Lengthening of QTc is the usual signal to indicate torsadogenic potential of a therapeutic agent. The ICH S7B guideline recommends that new chemical entities should be assessed for potential of delayed ventricular repolarization in animal models. The aim of this study was to determine a feasibility of using isolated failing heart rabbit to assess the QT-lengthening drugs in comparison with their effects on isolated normal heart rabbits. Heart failure was induced by ligation of the left anterior descending and descending branch of left circumflex coronary arteries. One month after ligation, all rabbits were anesthetized and the hearts were removed quickly, and they were perfused with the oxygenated Krebs-Henseleit solution to which escalating concentrations of QT-lengthening compounds were added. RR, QT, and QTc(F) were not significantly different, at rest, between failing and normal hearts. During baseline, dP/dtmax was lower and dP/dtmin was higher for failing hearts than for normals. In responses to all three QT-lengthening compounds, RR, QT and QTc(F) lengthened similarly in a dose-response manner in both the failing and normal hearts. Neither the failing nor the normal hearts developed fatal arrhythmias, torsades de pointes. Langendorff preparations of failing hearts are as good as normal isolated hearts and can be use to assess the potential of delayed ventricular repolarization of test articles.
Three female Crl:CD(SD) rats/group were dosed with single wall carbon nanotube (SWCNT) or multi wall carbon nanotube (MWCNT) four times by gavage at a total of 50 mg/kg bw or 200 mg/kg bw (four equally divided doses at one-hour intervals). Acute oral doses of SWCNT and MWCNT caused neither death nor toxicological effects, and thus the oral LD50 values for SWCNT and MWCNT were considered to be greater than 50 mg/kg bw and 200 mg/kg bw, in rats respectively. Five or ten Crl:CD(SD) rats/sex were dosed with SWCNT once daily by gavage at a dose of 0 (control), 0.125, 1.25 or 12.5 mg/kg bw/day for 28 days with a 14-day recovery period (0 and 12.5 mg/kg bw/day groups). Six or twelve Crl:CD(SD) rats/sex were dosed with MWCNT once daily by gavage at a dose of 0 (control), 0.5, 5.0 or 50 mg/kg bw/day for 28 days with a 14-day recovery period (0 and 50 mg/kg bw/day groups). Based on no toxicological effects, the no observed adverse effect levels (NOAELs) of repeated dose toxicity of SWCNT and MWCNT were considered to be 12.5 mg/kg bw/day and 50 mg/kg bw/day (the highest dose tested), respectively. It was suggested that SWCNT and MWCNT dosed by gavage reached the gastro-intestinal tract as agglomerates and were mostly excreted via feces.
Cochineal extracts (CE) is a coccid-derived natural food colorant containing carminic acid (CA) as an active ingredient that potentiates inhibition of tissue proteolysis mediated by activation of plasma hyaluronan-binding protein (PHBP). In our previous study, dietary administered CE (CA: 28.5% in CE) has shown to promote the macroscopic development of capsular invasive carcinomas (CICs) associated with up-regulation of angiogenesis-related genes in an intracapsular invasion model of experimental thyroid cancers using rats. However, the promoting effect of CE could not be confirmed histopathologically. The purpose of the present study was to confirm the promoting effect of CE through direct injections to animals on the development of CICs using this cancer invasion model. One week after initiation with N-bis(hydroxypropyl)nitrosamine, male F344/NSlc rats were administered CA-enriched CE (CA: 52.6% in CE) by intraperitoneal injections every other day (10 mg/kg body weight) during the promotion with 0.15% sulfadimethoxine in the drinking water for 8 weeks. The multiplicities of macroscopical CICs and the mean area of early capsular invasive foci estimated by Tenascin (TN)-C-immunoreactivity in the thyroid significantly increased with CE-treatment, while the number of TN-C-positive foci did not change with CE. Transcript level of Phbp and downstream genes unchanged; however, transcript level of angiogenesis-related genes, i.e, Vegfb and its transcription factor gene, Hif1a, those being downstream of phosphatase and tensin homolog (PTEN)/Akt signaling, up-regulated in the thyroid tissue with CE-administration. These results suggest that CE potentiates promotion activity by facilitating angiogenesis through activation of PTEN/Akt signaling without accompanying modification of PHBP-related proteolysis.
Mono-(2-ethylhexyl) phthalate (MEHP) is the most toxic metabolite of di-(2-ethylhexyl) phthalate (DEHP). It has been reported that DEHP causes abnormal reproductive development in women, and suppresses estradiol synthesis and ovulation in female rats with diminished size of preovulatory follicles. The present study was conducted to evaluate the ovarian toxicity of MEHP using cultured rat ovarian follicles. Secondary follicles were isolated from the ovaries of 14-day-old female rats and cultured for 48 hr with MEHP (0, 10, 30, and 100 µg/ml). At 0, 24, and 48 hr of MEHP treatment, follicular diameters were measured. After the culture, viability and apoptosis of follicles were assessed, and progesterone, androstenedione, testosterone, and estradiol levels in culture media were measured. At 100 µg/ml, suppression of follicular development was observed, which is associated with decreased viability of follicles and apoptosis of granulosa cells. At this concentration, progesterone level increased markedly, whereas androstenedione, testosterone, and estradiol levels decreased. At 10 and 30 µg/ml, follicular development was not suppressed, no apoptotic change was observed, and the levels of all measured steroid hormones tended to increase. The combined levels of all steroid hormones increased at all concentrations of MEHP, and the increase implies that MEHP activates the synthetic pathway from cholesterol to estradiol including de novo synthesis of cholesterol. However, the progesterone/androstenedione ratio increased extremely at 100 µg/ml, and the increase implies that MEHP inhibits the conversion of progesterone to androstenedione. In conclusion, MEHP induces ovarian toxicity via suppression of follicular development and abnormal steroid hormone synthesis in cultured rat ovarian follicles.
