Analysis for loss of heterozygosity (LOH) of p53 allele in tumors derived from p53+/- and CD-1 mice following repeated subcutaneous injections of solutions containing antioxidants.

Genomic DNA was isolated from subcutaneous masses observed in CD-1 and p53+/- heterozygous mice during the course of carcinogenicity studies in the vehicle control groups. These masses resulted after daily subcutaneous injection of an antioxidant vehicle with a pH adjusted to 3-4. The vehicle was 1.0% ascorbic acid plus 0.05% sodium metabisulfite in 0.75% saline in a dosing volume of 10 ml/kg/day. These masses were first palpable after 13 and 37 weeks of dosing among p53+/- and CD-1 mice, respectively. By week 26, the incidence of these masses was 89% and 80% in male and female p53+/- mice, respectively (n = 15 mice/sex) and was 0% in both male and female CD-1 mice (n = 60 mice/sex). These masses originated from panniculus carnosus muscle. Histopathological examination of the p53+/- mouse masses indicated the tumors to be sarcomas of spindle-cell origin. The histopathological examination of the masses in the CD-1 mice revealed fibrosarcomas. Five mice/sex/strain were randomly selected from a pool of mice that developed these masses in the course of the two studies. Frozen tissues from these masses were used to examine the DNA for loss of the functional p53 allele in the p53+/- mice (i.e., loss of heterozygosity, or LOH) or for loss of one of the alleles in the wild type (p53+/+) CD-1 mice by the polymerase chain reaction (PCR) technique. Loss of the functional allele was observed only in the tumor from one p53+/- male mouse. These results support a nongenotoxic mechanism for these injection site masses.

Evaluation of apomorphine HCl effects on reproductive endpoints: studies in male rats and dogs.

Apomorphine HCl is currently in phase III clinical trials for the treatment of erectile dysfunction. The potential for reproductive toxicity in males was assessed based on a 13-week rat study--a fertility study in male rats--and a 6-month study in dogs. The subcutaneous (s.c.) route was selected to simulate the sublingual route in humans. Dosages of apomorphine were 0.0 (vehicle), 0.8, 2, and 8 mg/kg/day in the 13-week study in rats (20/group), with 8 mg/kg/day used for only 9 weeks. In the fertility study, 24 males/group were cohabited with females, and doses were 0.0, 0.2, 0.8, and 2 mg/kg/day. Males were treated for 4 weeks prior to cohabitation and for 5 weeks throughout cohabitation. Organ weights, including testis and left epididymis, sperm count and morphology in the epididymis, and sperm motility in the vas deferens were evaluated. Male fertility index, and in females, the numbers of fetuses, implantation sites, and corpora lutea were counted. Male dogs (five/group) were dosed with 0.04, 0.1, or 0.4 mg/kg/day for 6 months. Epididymal and prostate weight, and testicular and epididymal histology were evaluated. Daily morbidity/mortality, weekly clinical observations, body weight, and food consumption were evaluated in all studies. No adverse effect was observed in any of the reproductive parameters in the studies. The NOEL for reproductive toxicity was approximately equal to 0.4 mg/kg/day in dogs and > or = 2 mg/kg/day in rats. These doses in rats and dogs correlated with plasma levels of approximately 240 and 130 ng/mL, and AUCs of 200 and 100 ng.h/mL, respectively. These levels suggest a safety margin for the evaluated male reproductive endpoints of at least 104 times based on the Cmax, and 44 times based on AUC of the clinical dose.

Mechanism for species-specific induction of Leydig cell tumors in rats by lansoprazole.

