Quantitation of glandular gastric changes in rats given a proton pump inhibitor for 3 months with emphasis on sampling scheme selection.

Proton pump inhibitors and H2-receptor antagonists suppress gastric acid secretion and secondarily induce hypergastrinemia. Sustained hypergastrinemia has a trophic effect on stomach fundic mucosa, including enterochromaffin-like (ECL) cell hypertrophy and hyperplasia. Histomorphometric quantitation of the pharmacologic gastric effects was conducted on 10 male and 10 female rats treated orally with LY307640 sodium, a proton pump inhibitor, at daily doses of 25, 60, 130, or 300 mg/kg for 3 mo. Histologic sections of glandular stomach, stained for chromogranin A, were evaluated by image analysis to determine stomach mucosal thickness, mucosal and nonmucosal (submucosa and muscularis) area, gastric glandular area, ECL cell number/area and cross-sectional area. Total mucosal and nonmucosal tissue volumes per animal were derived from glandular stomach volumetric and area data. Daily oral doses of compound LY307640 sodium caused slight to moderate dose-related mucosal hypertrophy and ECL cell hypertrophy and hyperplasia in all treatment groups as compared with controls. All observed effects were prominent in both sexes but were generally greater in females. The morphometric sampling schemes were explored to optimize the data collection efficiency for future studies. A comparison between the sampling schemes used in this study and alternative schemes was conducted by estimating the probability of detecting a specific percentage of change between the male control and high-dose groups based on Tukey's trend test. The sampling scheme analysis indicated that mucosal thickness and mass had been oversampled. ECL cell density quantitation efficiency would have been increased by sampling the basal mucosa only for short-term studies. The ECL cell size sampling scheme was deemed appropriate for this type of study.

The selective estrogen receptor modulator, raloxifene: reproductive assessments in adult male rats.

Raloxifene HCl is a nonsteroidal, selective estrogen receptor modulator developed for postmenopausal osteoporosis. Reproductive toxicity of raloxifene was examined in adult male CD rats after the oral administration of doses of 0, 10, 30, or 100 mg/kg/d. In the first study, males (12/group) were treated for 2 weeks followed by 2 weeks without treatment. After dose administration on Day 13, 6 males/group were cohabited with untreated females (1:2) for up to 7 d. Males were killed on Day 14 or 28 (6/group each day). Sperm were collected from the right cauda epididymis and evaluated for relative concentration, motion characteristics, and breakage. The kinetics of spermatogenesis were examined by DNA flow cytometry. The left testis and epididymis were preserved for histopathologic evaluation. Females were examined for reproductive status on Gestation Day 13. In a second study, males (20/group) were treated for 7 weeks (4 weeks prior to cohabitation during a 2-week cohabitation period, and for 1 additional week). Treated males were cohabited with untreated females (1:1). On Gestation Day 20, untreated females were examined for reproductive status and fetuses were examined for viability, weight, gender, and morphology. At necropsy, male reproductive tissues were collected, weighed, and preserved for histopathologic evaluation. In both studies, male body weight gain and food consumption were depressed at all dose levels. There was no indication in either study that raloxifene caused important changes in sperm production, sperm quality, or male reproductive performance at doses as high as 100 mg/kg/d.

The selective estrogen receptor modulator, raloxifene: reproductive assessments following premating exposure in female rats.

