IGF-1 values ranged from 1.2 to 2.0 ng/ml with minimal variation among three separate samples taken during the course of the study from each dog. Similarly, the CSF GH levels were very low (< 0.98 ng/ml to 2.4 ng/ml) in all dogs irrespective of treatment group. This study has demonstrated that there is no correlation between the circulating levels of IGF-1 or GH and the levels of these hormones in the CSF of normal dogs. An approximately 100-fold difference between serum and CSF IGF-1 levels in vehicle control dogs suggest that there is a blood-brain barrier for the circulating IGF-1. Similarly, failure to see an elevation in CSF GH levels despite increases in serum GH levels shows that there is a blood-brain barrier for GH in normal dogs. These results suggest that the likely source of GH and IGF-1 in the CSF of dogs is from the CNS.

Morphological and hormonal changes in the ventral and dorsolateral prostatic lobes of rats treated with finasteride, a 5-alpha reductase inhibitor.

BACKGROUND: In rats, the prostate is divided into three distinct lobes, and the lobes are dependent on androgens [testosterone (T) and dihydrotestosterone (DHT)] as trophic hormones. However, the reasons for the difference in the incidence of proliferative changes reported are not well-understood. Administration of finasteride, a 5-alpha reductase (5alphaR) inhibitor which selectively inhibits the conversion of T to DHT, results in elevated intraprostatic T levels. However, long-term (2 years) administration of finasteride results in no increase in proliferative changes in the ventral lobes of the rat prostate. Therefore, studies were designed to determine the differences in intraprostatic hormonal levels, morphology, and 5alphaR activity in different lobes of the rat prostate. METHODS: Sexually mature male Sprague-Dawley rats were used in all studies. Finasteride was administered orally to rats. The methodology included determination of intraprostatic T and DHT levels by radioimmunoassay, qualitative and quantitative evaluation of prostatic morphology, and in vitro determination of 5alphaR activities in rat prostatic lobes. RESULTS: A significant amount of 5alphaR activity was observed in the dorsal, ventral, and lateral lobes of the rat prostate. Both 5alphaR isozymes (types 1 and 2) were present in all lobes, based on 5alphaR activities observed at both acidic and neutral pH. Oral administration of finasteride (160 mg/kg/day) for 15 days resulted in significant (P < or = 0.001) decreases in intraprostatic DHT levels and increases in T levels; when compared to controls, the mean decrease in DHT levels in the ventral and the dorsolateral lobes was 86% and 94%, respectively, and the mean increase in T levels in the ventral and the dorsolateral lobes was approximately 3 times and 20 times, respectively, higher than in controls. Chronic administration of finasteride (80 mg/kg/day) for 6 months resulted in significant (P < or = 0.001) decreases in the weights of the prostatic lobes, which correlated with significant (P < or = 0.001) decreases in the total number of epithelial and stromal cells per gland in both the ventral and dorsolateral lobes of the prostate. There were no qualitative differences in prostatic morphology between the control and finasteride-treated groups. A short-term study in control rats exposed to bromodeoxyuridine (Brdu) showed that the number of Brdu-labeled cells in the dorsolateral lobe was significantly (P < or = 0.05) greater than in the ventral lobe. CONCLUSIONS: This first comparative study has highlighted some of the similarities and differences among the prostatic lobes of the rat. Inhibition of conversion of T to DHT with finasteride resulted in a significant increase in intraprostatic T levels and a significant decrease in DHT levels in rats; despite a significant increase in intraprostatic T levels, the prostate remained atrophic, indicating that DHT alone has a trophic effect on the prostate.

Pharmacological and toxicological effects of chronic porcine growth hormone administration in dogs.