Lansoprazole is a substituted benzimidazole which inhibits gastric acid secretion by inhibiting the hydrogen-potassium ATPase (proton pump) in the parietal cell. The finding of Leydig cell hyperplasia and Leydig cell tumors in 2-year oral studies in Sprague-Dawley rats but not in CD-1 mice prompted investigative studies to determine the mechanism for the Leydig cell changes. hCG challenge studies in Sprague-Dawley rats revealed decreased testosterone responsiveness in rats treated orally for 1 or 2 weeks with lansoprazole. After 4 weeks of daily oral treatment increases in serum LH and decreases in serum testosterone were detected within a few hours after dosing. In a study where 9-month-old male F344 rats were given testosterone supplementation via Silastic implants and then treated with lansoprazole for 6 months, a high incidence of Leydig cell tumors was seen in lansoprazole-treated, unsupplemented rats, whereas no Leydig cell tumors were seen in testosterone supplemented rats. This implied that reduction of the normal feedback inhibition at the level of the hypothalamus and/or pituitary due to reduced testosterone levels, thus giving rise to elevated levels of LH, was involved in the induction of Leydig cell tumors by lansoprazole. In vitro studies with Leydig cells from rats using various stimulators and precursors of testosterone biosynthesis demonstrated that the most sensitive site for inhibition of testosterone synthesis by lansoprazole is the transport of cholesterol to the cholesterol side chain cleavage enzyme. The IC50s for inhibition of LH or hCG-stimulated testosterone synthesis in Leydig cells from rats, mice, and monkeys were 11-12, 8, and 24.7 micrograms/ml, respectively. In vitro studies with metabolites of lansoprazole revealed that three metabolites were more potent inhibitors of testosterone synthesis than the parent drug, two of them being at least 10 times more potent. These metabolites are present in rats at substantial levels but are undetectable in humans. The lack of induction of Leydig cell tumors in mice, lower sensitivity of primate Leydig cells, and the absence of testosterone synthesis-inhibiting metabolites in man suggest that Leydig cell tumors found in rats represent a species-specific sensitivity and does not imply a risk for clinical use in man.

Genotoxic potency in Drosophila melanogaster of selected aromatic amines and polycyclic aromatic hydrocarbons as assayed in the DNA repair test.

Drosophila melanogaster stock consisting of meiotic recombination deficient (Rec-) double mutant mei-9a mei-41D5 males and Rec+ females was exposed at the larval stage to an aromatic amine or a polycyclic aromatic hydrocarbon. After emergence as adult flies, the males and the females were scored separately. When the treatment caused a dose-dependent reduction in the male to female ratio from the control level; the experiment was repeated with a larval stock consisting of Rec+ males and Rec+ females under comparable conditions. A preferential killing effect upon Rec- larvae was taken as evidence of DNA damaging effect of the test compound. Among 16 compounds tested, 1-AP, B(a)P, 2-AF, DAF, 4-AAF, 2-AAF, 1-AA, 2-AA, DMA, B(a)A and DMBA were registered as positive; Py and 3-MC were weakly positive; and B(e)P, Fluo and Ant were negative. The selective killing effects of the compounds in each of the pyrene, fluorene and anthracene series varied drastically as a function of structure in a way similar to that reported for the genotoxicity in Drosophila and the carcinogenicity in rodents. The Drosophila DNA repair assay will serve as a simple adjunct to the already available means for studying the genotoxic potency of aromatic amines and polycyclic aromatic hydrocarbons.

Fetal liver micronucleus assay in mice of 5-fluorouracil and related compounds.

The cytogenetic effects of 5-fluorouracil (5-FU), 1-hexyl-carbamoyl-5- fluorouracil (HCFU) and 1-(2-tetrahydrofuryl)-5-fluorouracil (TF) were examined with the fetal liver micronucleus assay in mice. The frequencies of micronucleated polychromatic erythrocytes (MNPCEs) in fetal liver peaked at 27, 24 and 27 h, respectively, after single intraperitoneal injections into pregnant mice on day 13 of gestation. The highest frequency of MNPCEs by 5-FU treatment in fetal liver was 13.6%, whereas the frequency in maternal bone marrow was only 0.4%. The micronucleus frequency and the number of micronuclei per individual polychromatic erythrocyte were clearly dose-dependent. These results suggest that the micronucleus test in fetal liver has particular advantages compared to maternal bone marrow for evaluating the cytogenetic effects of 5-FU and related compounds after a single treatment. The cytogenetic effect was ranked 5-FU = HCFU > TF, in both a time-course study and a dose-response study of micronucleus distribution.

Buflomedil: one-year oral safety evaluation in rats.

