Possible mechanisms for testicular focal necrosis induced by human chorionic gonadotropin (hCG) were examined in Fischer 344 rats. A single s.c. injection of 2000 IU/kg hCG produced focal necrosis 2 days later in testicular tissues such as the seminiferous tubules in the frontal lower part of the testis (FLPT) of 11-week-old F344/Jcl rats. This hCG-induced necrosis was suppressed by an oral treatment (concomitant or delayed by 3 hr) with cyclooxygenase inhibitors (indomethacin, rofecoxib) or prostaglandin (PG) receptor blocker (AA-2414). Focal necrosis was also induced by intratesticular injection of PGF2α or PGE2 with this necrosis suppressed by previous oral treatment with AA-2414, and the PGF2 level in the testis increased 4 hr after hCG treatment. These findings suggested that de novo synthesis of PGs beginning at 3-4 hr was responsible for induction of necrosis. No necrosis was induced by hCG in the Leydig cell-devoid testis produced by ethane dimethanesulfonate treatment. Necrosis of spontaneously-induced Leydig cell tumor mass was also induced by hCG, suggesting that Leydig cells are responsible for induction of necrosis. An injection of dye into the testicular artery and laser Doppler flowmetry revealed a continuous reduction of blood flow at the FLPT at 6-48 hr after hCG treatment; contrary to this, the upper part showed an early recovery from the reduced flow. From these results, the mechanism of the hCG-induced necrosis was concluded to be: 1) hCG stimulates Leydig cells to synthesize PGs de novo; 2) PGs induce the intratesticular arteries to contract in the FLPT; and 3) obstruction of blood flow (ischemia) for more than 12 hr induced focal necrosis in the testis.
This study describes the detection of high amount of 7-O-acyl-derivative dinophysistoxin-1 (Dinophysistoxin-3) in filter bivalves collected on February 2005 in the Seno de Reloncav?, Puerto Montt City, Southern Chile, in the same period of time where an intoxication episode was associated with the presence of Vibrio parahaemolyticus in shellfish. The Diarrhetic Shellfish Poisoning (DSP) mouse bioassay of mussel extract samples, performed as described for regulatory testing, were negative to DSP toxins. Therefore, the same mussel samples collected from 8 places of Seno de Reloncav? were then analyzed by the HPLC-FLD method with pre-column derivatization procedure for DSP toxins. The samples showed mainly 7-O-acyl-derivative dinophysistoxin-1 (Dinophysistoxin-3) in concentrations ranging from 190.3 ± 6.8 to 311.1 ± 4.8 ng of DSP toxin/g hepatopancreas and less amounts of Dinophysistoxin-1 ranging from 1.9 ± 1.5 to 11.7 ± 4.6 ng of DSP toxin/g hepatopancreas. After alkaline hydrolysis of the mussel extracts, 279.4 ± 7.2 ng of Dinophysistoxin-1 /g hepatopancreas (mean ± SEM, N=6) were found in mussel extracts (Zone 8). These data showed that these shellfish samples are contaminated with the ester form 7-O-acyl-derivatives of Dinophysisyoxin-1, far beyond the safe regulatory limit. This paper also shows a direct relation between lipid content in the mussel tissue extracts and the levels of Dinophysistoxin-3. The 7-O-acyl-derivative dinophysistoxin-1 ester was the only compound associated with DSP toxins detected in the shellfish samples, and in view of the fact that metabolic transformation of Dinophysistoxin-3 into Dinophysistoxin-1 in humans has recently been described in the literature, the consumption of shellfish contaminated with 7-O-acyl-derivatives dinophysistoxin-1 could be a major reason that explains the diarrhetic symptoms shown by the intoxicated patients.
Some of the principal requisites of toxicity screening methods in drug discovery are their ease to perform and high throughput, as well as the possibility to predict the occurrence of clinical events. Phospholipidosis is one of the toxicities often induced by potential drugs. Several physicochemical methods for the prediction of phospholipidosis have been reported. The purpose of the present study was to examine the predictability of methods based on lipophilicity and charge parameters. We employed a test set of 33 compounds including 11 in-house compounds. The phospholipidosis-inducing potential (PLIP) of the test set compounds was determined by the fluorescence-labeled lipid accumulation assay using isolated rat hepatocytes. This assay was verified by transmission electron microscopy (EM). The usefulness of the ClogP - most basic pKa (pKa -MB) plot to the PLIP of compounds was examined. This plot was unable to predict the PLIP of zwitterions. In order to improve its predictability, the net charge of a given molecule (NC) was introduced instead of pKa - MB, since the NC corresponds directly to the ionization state of compounds in the organelles. Compounds with high ClogP (> 1) and high NC (1≤NC≤2) tended to be positive. This finding was also confirmed using 30 additional validation set compounds obtained from the literature. The ClogP - NC plot differentiated positive and negative compounds with more than 98% accuracy (62/63), indicating its usefulness in drug discovery.