The purpose of this study was to evaluate the pharmacological and toxicological effects of exogenous GH administration in normal adult dogs. Because porcine GH (pGH) is structurally identical to canine GH, pGH was selected for a 14-wk study in dogs. Thirty-two dogs (< 2 yr) were randomized to 4 groups (4 dogs/sex/group); 1 group was treated with the vehicle and 3 groups received pGH at 0.025, 0.1, or 1.0 IU/kg/day subcutaneously. Daily clinical signs and weekly body weights were recorded. Hematology, serum biochemistry, urinalyses, electrocardiograms, and ophthalmoscopic examinations were done. Serum GH, insulin-like growth factor-1 (IGF-1), insulin, thyroxine (T4), triiodothyronine (T3), and cortisol levels were determined. Necropsies were performed, organs weighed, and tissues were fixed and processed for light microscopic examination. Porcine GH caused increased body weight gain (p < or = 0.05) through the mid dose; the mean weight gains at study termination in mid- and high-dose groups were 2.8 kg and 4.7 kg, respectively, compared to 0.4 kg and 0.8 kg in control and low-dose groups, respectively. Dose-related increased weights of liver, kidney, thyroid, pituitary gland, skeletal muscle, and adrenal gland were noted. In pGH-treated dogs, increased skin thickness seen grossly correlated histologically with increased dermal collagen. There was no gross or histomorphological evidence of edema. There were dose-related increased serum IGF-1 levels (approximately 2-10-fold; p < or = 0.05) that correlated with the elevated serum GH levels in pGH-treated dogs. Also, increased serum insulin levels (p < or = 0.05) through the mid dose were seen throughout the study. In high-dose dogs, the insulin levels remained elevated over 24 hr postdose. The serum glucose levels in fasted dogs remained within the control range and there was no chronic hyperglycemia based on glycosylated hemoglobin levels. Renal glomerular changes, significant polyuria with decreased urine specific gravity, and increased serum insulin levels suggested that the dogs had early insulin-resistant diabetes. There was minimal or no biologically significant effect of pGH on serum T3, T4, and cortisol levels in dogs. Other serum biochemical changes in pGH-treated dogs included decreased urea nitrogen and creatinine, and increased potassium, cholesterol, and triglycerides. Significant increases in serum calcium and phosphorous levels and alkaline phosphatase activity (bone isozyme) correlated with the histological changes in bone. In pGH-treated dogs, there was a dose-related normochromic, normocytic, nonregenerative anemia. The changes described above, except for the anemia, are related to either anabolic or catabolic effects of high doses of GH. Based on this study, it is concluded that the dog is a good model in which to evaluate the safety of GH secretagogues as well as compounds with GH-like activity.

Morphological changes in the pituitary gland of dogs chronically exposed to exogenous growth hormone.

Growth hormone (GH) synthesis and release from the pituitary is regulated by hypothalamic releasing hormone, insulin-like growth factor-1 (IGF-1), and somatostatin. However, the potential effects of pharmacological doses of exogenous GH on the pituitary are not well studied. To determine the potential chronic effects of exogenous GH on pituitary morphology in dogs, porcine GH (pGH) was administered subcutaneously to 3 groups of dogs (4 animals/sex/group) at doses of 0.025, 0.1, and 1.0 IU/kg/day for 14 wk. A group (4/sex) of dogs served as the vehicle control. The pituitaries from all dogs were weighed and fixed in appropriate fixatives for light and electron microscopic examination; in addition, cells of the pars distalis were quantitated by a point counting method following immunostaining to identify cells containing GH, prolactin (PRL), and adrenocorticotrophic (ACTH) hormones. Administration of pGH resulted in a statistically significant (p < or = 0.05) increased pituitary weight through the high dose. By light microscopy (LM), hypertrophy of pars distalis cells was evident in mid- and high-dose female dogs. The pituitaries of dogs given the lowest dose (0.025 IU/kg/day) of pGH were not remarkable based on weight and LM findings. In addition, transmission electron microscopic (TEM) examination of the pituitary gland of high-dose demonstrated, in both sexes, pituitary cells with variably dilated rough endoplasmic reticulum and decreased numbers of secretory granules; some of these cells reacted positively to GH immunostaining. Quantitative analysis of the pituitary gland of high-dose males and females showed an increase in the absolute volume of all cell populations studied: GH-, PRL-, and ACTH-positive cells. Based on the LM and TEM findings, the increased volume of the cell populations studied is likely related to cellular hypertrophy. The expected elevation in serum GH levels following repeated administration of pGH and an associated elevation in serum IGF-1 levels resulted in morphologic changes in the pituitary gland of dogs given high doses (> or = 0.1 IU/kg/day) of pGH; these observations differed from the reported findings in pituitaries of transgenic mice secreting large quantities of bovine GH.