Buflomedil was given as a dietary mix to Sprague-Dawley Rats for one year at dosages of 0, 25, 75, and 400 mg/kg/day. Each group contained thirty rats of each sex, of which 10/sex/group were preassigned to a withdrawal period of 3 months following the 1-year drug administration period. There were no treatment-related deaths or clinical signs. Dosage-related decreases in body weight gain occurred during the 1-year treatment period. Food consumption was comparable to controls, except for a 10% decrease in high-dosage males during the first week of treatment. During the withdrawal period, body weight gain was higher than controls in all drug-treated groups. Hematocrit (PCV) and hemoglobin were slightly decreased during week 52 in high-dosage males; hematology values were comparable to controls during weeks 13 and 26 and at the end of the withdrawal period. There were no treatment-related changes in blood biochemistry. Urine pH was decreased in females at 75 mg/kg/day and in both sexes at 400 mg/kg/day. Discolored urine was observed in males at 75 mg/kg/day and in both sexes at 400 mg/kg/day. Urine pH and color were comparable to controls after the 3-month withdrawal period. There were slight decreases compared to controls in urine volume at all dosages in females during week 52; these changes were still evident in the mid- and high-dosage groups at the end of the withdrawal period. The only possibly treatment-related observation at necropsy was dirty tails at the end of the treatment period in the high-dosage group which may have been related to the discolored urine. Liver and kidney weights were slightly increased in males at 400 mg/kg/day at the end of the 1-year treatment period; these changes were not evident after the 3-month withdrawal period. There were no treatment-related histopathological changes. The changes observed were thought to result from either pharmacologic activity or physiological adaptation to compound administration or were marginal in severity. None were considered toxicologically significant. Therefore, the no-toxic-effect dosage was 400 mg/kg/day which is 40 times the maximum clinical dosage.

Effects of a GnRH agonist on fertility following administration to prepubertal male and female rats.

Leuprolide, a GnRH agonist, was administered daily to male and female rats for 90 days. Animals were sexually immature (25 days old) at the outset. Dosages were 20 and 200 micrograms/kg/day. Five males and five females were euthanized on Day 91. Sex organs were weighed and evaluated for histopathologic changes. These procedures were repeated 140 days later. Following a recovery period lasting 45 days (onset of normal-appearing estrous cycles) in females and 140 days (two spermatogenic cycles) in males, the fertility of these rats was assessed by mating with untreated animals. Treated males gained less weight while treated females gained more weight than controls. Weights of primary and secondary sex organs were reduced below control, but returned to normal following 140 days of recovery. Treated males were fertile and produced normal litters. Reproductive performance of low-dosage (20 micrograms/kg/day) females was normal 45 days after treatment cessation, but half of the high-dosage (200 micrograms/kg/day) females failed to become pregnant. However, reproductive performance of this group compared well with control performance after an additional 6 weeks of recovery. Atrophic changes were noted in male and female sex organs. Following 140 days of recovery, ovaries, uterus, vagina, prostate, and seminal vesicle were normal. Although testes and epididymides showed partial recovery at this time, multifocal or segmental atrophy and mineralization were noted in portions of some seminiferous tubules.

Mutagenicity of quinolone antibacterials.

The literature is summarised on the activity of quinolone antibacterial compounds in assays which are commonly used for risk assessment of new pharmaceuticals. These include assays for DNA damage, sister chromatid exchanges, chromosome aberrations and mutation induction. The general pattern of activity exhibited by these compounds is induction of DNA damage in both prokaryotic and eukaryotic cells, and induction of mutations in DNA repair-proficient bacteria and at the thymidine kinase locus in mammalian cells. They do not appear as a class to induce mutations at the hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) or Na+,K(+)-ATPase loci or to cause chromosome aberrations. It is suggested that these actions may be the result of interference with eukaryotic topoisomerase and that this interference differs in some respects from the topoisomerase interference caused by certain antitumour compounds. The postulated mechanism of action has important implications for assessment of risk from consumption of quinolone antibacterials. The risk of adverse genotoxic events should vary directly with the concentration of drug reaching the intracellular enzyme target and the affinity of the drug for the target. Results of carcinogenicity studies conducted to date with the quinolone antibacterials suggest minimal risk from long term consumption of the newer, second-generation compounds.

Phototoxicity of tin protoporphyrin, tin mesoporphyrin, and tin diiododeuteroporphyrin under neonatal phototherapy conditions.