Raloxifene HCl is a nonsteroidal, selective estrogen receptor modulator developed as a therapeutic agent for postmenopausal osteoporosis. Two studies were conducted that examined the effects of premating exposure to raloxifene HCl. In the first study, adult female CD rats (20/group) were given diets containing 0, 0.01, or 0.1% raloxifene (providing an average of 0, 6, or 63 mg/kg/d, respectively) for 2 weeks, after which the treated diets were replaced with control diet. Following a 2-week period without treatment, each female that had displayed at least three conversions in vaginal cytology from cornified cells to leukocytes was cohabited for 1 to 2 d with an untreated male as she entered proestrus. Females were killed at midgestation and examined for evidence of pregnancy. In the second study, adult female CD rats (40/group) were given oral gavage doses of raloxifene (0, 0.1, 1, or 10 mg/kg/d) for 4 weeks. Immediately or following a 2-week period without treatment, 20 females/group were cohabited with untreated males (1:1) for up to 3 weeks. The females were allowed to deliver and rear their offspring until Postpartum Day 21. Progeny survival, growth, and development were evaluated. Maternal body weight, body weight gain, and food consumption were depressed in all raloxifene treatment groups. Doses > or =1 mg/kg caused disruptions in estrous cycles. In Study 1, 90% of the females treated with raloxifene resumed normal cycling, and fertility was not significantly affected. Although there were no statistically significant differences in time-to-mating, fertility, or liveborn indices in Study 2, females in the 10-mg/kg immediate-cohabitation group had slightly increased gestation lengths and smaller litter sizes. Progeny from these litters were larger on Postpartum Day 1 and had advanced incisor eruption and eye opening. In addition, slight delays were seen in physical landmark appearance in the 0.1- and 1-mg/kg immediate-cohabitation groups and in the 1- and 10-mg/kg delayed-cohabitation groups. Progeny viability, growth, and negative geotactic performance were not adversely affected. In these studies of maternal premating exposure to raloxifene, findings were consistent with established pharmacologic activity of the test chemical. Reproductive effects (disrupted estrous cycles and decreased litter size) occurred at doses > or =1 mg/kg and were generally reversible. Effects on offspring were seen at doses > or =0.1 mg/kg, were of minor importance, and were resolved during the lactation period.

The selective estrogen receptor modulator, raloxifene: a segment II/III delivery study in rats.

Raloxifene is a nonsteroidal, selective estrogen receptor modulator developed by Eli Lilly and Company as a therapeutic agent for postmenopausal osteoporosis. Raloxifene was administered orally by gavage at doses of 0, 0.1, 1, or 10 mg/kg/d to female CD rats (25/group) on Gestation Day 6 (GD 6) through Postpartum Day 20 (PD 20). Females were allowed to deliver and maintain their progeny until PD 21. All dead pups and pups culled on PD 1 were given internal and external examinations. One pup/sex/litter was assigned to each of the following assessment groups: 1) the primary pair for the F1 generation study, in which survival, growth, development, behavior, indicators of sexual maturation, and reproductive performance were evaluated; 2) terminal necropsy evaluations at PD 21; 3) terminal necropsy evaluations at 60 d of age; and 4) assessments of immune function at 5 to 6 weeks of age. At termination on PD 21, 60, or approximately 140, a necropsy was performed; crown rump and tibia lengths were measured; pituitary weights were taken; and a portion of the anterior pituitary was retained for growth hormone, luteinizing hormone, and prolactin content determinations (control and 10-mg/kg groups only). The remainder of the pituitary and reproductive tissues were retained for histologic evaluations. Dose-related depressions in maternal body weight and food consumption occurred during gestation. Mean gestation length was increased at 1 and 10 mg/kg. Delayed, extended, and/or disrupted parturition occurred in dams given 10 mg/kg, which resulted in a high incidence of maternal morbidity and/or death, increased numbers of dead pups, and the survival of only 66% of live pups to PD 21. Progeny body weights were not decreased at birth, but were depressed progressively in a dose-related manner during the 3-week lactation period. Negative geotaxis and incisor eruption were apparently accelerated in the 1- and 10-mg/kg groups, but eye opening was delayed at 10 mg/kg. Postweaning activity levels, auditory startle, and passive avoidance performance were not affected in the raloxifene groups. Dose-related decreases in spleen cellularity and thymus weights occurred in both sexes, but immune system function, as measured by splenic natural killer cell activity and antibody response to sheep red blood cells, was not affected. Postweaning body weights and growth parameters, as well as pituitary hormone content, were affected in both an age- and sex-specific manner. Preputial separation was not affected, but vaginal patency occurred ca 2 d earlier than controls in females from the 10-mg/kg group. Estrous cycles of the F1 females were not affected during the first two weeks after vaginal opening, but were disrupted at 12 to 14 weeks of age in the 10-mg/kg group. These females showed poorer mating and fertility indices, and litter size was reduced in the two females that were pregnant. Histologically, reproductive organs were not affected in males at any age or in females at PD 21. At PD 60, vaginal mucification occurred in females from the 0.1- and 1-mg/kg groups. At PD 140, the only finding was a high rate of uterine hypoplasia in the 10-mg/kg group, and this finding occurred in the absence of any concomitant ovarian or vaginal changes. These reproductive and developmental findings are consistent with estrogen antagonist activity of raloxifene.