Effect of chronic growth hormone administration on skeletal muscle in dogs.

Administration of growth hormone (GH) results in increased body weight gain in dogs. Increased body weight gain is believed to be a result of the trophic effect of GH on the musculoskeletal system. However, edema is one of the side effects described in man following exogenous GH administration. Thus, the objective of this study was to determine if the expected increased weight gain in GH-treated dogs is a result of increased muscle mass. Porcine growth hormone (pGH), administered subcutaneously to beagle dogs at doses of 0.025, 0.1, and 1 IU/kg/day for 14 wk, resulted in elevated serum GH and insulin-like growth factor-1 (IGF-1) levels (see accompanying paper, Prahalada et al). This was associated with a significant increase in body weight gain and weights of the cranial tibialis muscle in both male and female dogs. The increased muscle mass likely contributed to the significant increase in body weight gain seen in both sexes. Quantitative analysis of skeletal muscle sections stained for ATPase activity showed increases in type I (slow twitch) and type II (fast twitch) myofiber sizes in mid- and high-dose males and in high-dose females. The ratio of type I and type II muscle fibers remained unchanged. Hypertrophic myofibers were enlarged but had a normal histologic and ultrastructural organization when observed by light and transmission electron microscopy. The results of this study have demonstrated that increased muscle mass in pGH-treated dogs is related to hypertrophy of muscle fibers and not due to edema. Exogenous GH administration has an anabolic effect on skeletal muscle in dogs.

Quantitative evaluation of glandular and stromal compartments in hyperplastic dog prostates: effect of 5-alpha reductase inhibitors.

The objective of this study was to determine the effects of 2 different 5-alpha reductase inhibitors (finasteride and MK-0434) on the glandular and stromal compartments of hyperplastic canine prostates. In this study, dogs received 1 of the 2 compounds orally, at a dose of 1 mg/kg/day for 16 weeks; control dogs received a placebo. The morphological changes in the glandular and stromal compartments in the prostate were quantitated by a point-counting method on Masson's trichrome-stained sections. Treatment with 5-alpha reductase inhibitors resulted in significant (P < or = 0.05) decreases in mean prostatic volumes, microscopic evidence of prostatic atrophy, and significant (P < or = 0.05) decreases in the absolute volumes of the prostatic glandular and stromal compartments compared to controls. In finasteride-treated dogs, the mean percent change from baseline was: epithelium, -52; lumens, -58; fibrovascular stroma, -41; and smooth muscle, -29. In MK-0434-treated dogs, the mean percent change from baseline was: epithelium, -77; lumens, -58; fibrovascular stroma, -38; and smooth muscle, -42. The effect on the glandular compartment in dogs treated with MK-0434 was slightly greater than in dogs treated with finasteride; however, the effect on the stroma was similar. These results clearly demonstrate that inhibition of 5-alpha reductase enzyme activity affects growth and maintenance of both glandular and stromal compartments of dog hyperplastic prostates. It is likely that the decrease in size of the prostate in finasteride-treated (Proscar) men is due to shrinkage of both glandular and stromal compartments.

Dose-related changes in the profile of ras mutations in chemically induced CD-1 mouse liver tumors.