Tin metalloporphyrins are being considered as therapeutic agents for neonatal hyperbilirubinemia, and it is possible that concurrent exposure to phototherapy will occur during their use. Euthymic hairless guinea pigs, Crl:IAF(HA)BR, were given daily intraperitoneal injections of tin protoporphyrin (SnPP), tin mesoporphyrin (SnMP), or tin diiododeuteroporphyrin (SnI2DP) for 3 successive days. They were concurrently exposed to ambient light or two different kinds of phototherapy light under conditions similar to that found in neonatal intensive care units. Phototherapy light exposure was for a continuous period of approximately 72 hours following the first injection of metalloporphyrin. The presence or absence of phototoxicity under these conditions was determined by observations for an erythematous response on the back and ears of the guinea pigs. The dosages used were 0.75, 3.75, and 7.5 mg/kg per day of SnPP, 0.075, 0.375, and 0.75 mg/kg per day of SnMP, and 0.9, 4.5, and 9.0 mg/kg per day of SnI2DP. These dosages for each drug were approximately 1 times, 5 times, and 10 times, respectively, the maximum anticipated clinical dosage. At equal multiples of the clinical dosages, SnI2DP was less phototoxic than SnPP, and SnMP was the least phototoxic of the three compounds. SnPP was marginally phototoxic at the lowest dosage. SnMP was phototoxic only at the highest dosage under phototherapy light emitting ultraviolet A irradiation, but when phototherapy light not emitting ultraviolet A irradiation was used, SnMP was not phototoxic. In all cases, the phototoxic response was reversible when the drug and phototherapy treatment were discontinued.

Carcinogenic evaluation of estazolam via diet in CD strain Sprague-Dawley rats and B6C3F1 mice for 2 years.

A comparative toxicity and carcinogenicity study was carried out for 2 years with estazolam, a benzodiazepine, via diet in Sprague-Dawley rats (0.5, 2, and 10 mg/kg/day) and in B6C3F1 mice (0.8, 3, and 10 mg/kg/day). In rats, no biologically significant changes were seen with respect to mortality, clinical signs, food consumption, or occurrence of palpable masses. Body weight gain in females (10 mg/kg/day) was depressed 12.6% and reflected a maximum-tolerated dosage (females). Spontaneous and incidental nonneoplastic lesions were consistent with aging in this species and unrelated to drug treatment. No biologically significant differences in tumor incidences occurred. Mice were more responsive to estazolam as suggested by (1) increased mortality (males) at 10 mg/kg/day, (2) increased food consumption and body weight gains (females), (3) withdrawal signs characterized by hyperactivity/aggressiveness and convulsions, and (4) appearance of dose-related nodular hyperplasia of the liver due to the relatively high dosages used coupled with the propensity of benzodiazepines to enhance liver enzyme induction. Several spontaneous benign and malignant tumors observed in all groups were not considered to be drug related. Based on the findings in these studies, estazolam was not considered to be carcinogenic when administered via diet to either rats at 0.5, 2, and 10 mg/kg/day or to mice at 0.8, 3, and 10 mg/kg/day for 2 consecutive years.

Heinz bodies, methemoglobinemia, and hemolytic anemia induced in rats by 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline.

Sprague-Dawley CD strain rats were given 18, 35, 70, or 140 mg/kg/day of 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline by gavage for 2 weeks. Heinz bodies were seen in the erythrocytes of rats given 140 mg/kg/day. Dose-related increases in methemoglobin were found at 35 mg/kg/day or more. Hemolytic anemia was characterized by dose-related decreases in hematocrit, hemoglobin, and total erythrocyte count. Reticulocytosis, decreased myeloid:erythroid ratio, splenomegaly, extramedullary hematopoiesis, increased serum total bilirubin, and icterus were also observed. This compound was found to oxidize oxyhemoglobin to methemoglobin in vitro, suggesting that the parent compound is capable of causing the hematological changes observed in vivo without conversion to active metabolites.

Terazosin: intravenous safety evaluation in rats.

Terazosin, an alpha-adrenergic antagonist, was administered as a 15 mg/ml solution to rats intravenously at a rate of 2 ml/min. Under these conditions the LD50 was 277 mg/kg for males and 293 mg/kg for females. When administered daily for 1 month at dosages of 0, 10, 40 or 150 mg/kg/day, the no-toxic-effect dosage was 40 mg/kg/day. Evidence of toxicity at 150 mg/kg included hypothermia and deaths. Death resulted from acute, exaggerated pharmacologic effects leading to cardiorespiratory failure. Evidence of sympatholytic activity observed at lower dosages included hypoactivity, blepharoptosis, ptyalism and splenic congestion.