Short-term male reproductive toxicity study with sulfasalazine in the rat.

Sulfasalazine (2-hydroxy-5-[[4-[(2-pyridinylamino) sulfonyl] phenyl]azo]benzoic acid; SASP) was administered to rats in a short-term male reproductive toxicity study to further examine the utility of this grouping of techniques and to generate reference data with a substance that is known to cause reversible infertility in men. Adult male CD rats (10/group) were orally administered 0, 150, 300, or 600 mg SASP/kg body weight in divided doses for 14 d followed by a 2-week period without treatment. Males were killed on test day (TD) 15 or 29. At each time point, the reproductive system was evaluated by comparing testicular and epididymal weights, DNA ploidy distributions of testicular cell suspensions, testicular and epididymal histopathology, and epididymal sperm concentrations, motion, morphology, and breakage. Adding time as a factor in the protocol aids in distinguishing testicular from posttesticular effects. Changes in sperm quality after 2 weeks of test article administration (TD 15) predominantly reflect effects that occurred after the sperm entered the epididymis, while testicular effects predominated on TD 29. Beginning on TD 14, males to be killed on TD 29 were cohabited with untreated females (1:2). Females were killed at midgestation and examined for pregnancy status. Body weight gain was depressed in all SASP groups during the first 3 d of test article administration. Food consumption was depressed at the 300- and 600-mg/kg dose levels. No changes were seen in testicular weight, but epididymal weight was depressed at the 600-mg/kg dose level. DNA ploidy distributions determined by flow cytometry did not indicate that the kinetics of spermatogenesis were disturbed. However, alterations in sperm release, which have not previously been reported, were seen at all SASP dose levels. On TD 29, the percentage of progressively motile sperm was depressed and beat/cross frequency was increased at the 600-mg/kg dose level. No changes were observed in sperm morphology or breakage. Fertility was slightly depressed at the 600-mg/kg dose level. In this study, testicular histopathology provided the most sensitive endpoint for reproductive toxicity. The impairment of fertility immediately after treatment was stopped, when no changes were apparent in sperm release or sperm motion, suggested that decreased sperm concentrations and altered motility, while contributory, may not be the primary causes of SASP-mediated infertility.

Utilization of a short-term male reproductive toxicity study design to examine effects of alpha-chlorohydrin (3-chloro-1,2-propanediol).