We investigated the role of dosing regimen on ras mutations in chemically induced CD-1 mouse liver tumors. The spectra of ras gene mutations in liver tumors that were induced by 15 daily i.p. injections of 7,12-dimethylbenz[a]anthracene (DMBA), 4-aminoazobenzene (AAB), N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or N-nitrosodiethylamine (DEN) were compared to those previously obtained for tumors induced by a single but higher dose of each carcinogen. The principal assay used was a direct tumor analysis involving sequencing of polymerase chain reaction (PCR)-amplified tumor DNA; additional mutations that were present in only a small fraction of tumor cells were detected using a transfection assay or a PCR-engineered restriction fragment length polymorphism method. Spontaneous liver tumors had a relatively low frequency of ras mutations, all found in Ha-ras codon 61, and most of these mutations were present in only a small fraction of tumor cells. With the exception of multiple-dose DEN, each group of single- and multiple-dose carcinogen-induced tumors exhibited a higher frequency of ras mutations compared with spontaneous tumors. For AAB, N-OH-AAF and DEN, the dosing regimen was found to affect significantly the profile of ras mutations. For each of these carcinogens, the multiple-dose tumor group (versus single-dose group) had fewer Ki-ras and N-ras mutations and more tumors in which the Ha-ras codon 61 (C-->A) mutation was present in a large fraction of cells. Our results demonstrate that the dosing procedure can materially affect the pattern of ras gene mutation in mouse liver tumors.

Induction of minisatellite DNA rearrangements by genotoxic carcinogens in mouse liver tumors.

We investigated whether somatic rearrangements in minisatellite DNA are more frequent in chemically induced mouse liver tumors than they are in spontaneous tumors. CD-1 mouse liver tumors were induced by either a single dose or 15 consecutive daily doses of 7,12-dimethylbenz[alpha]anthracene, 4-aminoazobenzene, N-hydroxy-2-acetyl-aminofluorene or diethylnitrosoamine (DEN). Using DNA fingerprinting analysis, we found that the single- and multiple-dose carcinogen treatments caused a 2- to 5-fold higher frequency of minisatellite DNA rearrangements compared with that found in spontaneous tumors--with the exception of single-dose DEN tumors, which showed no increase in rearrangements. Our results suggest that DNA fingerprinting may be a valuable assay for differentiating certain chemically induced tumors from spontaneous tumors.

Qualitative and quantitative evaluation of prostatic histomorphology in rats following chronic treatment with finasteride, a 5-alpha reductase inhibitor.

OBJECTIVE: To determine any potential direct and/or indirect effects of elevated intraprostatic T levels on the prostates of rats chronically (1-2 years) exposed to high doses (160 mg/kg/day) of finasteride, a selective inhibitor of 5-alpha reductase. METHODS: Sprague-Dawley male rats were administered daily finasteride by oral gavage. Prostates from all rats were weighed, fixed in 10% neutral buffered formalin, and processed for light microscopic examination. The volume fractions of the prostatic glandular and stromal compartments were quantitated by morphometric analysis. RESULTS: Administration of finasteride at doses of 20, 40, and 80 mg/kg/day for one year resulted in a significant (P < or = 0.05) decrease in prostatic weight; prostatic atrophy was evident by light microscopy. Morphometric analysis of the prostate showed that chronic finasteride administration resulted in a significant (P < or = 0.001) decrease in the absolute volume of both glandular (-65.2%) and stromal (-57.1%) compartments of the prostate. Furthermore, the total number of epithelial and stromal cells per gland were significantly (P < or = 0.002) decreased in finasteride-treated rats compared with vehicle controls; the magnitude of mean decrease was 69.8 percent and 50.6 percent of controls in epithelial and stromal cells, respectively. In addition, prostates from all two hundred fifty rats in a two-year study were qualitatively evaluated by light microscopy. Administration of finasteride at doses ranging from 2.5 mg/kg/day to 160 mg/kg/day for two years did not result in an increase over the background incidence of prostatic focal hyperplasia or adenoma. No malignant tumors of the prostate were seen in any of the groups. CONCLUSIONS: These studies have demonstrated that the expected pharmacologic effects of finasteride on the prostate are maintained following chronic treatment and that there was no evidence of a direct and/or an indirect effect of elevated intraprostatic T on prostatic morphology in rats.

Leydig cell hyperplasia and adenomas in mice treated with finasteride, a 5 alpha-reductase inhibitor: a possible mechanism.

