Phenytoin (DPH) is known to affect bone formation. However, the mechanism of this effect has not been well understood. In this study, we evaluated the effects of DPH on cartilage formation in a model system using ATDC5 cells, a clonal murine chondrogenic cell line. Alcian blue staining for cartilage nodules and real-time reverse-transcription polymerase chain reaction for the expression of genes encoding type II collagen, aggrecan, transforming growth factor (TGF)-β1, bone morphogenetic protein (BMP)-4, parathyroid hormone-related peptide (PTHrP), indian hedgehog (Ihh), and patched (Ptc) were performed in ATDC5 cells cultured with DPH. The ATDC5 cells demonstrated enhanced cartilage formation in cultures with DPH. During promoted chondrogenic differentiation, it was observed that DPH increased the mRNA expression of TGF-β1, BMP-4, Ihh, and Ptc, in a dose-dependent manner on Days 5 to 15. In contrast, other antiepileptic drugs, phenobarbital and valproic acid had no effects on chondrogenesis in ATDC5 cells and osteogenesis in MC3T3-E1 cells. Our results provide fundamental evidence that DPH has a direct stimulatory effect on cartilage formation by regulating TGF-β and hedgehog signaling molecules, and that DPH effect on bone formation, including chondrogenesis and osteogenesis, is distinct from other antiepileptic drugs as suggested in clinical settings.

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We investigated the effects of a protein kinase C inhibitor and a tyrosine kinase inhibitor on the cellular injury induced by cephaloridine in an established renal epithelial cell line, LLC-PK₁. Cephaloridine increased the leakage of lactate dehydrogenase (LDH) from LLC-PK₁ cells into the medium and also caused an increase in the level of lipid peroxide (index of oxidative stress) in the cells. Treatment of the cells with a hydroxyl radical scavenger, dimethylthiourea (DMTU), inhibited the increases in LDH leakage and lipid peroxidation in LLC-PK₁ cells exposed to cephaloridine. A protein kinase C inhibitor, H-7, and tyrosine kinase inhibitors, genistein and lavendustinA, inhibited the increases in LDH leakage and lipid peroxidation in LLC-PK₁ cells exposed to cephaloridine. These results suggest that a signaling pathway which involves protein kinase C and tyrosine kinase plays a role in the generation of reactive oxygen species in LLC-PK₁ cells damaged by cephaloridine.

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Indole-3-acetic acid (IAA), a natural auxin, induces microencephaly in rats exposed to IAA during gestation days (Days) 12−14, corresponding to the early stage of cerebral cortex development. The purpose of this study was to examine the effects of 5 IAA derivatives administration in pregnant rats on neuroepithelial cells in the embryos. N-Methylindole-3-acetic acid (1Me-IAA), 2-Methylindole-3-acetic acid (2Me-IAA), 2-Methyl-5-methoxyindole-3-acetic acid (2Me-5MeO-IAA), 5-Methoxyindole-3-acetic acid (5MeO-IAA), Indole butyric acid (IBA), and IAA were administered at 1,000 mg/kg except for 2Me-IAA at 500 mg/kg on Days 12, 13 and 14, and then embryos/fetuses were harvested on Day 14.5 or 21. The dams in the 1Me-IAA and 2Me-IAA groups exhibited rigidity and a decrease in locomotor activity. Although a decrease in the absolute brain weight was observed in the 1Me-IAA, 5MeO-IAA, IBA and IAA groups, a decrease in the relative brain weight was observed in only the IAA group. Histopathologically, apoptotic cells were observed mainly in the medial and dorsal layer of the neuroepithelium in the 5MeO-IAA and IAA groups on Day 14.5. The degree of induced neuroepithelial cell apoptosis was less in the 5MeO-IAA group than in the IAA group. However, it was confirmed that the histopathological changes induced by 5MeO-IAA were quite similar to the lesions induced by IAA and may have resulted from the same mechanisms.