alpha-Chlorohydrin (ACH) was administered to rats in a short-term male reproductive toxicity study to examine the usefulness of the method and to provide reference data with a substance that is known to elicit adverse effects on both sperm production and sperm quality within or following a 2-week treatment period. Adult male CD rats (10 per group) were administered ACH orally by gavage at doses of 0, 1, 5, or 25 mg/kg/day for 14 days. Males were killed on Test Day (TD) 15 or 29. A 2-week period without treatment was included to distinguish between testicular and posttesticular effects. At each time point, the reproductive system was evaluated by comparing testes weight, DNA ploidy distributions of testicular cell suspensions, testicular and epididymal histopathology, and epididymal sperm concentration, motility, morphology, and breakage. Beginning on TD 14, males to be killed on TD 29 were cohabited with untreated females (1:2). Females were killed on Gestation Day 13 and examined for pregnancy status. During the treatment period, minor depressions in body weight and relative food consumption occurred in rats administered 25 mg/kg ACH. Testicular and epididymal lesions also occurred at this dose level. DNA ploidy distributions determined by flow cytometry were predictive of testicular damage, with effects more pronounced on TD 29 than on TD 15. The preparation methods used selected for the most motile and vigorous population of epididymal sperm. Sperm motion was altered at the 5- and 25-mg/kg dose levels on TD 15. The percentage of motile sperm and the percentage of progressively motile sperm were markedly depressed at both the 5- and 25-mg/kg dose levels where antifertility effects occurred. Sperm velocities and amplitude of lateral head displacement were depressed at the 25-mg/kg dose level on both TD 15 and 29. Additionally, decreased epididymal sperm concentrations and increased breakage were recorded at this dose level. The findings in this study are consistent with the scientific literature for ACH and demonstrate posttesticular effects on epididymal sperm and delayed expression of testicular lesions. They also support the use of this methodology for an initial assessment of male reproductive effects.

Expression of bovine myf5 induces ectopic skeletal muscle formation in transgenic mice.

myf5 is one of a family of four myogenic determination genes that control skeletal muscle differentiation. To study the role of myf5 in vivo, we generated transgenic mice harboring the bovine homolog, bmyf, under control of the murine sarcoma virus promoter. Ectopic expression of the full-length bmyf transgene was detected in brain and heart tissue samples of F1 progeny from transgenic founder mice. Ectopic bmyf expression activated endogenous skeletal myogenic determination genes in the hearts and brains of transgenic animals. Incomplete skeletal myogenesis in most hearts gave rise to cardiomegaly and focal areas of cardiomyopathy. In brains in which ectopic expression led to a more complete myogenesis, focal areas of multinucleated, striated myotubes containing actin, desmin, and myosin were observed. These unexpected results show that myf5 can initiate myogenic differentiation in vivo, supporting the hypothesis that myf5 is responsible for determination of cells to the myogenic lineage in normal embryogenesis.

Escherichia coli K12 does not colonize the gastrointestinal tract of Fischer-344 rats.

The colonizing potential of Escherichia coli K12 containing a vector coding for somidobove (bovine somatotropin) was determined. Treated male and female Fischer-344 rats were given a single oral gavage inoculum of sucrose with/without tetracycline (15 micrograms/ml). Untreated control animals received similar drinking water regimes. All animals survived until termination. There were no clinical signs of toxicity observed and no treatment-related effect upon body weight, food consumption, or efficiency of food utilization. Fresh fecal samples were collected from each rat every 24 h following inoculation and the population of the marked strain was quantitated until no bacterial colonies were observed for two consecutive days. While all inoculated rats were positive at 24 h, by 72 and 96 h all had become negative for the test (marked) strain, as were the corresponding control group throughout the test. The frozen stock of the marked strain used as the positive control demonstrated that the agar plates were selective for the test strain. Fourteen days following inoculation, all groups of rats were killed and the gastrointestinal tracts removed and treated to recover the marked strain. There was no evidence of the marked strain in the gastrointestinal tract of any from any group. Thus, the E. coli K12 host/vector system used in this experiment does not colonize the gastrointestinal tract of Fischer-344 rats.

Preclinical toxicology studies with nizatidine, a new H2-receptor antagonist: acute, subchronic, and chronic toxicity evaluations.