Finasteride is a selective inhibitor of the enzyme 5 alpha-reductase which is responsible for the conversion of testosterone (T) to dihydrotestosterone (DHT). Finasteride is indicated for the treatment of benign prostatic hyperplasia in man (approximately 0.1 mg/kg/day). The effect of long-term treatment was studied in mice given high doses (2.5, 25, and 250 mg/kg/day) of finasteride for 83 weeks. In finasteride-treated mice, increased incidences of testicular Leydig cell hyperplasia (52% compared to 24% in control group) at doses equal to or greater than 25 mg/kg/day and Leydig cell adenomas (32% compared to 0.5% in control group) at 250 mg/kg/day were observed. There were no drug-related effects on the seminiferous tubules. Since luteinizing hormone (LH) is a trophic hormone for Leydig cells, short-term studies (5 to 14 weeks) were done to investigate the relationship between Leydig cell hyperplasia and serum LH levels in finasteride-treated mice. In these studies, there was a positive correlation between the drug-related increased incidence of Leydig cell hyperplasia and a statistically significant (p < or = 0.05) increase in serum LH levels in finasteride-treated (250 mg/kg/day) mice. Furthermore, studies in castrated male mice showed that the suppression of serum LH levels by T is reversible by inhibition of conversion of T to DHT with finasteride (250 mg/kg/day), supporting the hypothesis that DHT is involved in the regulation of LH release in mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chronic oral administration of a selective 5 alpha-reductase inhibitor, finasteride, on the dog prostate.

Young mature dogs received finasteride, a selective 5 alpha-reductase inhibitor, orally at 0, 5, 15, and 45 mg/kg/day for 27 or 53 weeks. The effect of finasteride administration on prostatic size and morphology was evaluated macroscopically and microscopically. Changes in glandular and fibromuscular compartments were quantitated by a point counting method on trichrome-stained sections. Finasteride administration induced a decrease of mean prostatic weights and epithelial atrophy in all treated groups. No changes in testicular weights and morphology were observed. The greatest prostatic shrinkage was obtained in the group receiving 45 mg/kg/day for 53 weeks; compared to placebo controls, the percent decreases in absolute volumes occupied by epithelium, lumens, fibrovascular stroma, and smooth muscle were 88, 97, 51 and 72, respectively. These results clearly demonstrate that prostatic shrinkage following finasteride administration results from a decrease in both glandular and fibromuscular compartments.

Enantioselective induction of peroxisomal proliferation in CD-1 mice by leukotriene antagonists.

The effects of a racemic leukotriene antagonist (MK-0571) and its component enantiomers (L-668,018 and L-668,019) on hepatic peroxisome proliferation were examined in mice, rats, and rhesus monkeys. Administration of racemic MK-0571 to mice resulted in increased liver weights, increased peroxisomal volume density, and a pleiotropic induction of characteristic peroxisomal and nonperoxisomal enzyme activities associated with peroxisomal proliferation. When the individual enantiomers of MK-0571 were administered to mice, a pronounced enantioselective induction of peroxisome proliferation was observed. Toxicokinetic studies showed that the levels of each enantiomer in the liver or plasma after separate administration were similar. Thus, the enantioselectivity in the induction of peroxisome proliferation could not be explained on the basis of pharmacokinetic differences between the enantiomers. The hepatic peroxisomal response of the rat to MK-0571 was greatly attenuated compared to the mouse. As has been seen with other peroxisome-proliferating agents, MK-0571 had no effect on either peroxisomal volume density or peroxisomal enzyme activity in monkeys. Due to the high degree of enantiomeric discrimination toward the induction of peroxisomal proliferation by these enantiomers, compounds of this type may prove useful as probes to examine the mechanisms by which peroxisomal proliferating agents induce their effects.

Activation of the Ha-, Ki-, and N-ras genes in chemically induced liver tumors from CD-1 mice.