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Influence of di-(2-ethylhexyl)phthalate (DEHP) on testicular development was studied by oral administration of DEHP at doses of 500 and 1000 mg/kg/day to pregnant rats on gestational days (G) 7 to 18. Ethinyl estradiol (EE) at dose levels of 0.25 and 0.5 mg/kg/day was used as a reference substance. Each 5-6 pregnant rats were sacrificed and their fetuses were examined on G12, 14, 16, 18 and 20. Fetal deaths averaging 20-36% were observed at every examination in the group receiving 1000 mg/kg of DEHP. Increases of fetal deaths over 50% were also observed in the reference group that received 0.5 mg/kg of EE. Microscopic examination of the fetal testis in groups treated with DEHP revealed degeneration of germ cells in G16 fetuses and localized proliferation or hyperplasia of interstitial cells in G18 and 20 fetuses. Germ cells having more than two nuclei were observed in a few cases including the control testes of G14 fetuses. These multinucleated cells were observed frequently in G20 fetuses treated with DEHP. Examination of testes of naturally delivered offspring of dams treated with 1000 mg/kg of DEHP at 7 weeks of age revealed scattered atrophy or dilatation of seminiferous tubules.
Another experiment was carried out to confirm the dose of DEHP affecting testicular development and spermatogenesis. DEHP was given to pregnant rats at doses of 125, 250 and 500 mg/kg/day during G7-18. Similar histopathological changes were observed in fetal testes of the group exposed to 500 and 250 mg/kg of DEHP, but not in those exposed to 125 mg/kg. In postnatal examinations, however, no abnormality was found in the testes at 5 and 10 weeks after birth in any of the treated groups. Furthermore, no abnormal findings were observed in the function of sperm, sperm counts and sperm morphology in the offspring of the group treated with DEHP during the fetal period at 10 weeks of age. Thus, 125 mg/kg/day is considered the no-observed-effect-level of DEHP on testicular development of rats by exposure in utero during the period of organogenesis.

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Predictive biomarkers of testicular toxicity are needed for an efficient development of drugs. The purpose of the present study was to obtain further insight into the toxicity mechanisms of various male reproductive toxicants and to detect genomic biomarkers for rapid screening of testicular toxicity. Four reproductive toxicants, 2,5-hexanedione (Sertoli cells toxicant), ethylene glycol monomethyl ether (EGME; spermatocytes toxicant), cyclophosphamide (spermatogonia toxicant) and sulfasalazine, were orally administered to male rats once. Six hours after the single dosing, gene expression in the testes was monitored by cDNA microarray and real-time RT-PCR and the testes were histopathologically examined. No histopathological abnormality was detected except for slight degeneration of spermatocytes in the EGME-treated testes. cDNA microarray analysis revealed differential gene expression profiles, and it was possible based on the profiles to characterize the action of the compounds in the testes. Interestingly, 3 spermatogenesis-related genes - heat shock protein 70-2, insulin growth factor binding protein 3 and glutathione S transferase pi - were affected by all the compounds. The above changes of gene expression were detectable within a short period after the dosing prior to the appearance of obvious pathological changes. These data suggest that cDNA microarray is a useful technique for evaluation of primary testicular toxicity. Furthermore, we propose the above 3 spermatogenesis-related genes as potential biomarkers of testicular toxicity.

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Previous studies revealed that atropine reduced male fertility in rats without any effects on mating performance, sperm production and motility, and testicular morphology. The present study was conducted to investigate whether the impairment of male fertility induced by atropine was related to the inhibition of sperm and semen transports from the vas deferens and seminal vesicle to the urethra during the process of emission. Male rats were treated with atropine at 125 mg/kg/day for 10-17 days prior to mating with untreated females. After confirmation of mating, male rats were euthanized and sperm number in the vas deferens and weights of the seminal vesicle and copulatory plug were determined as indicators of inhibition of sperm and semen transports, respectively. Reproductive status of mated females was determined on gestation days 15-17. A low pregnancy rate associated with a decreased number of implants was observed in females that mated with the atropine-treated males. The average number of sperm in the vas deferens was increased in the atropine-treated males. The average seminal vesicle weight in the atropine-treated males was greater than that of controls. The copulatory plug weights were decreased in the atropine-treated males. These results suggest that inhibitions of sperm and semen transports from the vas deferens and seminal vesicle to the urethra during the process of emission result in reduced male fertility in rats.

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We investigated the effects of three hepatotoxicants, acetaminophen (APAP), amiodarone (AD) and tetracycline (TC), on protein expression in primary cultured rat hepatocytes with toxicoproteomic approach, which is two-dimensional gel electrophoresis (2DE) and mass spectrometry. The objectives of this study were to search for alternative toxicity biomarkers which could be detected with high sensitivity prior to the appearance of morphological changes or alterations of analytical conventional biomarkers. The related proteins in the process of cell degeneration/necrosis such as cell death, lipid metabolism and lipid/carbohydrate metabolism were mainly affected under exposure to APAP, AD and TC, respectively. Among the differentially expressed proteins, several oxidative stress-related proteins were clearly identified after 24-hr exposure, even though they were not affected for 6-hr exposure. They were glutathione peroxidase (GPX) as a down-regulated protein as well as peroxiredoxin 1 (PRX1) and peroxiredoxin 2 (PRX2) as up-regulated proteins, which are known to serve as antioxidative enzymes in cells. These findings suggested that the focused proteins, GPX and PRXs, could be utilized as biomarkers of hepatotoxicity, and they were useful for setting high throughput screening methods to assess hepatotoxicity in the early stage of drug discovery.