To investigate the contribution of intestinal calcium (Ca) absorption to 1,25-dihydroxyvitamin D3 (1,25(OH) 2D3)-induced Ca action, we assessed parameters related to Ca metabolism after a single dosing of 1,25(OH)2D 3 in the total parenteral nutrition (TPN) solution or 5% D-mannitol (MAN) solution treatment with rats. Animals were divided into 6 groups (vehicle, 100 μg/kg p.o. and 25 μg/kg i.v.; n=8) in Experiment 1 and 8 groups (vehicle, 1, 10 and 100 μg/kg p.o.; n=6) in Experiment 2 at TPN or MAN solution treatment. In both experiments, the parameters related to Ca metabolisms, urinary Ca and urinary deoxypyridinoline on 0-24 hr or serum Ca, osteocalcin and parathyroid hormone at 24 hr were measured after 1,25(OH)2D 3 dosing. 1,25(OH)2D3-related increased urinary Ca or serum Ca were observed in both experiments. Decrease rates in change of urinary Ca in TPN solution treatment rats were 36.3% (100 μg/kg p.o.) or 47.1% (25 μg/kg i.v.) of MAN solution treatment rats in Experiment 1, and 29.0% (1 μg/kg), 56.2% (10 μg/kg) or 35.3% (100 μg/kg) of MAN solution treatment rats in Experiment 2. Decrease rates in change of serum Ca at 72 hr in TPN solution treatment rats were 57.3% (100 μg/kg p.o.) or 44.5% (25 μg/kg i.v.) of MAN solution treatment rats in Experiment 1, and were 57.0% (100 μg/kg) of MAN solution treatment rats in Experiment 2. There were no differences in the change of serum Ca in the 1,25(OH)2D3 1 or 10-μg/kg group in Experiment 2. Our results suggest that differences in urinary Ca or serum Ca between MAN solution treatment rats and TPN solution treatment rats express the contribution of intestinal Ca absorption to 1,25(OH)2D3-induced Ca action in the conditions of the study.
Although paraquat (PQ) is known to induce pulmonary fibrosis, how it does so is not entirely clear. To elucidate the mechanisms involved, the profile of gene expression in the lung at three months after exposure to PQ (7 mg/kg, s.c., daily for eight administrations) was investigated in rats using a DNA microarray. Changes in gene expression that were considered to reflect damage to the lung, a change in the balance of electrolytes and fluid, and alveolar remodeling were observed. The products of these genes were: CSF-1 receptor, which is a receptor of inflammatory cytokines that activates monocyte/macrophages; TGF-beta type II receptor, which is a receptor of TGF-betas involved in wound healing and fibrosis; a subunit of Na+/K+-ATPase, an amiloride-sensitive cation channel, and a subunit of the potassium channel, all of which regulate the alveolar fluid balance and play a role in clearing lung edema; the adenosine A2a receptor, which has a protective function in the lung and interacts with dopamine D1 and D2 receptors to regulate the function of amiloride-sensitive cation channels; cofilin, which is involved in the depolymerization and cleavage of actin filaments; LIM motif-containing protein kinase 1, which negatively regulates the activity of cofilin; SHPS-1, which regulates the integrin-mediated reorganization of the cytoskeleton; and sodium channel beta 2, which is involved in cell adhesion and migration. These results indicate that PQ-induced pulmonary fibrosis does not merely terminate as cicatrices three months after the discontinuation of PQ treatment, but that dynamic functional change continues in the lung.
Chronic toxicity and carcinogenicity of hinokitiol (beta-thujaplicin), used as an antibiotic and fungicidal agent of a food additive, was examined in both sexes of F344/DuCrj (F344) rats. In this chronic toxicity study, groups of 10 rats of each sex were given a diet containing hinokitiol at doses of 0, 0.005, 0.015 and 0.05% for 52 weeks. No treatment-related adverse effects were noted in the survival rate, general condition, body weights, food consumption, urinalysis, hematology and clinical chemistry. Slight but significant elevation of spleen and liver weights was noted in both sexes given 0.05% hinokitiol, along with an increase in hemosiderin deposits in male spleens, related to chelator binding of iron, together with slight centrilobular hypertrophy of male hepatocytes. However, these alterations were negligible and not toxicologically significant. In the carcinogenicity study, groups of 50 female and 50 male rats were given a diet containing hinokitiol at doses of 0, 0.005, 0.015 and 0.05% (excluding 0.005% in females). No treatment-related changes in survival rate, general condition, body weight, food consumption, hematology and organ weights were noted. Detailed histopathological examination revealed no treatment-related increase in the incidences of any neoplastic lesions. The results demonstrate that hinokitiol is not carcinogenic in F344 rats of either sex.