We compared the profile of ras gene mutations in spontaneous CD-1 mouse liver tumors with that found in liver tumors that were induced by a single i.p. injection of either 7,12-dimethylbenz(a)anthracene (DMBA), 4-aminoazobenzene, N-hydroxy-2-acetylaminofluorene, or N-nitrosodiethylamine. By direct sequencing of polymerase chain reaction-amplified tumor DNA, the carcinogen-induced tumors were found to have much higher frequencies of ras gene activation than spontaneous tumors. Furthermore, each carcinogen caused specific types of ras mutations not detected in spontaneous tumors, including several novel mutations not previously associated with either the carcinogen or mouse hepatocarcinogenesis. For example, the model compound DMBA is known to cause predominantly A to T transversions in Ha-ras codon 61 in mouse skin and mammary tumors, consistent with the ability of DMBA to form bulky adducts with adenosine. Our results demonstrate that the predominant mutation caused by DMBA in mouse liver tumors is a G to C transversion in Ki-ras codon 13 (DMBA is also known to form guanosine adducts), illustrating the influence of both chemical- and tissue-specific factors in determining the type of ras gene mutations in a tumor. 4-Aminoazobenzene and N-hydroxy-2-acetylaminofluorene also caused the Ki-ras codon 13 mutation. In addition, we found that N-nitrosodiethylamine, 4-aminoazobenzene, and N-hydroxy-2-acetylaminofluorene all caused G to T transversions in the N-ras gene (codons 12 or 13). This is the first demonstration of N-ras mutations in mouse liver tumors, establishing a role for the N-ras gene in mouse liver carcinogenesis. Finally, comparison of the ras mutations detected in the direct tumor analysis with those detected after NIH3T3 cell transfection indicates that spontaneous ras mutations (in Ha-ras codon 61) are often present in only a small fraction of the tumor cells, raising the possibility that they may sometimes occur as a late event in CD-1 mouse hepatocarcinogenesis.

Polymerase chain reaction/sequencing analysis of ras mutations in paraffin-embedded tissues as compared with 3T3 transfection and polymerase chain reaction/sequencing of frozen tumor deoxyribonucleic acids.

The efficiency of detection of H- and K-ras mutations in 27 CD-1 mouse liver tumors by direct sequencing of polymerase chain reaction (PCR)-amplified DNA isolated from formalin-fixed and paraffin-embedded tissues was compared with that after assay by both NIH 3T3 transfection (followed by sequencing of amplified transformant DNA) and direct sequencing of PCR-amplified DNA isolated from frozen tumors. Some tumor samples were chosen for comparison because they contained ras mutations that were detected by either NIH 3T3 transfection or sequencing of PCR-amplified DNA derived from frozen tumors, but were not detected by both techniques. The efficiency of detecting K-ras mutations was similar for sequencing of amplified fragments derived from both paraffin-embedded tissues and from frozen tumors. However, these two techniques differed in their efficacy for detection of H-ras codon 61 mutations. Furthermore, this difference appeared to be mutation-specific: the sequencing of amplified products from paraffin-embedded tumor tissues allowed increased detection of CAA to AAA mutations but decreased detection of CAA to CTA mutations relative to sequencing of amplified fragments derived from frozen tumor DNA. Direct sequencing of PCR products from paraffin-embedded sections was more sensitive than NIH 3T3 transfection for detection of activated K-ras genes containing codon 13 mutations but less sensitive for detection of activated H-ras genes containing codon 61 mutations. In summary, direct sequencing of amplified DNA from either frozen tumors or formalin-fixed, paraffin-embedded tissues can be more sensitive than NIH 3T3 transfection for detection of codon 13-activated K-ras genes. However, it appears to be less sensitive than NIH 3T3 transfection for detection of certain activating H-ras mutations. Depending upon the questions being asked of the data, each of the methods can provide useful information about ras gene mutations in tumor samples. The apparent differences in sensitivities between the methods is not yet understood, but such differences should be considered in the analysis of data obtained when only one method is used.

Murine p53 intron sequences 5-8 and their use in polymerase chain reaction/direct sequencing analysis of p53 mutations in CD-1 mouse liver and lung tumors.