Tulobuterol: one-month intravenous safety studies in rats and dogs.

Tulobuterol was given intravenously to rats and dogs at dosages of 1, 5, or 25 mg/kg/day and 0.6, 2, or 6 mg/kg/day, respectively. The no-toxic-effect dosages were 5 mg/kg/day in rats and 6 mg/kg/day in dogs. Two rats died at 25 mg/kg/day. Convulsions, jerking movements, hyperactivity, tremors, hypoactivity and ptyalism were observed in rats given 25 mg/kg/day. Restlessness, ptyalism and hypoactivity were also observed in dogs at 2 and 6 mg/kg/day. Cutaneous and/or mucosal erythema were observed in rats and dogs at all dosages. Increased body weight gain occurred in drug-treated rats and in mid- and high-dose female dogs. Slight elevations in serum creatinine and BUN were seen in rats and dogs at the highest dosages. Heart weights were increased in rats at all dosages after 1 month of treatment and in rats given 25 mg/kg/day after 2 weeks of recovery. There were no treatment-related morphologic changes in either species.

Cetaben versus clofibrate: comparison of toxicity and peroxisome proliferation in rats.

Cetaben and clofibrate were each administered to groups of 6 rats (3 male and 3 female) by gavage at dosages of 50 mg/kg per day and 200 mg/kg per day for 2 weeks. Cetaben caused 1 death at 200 mg/kg per day and decreases in body weight gain and food consumption at 50 mg/kg per day or more. There were no effects on body weight or food consumption in the clofibrate-treated groups. At 200 mg/kg per day cetaben and clofibrate induced comparable increases in liver weight and in numbers of liver peroxisomes while cetaben caused increases in liver catalase activity as well.

Buflomedil: three month intravenous safety evaluation in rats.

Buflomedil was administered intravenously to rats at dosages of 1, 4, 12 or 30 mg/kg/day for up to three months. The no-adverse-effect dosage was considered to be 12 mg/kg/day. At 30 mg/kg/day several deaths and clinical signs, including ataxia, decreased activity, dyspnea and jerking movements after dosing, were observed. These were considered to result from the acute, exaggerated pharmacologic effects of buflomedil. Body weight gain and food consumption were decreased after six weeks in males at 30 mg/kg/day. Increases in the relative weights of the kidneys, brain and testes of males at 30 mg/kg/day were correlated with decreased body weight gain in this group. There were no effects on hematology or serum chemistry parameters, and no morphologic changes were found.

Altered DNA repair in fibroblasts from aged rats.

The role of DNA repair in the aging process was studied using fibroblast cultures derived from rats at the beginning (3 weeks) and at the end (2 years) of their potential lifespan. The effect of treatment of the cultures with the DNA-damaging agent, ethylnitrosourea, on the integrity of the pre-existing parental DNA and on de novo DNA synthesis was determined using the alkaline elution method. It was found that drug treatment resulted in lower levels of fragmentation of parental DNA and in decreased fractions of short daughter strands in cells derived from old relative to young animals. These results indicate an alteration of the processing of DNA damage in cells derived from aged rats.

Endometrial estrogen and progesterone receptors in a uterus didelphys.

A 23-year-old woman with a uterus didelphys and a totally occluded left tube had a hysterectomy one year after having a child. Endometrial estrogen and progesterone receptors, both cytoplasmic and nuclear, were determined in five longitudinal sections of each horn. The amount and distribution of these receptors were normal, but the receptor content of the right horn was higher than that of the left.

PIP: A 23-year-old white woman was admitted with a complaint of increasing dysmenorrhea since the birth of her child 1 year before. She was diagnosed as having a uterus didelphys and a totally occluded left tube, and hysterectomy was performed. 5 longitudinal sections of each uterine horn was examined for both progesterone and estrogen endometrial nuclear and cytoplasmic hormone receptors. Cytoplasmic receptor values were in the expected normal range, and in general descended from the fundus to the cervix as in normal uteri. Nuclear receptor values were also in the expected range for secretory nuclei and their distrubtion was as seen in normal uteri. Both receptors (estrogen + progesterone; cytoplasmic + nuclear) however, were higher in the right horn vs. the left horn; this difference is ascribed to the woman's earlier pregnancy.