Nizatidine (NIZ), a new antiulcer drug, was evaluated for toxicity in acute, subchronic, and chronic tests. Acute toxicity studies were conducted in rats, mice, dogs, and monkeys. Median lethal doses (MLD) in rodents were greater than 1600, 230, and 1000 mg/kg by oral (po), iv, and sc administration, respectively. No deaths occurred in dogs given single doses of 800 mg/kg (po), 75 mg/kg (iv), or 225 mg/kg (im) or in monkeys given 1200 mg/kg (po) or 200 mg/kg (iv). Rats survived up to 1.0% dietary NIZ (daily intake ranging from 24 to 800 mg/kg/day) for 1 year. Slight decreases in body weight gain and increases in liver and kidney weights occurred. Slight decreases in erythrocytic parameters at 3 months were not present at 6 or 12 months. Mice survived up to 1.5% dietary NIZ for 3 months and effects were limited to slight decreases in body weight gain and increases in relative liver weight. Dogs survived oral doses up to 800 mg/kg/day for 3 months but had numerous clinical signs of toxicity and body weight loss. All dogs given oral NIZ doses up to 400 mg/kg/day survived except for one high-dose dog that was killed in a moribund condition following convulsions in the 41st week of treatment. Effects in dogs included miosis, body weight loss, increased thrombocyte counts, and decreased hepatic microsomal enzyme activity and P450 content. The increase in thrombocyte counts was unaccompanied by changes in thrombocyte function and did not reoccur in a subsequent study. A decrease in plasma testosterone in two of three surviving male dogs given 400 mg/kg/day for 1 year was unaccompanied by effects on the size or morphology of testes or prostate. Peak plasma levels of NIZ in all species tested were in excess of human plasma levels after therapeutic doses. In conclusion, there was no evidence of significant toxicity in organs or tissues including those sites (gastric mucosa, male sex organs, and liver) that have been affected by some agents of this therapeutic class.

Evidence for suppression or incomplete maturation of cell-mediated immunity in neonatal calves as determined by delayed-type hypersensitivity responses.

Fifteen bovine fetuses were inoculated in utero 20 to 123 days before birth with a mixture of killed Mycobacterium bovis, tetanus toxoid, and ferritin in Freund's complete adjuvant. On the day of birth (day 0) and when the calves were 21 days of age, the calves were skin-tested to each of the antigens for delayed-type hypersensitivity. Nine delayed-type hypersensitivity responses to the various antigens were obtained at the 0-day test, whereas 28 responses were obtained at the 21-day test. Of those responses that were positive, the mean differences in the double skin-fold thickness before testing and 48 hours later were 5.4 mm for the 0-day and 21-day test and 9.4 mm for the 21-day test. Six control calves that were not inoculated in utero were skin tested on days 0 and 21 and did not exhibit any positive reactions. There was no indication that the interval between immunization and birth had any effect on the immune response. Cellular characteristics of the reactions at 0 and 21 days were the same.

Shedding of Treponema hyodysenteriae, transmission of disease, and agglutinin response to pigs convalescent from swine dysentery.

Fecal shedding of Treponema hyodysenteriae, transmission of disease, and humoral antibody production against the large spirochete were evaluated in pigs convalescent from experimentally induced swine dysentery. Untreated pigs (n = 21) and 5 pigs that had been treated with virginiamycin were included in the study. Treponema organisms were culturally detected in the feces of 2 untreated pigs as long as 70 and 71 days, and in the feces of 1 treated pig as long as 83 days after the last clinical evidence of disease. Of 8 convalescent pigs that intermittently discharged T hyodysenteriae in their feces, 4 transmitted clinical disease to exposed susceptible pigs. One of the convalescent animals has been free of clinical signs of disease for 57 days before introduction of the susceptible pigs. Treated and untreated convalescent pigs developed similarly elevated agglutinin titers that were maintained as long as 150 days after infection. There was no apparent correlation between the frequency or duration of fecal shedding of the spirochetes and the magnitude of the agglutinin titers of the convalescent pigs.

Renal styphlodoriasis in a boa constrictor.

Renal styphlodoriasis was diagnosed in a 2 1/2-year-old male boa constrictor (Constrictor constrictor). The snake had been anorexic for 6 months prior to its death. Necropsy revealed numerous hard, white foci of mineralization in the kidneys. Histologic examination revealed distorted renal tubules containing cross sections of trematodes or mineralized debris, tubular epithelial hyperplasia, and chronic interstitial nephritis. Several adult trematodes were recovered and identified as Styphlodora horrida.