Inactivating point mutations and small deletions in the p53 tumor suppressor gene have been found in human liver and lung tumor--derived cell lines and tumors. However, little evidence has been reported concerning inactivation or mutation of the p53 gene in mouse primary tumors. To examine CD-1 mouse liver and lung tumors for mutations in the p53 gene, we first sequenced p53 introns 5-8 so that polymerase chain reaction amplification and sequencing primers located within the introns could be prepared. Use of these primers prevented amplification of the mouse p53 pseudogene and allowed sequencing of exons 5-8 in their entirety as well as their intron-exon junctions. DNA isolated from CD-1 mouse tumors was amplified and directly sequenced using nested primers. Nine spontaneous hepatocellular carcinomas (HCCs) and 34 chemically induced HCCs (induced by single intraperitoneal injections of N-nitrosodiethylamine [DEN] [8 HCCs], 7,12-dimethylbenz[a]anthracene [DMBA] [8 HCCs], 4-aminoazobenzene [8 HCCs], and N-OH-2-acetylaminofluorene [10 HCCs]) were examined for mutations in exons 5-8 of the p53 gene. In addition, 12 spontaneous, 10 DMBA-induced, and 13 DEN-induced lung adenocarcinomas or adenomas were analyzed for mutations. No mutations were found in any of the tumors examined. However, a mutation was demonstrated at codon 135 in the positive-control plasmid LTRp53cG(val). The results of this study suggest that inactivation of p53 is unlikely to play a major role in murine lung or liver carcinogenesis. However, inactivation of p53 may occur at a very low frequency, or it may occur as a late event and therefore be present in only a very small number of the tumor cells, rendering it undetectable by this method. Lastly, although few p53-inactivating mutations are found outside of exons 5-8 in human tumors, it is possible that these murine tumors contained mutations outside of this region and were therefore missed by our approach.

Activation of the Ki-ras gene in spontaneous and chemically induced lung tumors in CD-1 mice.

As part of an evaluation of the effectiveness of using ras mutation analysis for distinguishing carcinogen-induced from spontaneous tumors, we examined the profile of ras gene point mutations in spontaneous, 7,12-dimethylbenz[a]anthracene (DMBA)-induced, and N-nitrosodiethylamine (DEN)-induced lung tumors from Crl:CD-1(ICR)BR (CD-1) mice. Although all of the lung tumors were assayed for mutations in the Ha-ras, Ki-ras, and N-ras genes (codons 12, 13, and 61), only Ki-ras mutations were found, which is consistent with other studies that have noted a strong preference for Ki-ras gene activation in mouse, rat, and human lung tumors. We found that spontaneous CD-1 mouse lung tumors had a very high frequency of Ki-ras gene activation (17 of 20 tumors; 85%), distributed among codons 12 (5 of 20), 13 (1 of 20), and 61 (11 of 20). DMBA-induced lung tumors had a slightly higher frequency of Ki-ras gene mutations (16 of 16; 100%), again distributed among codons 12 (5 of 16), 13 (2 of 16), and 61 (9 of 16). However, seven of the DMBA tumors had mutations qualitatively different from those found in spontaneous tumors. In contrast to DMBA-induced tumors, DEN-induced tumors had a lower frequency of Ki-ras mutations (36%) when compared with spontaneous lung tumors, suggesting that DEN primarily induces lung carcinogenesis by a mechanism other than ras gene activation. Thus, although spontaneous and induced CD-1 mouse lung tumors have a strong tissue-specific preference for carrying an activated Ki-ras gene, the nature of the initiating carcinogen can influence the frequency or profile of Ki-ras mutations.

Monoclonal gammapathies in long-term surviving rhesus monkeys after lethal irradiation and bone marrow transplantation.

Late effects of total body irradiation and subsequent autologous bone marrow transplantation on the development of age-related monoclonal gammapathies (MG) were studied in 14 long-term surviving Rhesus monkeys. Together with 27 untreated control monkeys, they have been followed up for more than 20 years. In contrast with the control group, the experimental monkeys developed MG with aging in higher frequencies, earlier and mainly of the benign MG category. One experimental monkey developed a multiple myeloma, the first observed in the nonhuman primates so far. These results indicate an accelerated senescence of the immune system in the experimental monkeys as a late consequence of tissue or cell damage during irradiation.