An in vitro crystal violet staining method using the rabbit cornea-derived cell line (SIRC-CVS) has been developed as an alternative to predict acute systemic toxicity in rodents. Seventy-nine chemicals, the in vitro cytotoxicity of which was already reported by the Multicenter Evaluation of In vitro Toxicity (MEIC) and ICCVAM/ECVAM, were selected as test compounds. The cells were incubated with the chemicals for 72 hrs and the IC50 and IC35 values (μg/mL) were obtained. The results were compared to the in vivo (rat or mouse) "most toxic" oral, intraperitoneal, subcutaneous and intravenous LD50 values (mg/kg) taken from the RTECS database for each of the chemicals by using Pearson's correlation statistics. The following parameters were calculated: accuracy, sensitivity, specificity, prevalence, positive predictability, and negative predictability. Good linear correlations (Pearson's coefficient; r>0.6) were observed between either the IC50 or the IC35 values and all the LD50 values. Among them, a statistically significant high correlation (r=0.8102, p<0.001) required for acute systemic toxicity prediction was obtained between the IC50 values and the oral LD50 values. By using the cut-off concentrations of 2,000 mg/kg (LD50) and 4,225 μg/mL (IC50), no false negatives were observed, and the accuracy was 84.8%. From this, it is concluded that this method could be used to predict the acute systemic toxicity potential of chemicals in rodents.
Several QT correction (QTc) formulas have been used for assessing the QT liability of drugs. However, they are known to under- and over-correct the QT interval and tend to be specific to species and experimental conditions. The purpose of this study was to determine a suitable formula for halothane-anesthetized dogs highly sensitive to drug-induced QT interval prolongation. Twenty dogs were anesthetized with 1.5% halothane and the relationship between the QT and RR intervals were obtained by changing the heart rate under atrial pacing conditions. The QT interval was corrected for the RR interval by applying 4 published formulas (Bazett, Fridericia, Van de Water, and Matsunaga); Fridericia's formula (QTcF = QT/RR0.33) showed the least slope and lowest R2 value for the linear regression of QTc intervals against RR intervals, indicating that it dissociated changes in heart rate most effectively. An optimized formula (QTcX = QT/RR0.3879) is defined by analysis of covariance and represents a correction algorithm superior to Fridericia's formula. For both Fridericia's and the optimized formula, QT-prolonging drugs (d,l-sotalol, astemizole) showed QTc interval prolongation. A non-QT-prolonging drug (d,l-propranolol) failed to prolong the QTc interval. In addition, drug-induced changes in QTcF and QTcX intervals were highly correlated with those of the QT interval paced at a cycle length of 500 msec. These findings suggest that Fridericia's and the optimized formula, although the optimized is a little bit better, are suitable for correcting the QT interval in halothane-anesthetized dogs and help to evaluate the potential QT prolongation of drugs with high accuracy.
Hepatic stellate (HS) cells were isolated from the livers of metallothionein (MT)-null and control mice and used to establish IMS/MT(-) and IMS/N cell lines, respectively, using SV40 virus transformation. Cellular morphology, incorporation of vitamin A and expression of α-SMA, desmin and SV40 T-antigen were used to confirm that both cell lines were immortal HS cells. The growth rates of both cell lines were similar and there was little difference between cell line sensitivity to zinc. MT-null IMS/MT(-) cells were more sensitive to cadmium and mercury, although both cell lines accumulated almost equal amounts of cadmium during a 24-hr culture period. As HS cells play an important role in hepatic fibrosis and are activated by heavy metals such as cadmium or reactive oxygen, the MT-null HS cell line derived in this study should be a useful experimental model for examination of the role of MT in HS cell activation.
The potential genotoxicity of the rodent liver carcinogen p-dimethylaminoazobenzene (DAB) was evaluated in compliance with the guidelines for genotoxicity studies of drugs (Notification No. 1604, Nov. 1, 1999, Ministry of Health and Welfare, Japan) and the OECD guidelines for testing chemicals. DAB was clearly positive in both the bacterial reverse mutation test (Ames test) and in vitro chromosomal aberration test in the presence of metabolic activation, whereas it was weakly positive at toxic doses in the rat bone marrow micronucleus test. It has been reported that DAB was clearly positive in in vivo genotoxicity tests, i.e., a mouse alkaline single cell gel electrophoresis (comet) assay and a young rat liver micronucleus test. These results suggest that the test system using the liver is effective for in vivo genotoxicity assessment of chemicals that show mutagenicity in in vitro genotoxicity tests in the presence of metabolic activation.