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The effects of drugs on the QT interval should be evaluated precisely in the early stages of drug development because QT prolongation can trigger the so-called torsades de pointes, a life-threatening polymorphic ventricular tachycardia. It has been reported that the QT interval is affected by autonomic nervous tone besides the heart rate. In this study, we investigated the direct effect of autonomic nervous tone on the QT interval using the parameters of heart rate variability in dogs, when the RR interval was constant (400 or 700 msec). Our results showed that the QT interval at the high HF (high vagal tone) or low LF/HF ratio (low sympathetic tone) was longer than that at the low HF (low vagal tone) or high LF/HF ratio (high sympathetic tone), when the RR intervals were constant, and that the effect of vagal tone on the QT interval might be somewhat stronger than that of the sympathetic tone. The present observations would support the idea that sympathetic as well as parasympathetic tone regulates QT interval and that QT interval may be controlled physiologically by myocardial autonomic nerves via and not via a sinus node. Therefore, a more precise correction formula of QT interval could be established using autonomic parameters other than RR interval (heart rate), while the QT interval is widely known to be dependent on the RR interval or heart rate.

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The purpose of this study was to evaluate a telemetry system for examining QT evaluation in the conscious free-moving guinea pig using 10 reference compounds whose effects on human QT interval are well established: 8 positive references (bepridil, terfenadine, cisapride, haloperidol, pimozide, quinidine, E-4031 and thioridazine), and 2 negative references (propranolol and nifedipine). Pharmacokinetic experiments were also performed for the 8 positive references. Telemetry transmitters were implanted subcutaneously in male Hartley guinea pigs, and the RR and QT intervals were measured. All 8 positive references prolonged QTc (QTc = k × QT/RR^1/2) 10% or more during the 60 min observation period. When the values of the QTc changes were plotted against the serum concentrations, the resulting curves exhibited an anticlockwise hysteresis loop for all 8 references. In guinea pigs treated with haloperidol, changes of the T-wave shape from positive to flat were observed. The 2 negative references did not prolong the QTc. These findings suggest that the present telemetry guinea pig model is useful for QT evaluation in the early stages of drug development, because of the small body size of guinea pigs and their action potential configuration, which is similar to that of humans.

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Flutamide is a drug with antiandrogen effects that are mediated through androgen receptors (ARs). In this study, flutamide was subcutaneously administered to female rats (3, 10 or 30 mg/kg/day) on gestation Days 16-21 to evaluate effects on memory and learning performance in F1 offspring. Brain sexual differentiation was also evaluated by measuring the volume of the sexual dimorphic nucleus of the preoptic area (SDN-POA) and analyzing levels of androgen receptor (AR) mRNA expression in the prostate, hypothalamus and hippocampus. In F1 offspring exposed in utero to flutamide, evaluation of motor activity, learning performance and spatial perception showed that flutamide tended to exert a dose-dependent increase on the motor activity in F1 males, but no significant differences were identified in the other measurements. Prominent changes in development of the SDN-POA were apparent in males after maturation. Doses of ≥3 mg/kg/day resulted in significantly decreased length and volume of the SDN-POA compared to controls. These differences tended to become more marked at higher doses. Volumes of the SDN-POA did not differ significantly between F1 males and females exposed to flutamide at 30 mg/kg/day. AR mRNA was assayed using the dot-blotting method in F1 animals. In flutamide dose groups, AR mRNA expression tended to be increased in the prostate gland and decreased in the hippocampus. These results might suggest that exposure to flutamide in utero might affect controlling AR expression on a hormonal signal transduction system mediated by testosterone. However, these changes were not clearly correlated to learning performance in male offspring other than motor activity.

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A simplified gravimetric marrow cell counting method for rats is proposed for a regular screening method. After fresh bone marrow was aspirated by an injection needle, the marrow cells were suspended in carbonate buffered saline. The nucleated marrow cell count (NMC) was measured by an automated multi-blood cell analyzer. When this gravimetric method was applied to rats, the NMC of the left and right femurs had essentially identical values due to careful handling. The NMC at 4 to 10 weeks of age in male and female Crj:CD(SD)IGS rats was 2.72 to 1.96 and 2.75 to 1.98 (×10⁶ counts/mg), respectively. More useful information for evaluation could be obtained by using this gravimetric method in addition to myelogram examination. However, some difficulties with this method include low NMC due to blood contamination and variation of NMC due to handling. Therefore, the utility of this gravimetric method for screening will be clarified by the accumulation of the data on myelotoxicity studies with this method.

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