Omeprazole (OPZ), a proton pump inhibitor, is a cytochrome P450 (CYP) 1A1/2 inducer. Some CYP1A inducers are known to have liver tumor promoting effects in rats and the ability to enhance oxidative stress. In this study, we performed a two-stage liver carcinogenesis bioassay in rats to examine the tumor promoting effect of OPZ (Experiment 1) and to clarify a possible mechanism of action (Experiment 2). In Experiment 1, male F344 rats were subjected to a two-third partial hepatectomy, and treated with 0, 138 or 276 mg/kg OPZ by oral gavage once a day for six weeks after an intraperitoneal injection of N-diethylnitrosamine (DEN). Liver weights significantly increased in the DEN+OPZ groups, and the number and area of glutathione S-transferase placental form (GST-P) positive foci significantly increased in the DEN+276 mg/kg OPZ group. In Experiment 2, the same experiment as Experiment 1 was performed, but the dosage of OPZ was 0 or 276 mg/kg. The number and area of GST-P positive foci as well as liver weights significantly increased in the DEN+276 mg/kg OPZ group. The number of proliferative cell nuclear antigen (PCNA)-positive cells also significantly increased in the same group. Real-time RT-PCR showed that the expression of AhR battery genes including Cyp1a1, Cyp1a2, Ugt1a6 and Nqo1, and Nrf2 battery genes including Gpx2,Yc2, Akr7a3, Aldh1a1 Me1 and Ggt1 were significantly upregulated in this group. However, the production of microsomal reactive oxygen species (ROS) and formation of thiobarbituric acid-reactive substances (TBARS) decreased, and 8-hydroxydeoxyguanosine (8-OHdG) content remained unchanged in this group. These results indicate that OPZ, CYP1A inducer, is a liver tumor promoter in rats, but oxidative stress is not involved in the liver tumor promoting effect of OPZ.
Categorizing chemicals is an approach with the potential to reduce animal testing for hazard assessment of chemicals. In this study we investigated the category approach for testing the hemolytic effects of ethylene glycol alkyl ethers (EGAEs) for repeated-dose toxicity (RDT). Using mechanistic information on the hemolytic effects of ethylene glycol butyl ether, a toxicologically meaningful category was built on the basis of similarity of metabolism, mode of action and the hemolytic effects of several EGAEs and related chemicals. The developed category was then evaluated for analogs from a different data source. Given all structural information on category chemicals, the category can be finally defined as EGAEs (alkyl chain carbon number: 1-4) and their acetates. Current RDT test data suggest that EGAEs with 3 and 4 alkyl carbons primarily cause hemolytic effects, while EGAEs with 1 and 2 alkyl carbon(s) show toxicity to the testis before demonstrating any hemolytic effects. Hence, the category approach appears to be applicable to hemolytic effects of EGAEs with 3 and 4 alkyl carbons and their acetates to estimate the no observable adverse effect level (NOAEL) for RDT. It consists of three steps: structure-based primary screening of untested chemicals, categorization of compounds that form hemolytic alkoxyacetic acids by predicting how they are metabolized, and finally estimation of hemolytic levels by employing read-across. Our results clearly demonstrate the usefulness of the category approach for predicting the hemolytic effects of untested EGAEs and their acetates in RDT.
To determine the threshold dose of β-Naphthoflavone (BNF) that induces hepatocellular tumor promoting effects, reactive oxygen species (ROS) generation and thiobarbituric acid-reactive substance (TBARS) formation, and drug-metabolizing enzymes that protect against ROS generation, two-stage liver carcinogenesis model was used. Partial hepatectomized rats (n = 11 to 12) were fed diets containing 0, 0.03, 0.06, 0.125 or 0.25% BNF for 6 weeks after an intraperitoneal injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Histopathologically, glutathione S-transferase placental form (GST-P)-positive foci significantly increased in rats given 0.25% BNF. No marked changes in ROS production and TBARS contents were observed between the BNF treated and DEN alone groups. Real-time RT-PCR showed that the expression of Cyp1a1, Cyp1a2, Cyp1b1 and Nqo1 significantly increased in the groups given 0.03% BNF or more, but Ugt1a6, Akr7a3 and Gstm1 significantly increased in the groups given 0.125% BNF or more. Gpx2 and Yc2 significantly increased in the groups given 0.06% BNF or more and 0.25% BNF, respectively. Inflammation-related genes such as Ccl2, Mmp12, Serpine1 and Cox-2 significantly increased in the 0.25% BNF group. In immunohistochemistry, the number of cyclooxygenase-2 (COX-2)-positive cells increased in rats given 0.25% BNF. These results suggest that 0.25% BNF is the threshold dose for liver tumor promotion, and the fact that inflammation-related genes and COX-2 protein increased in the 0.25% BNF group strongly suggests that inflammation is involved in the liver tumor promoting effect of BNF in rats.
The 26-week oral toxicity of diheptyl phthalate (DHP), a peroxisome proliferator-activated receptor α (PPARα) agonist, with special emphasis on the potential induction of hepatocellular proliferative lesions was investigated in this study. DHP was administered to male F344 rats via gavage at 0 (control), 1,000 or 2,000 mg/kg/day for 26 weeks. Body weight gain was significantly lower, whereas food and water consumption was significantly higher in DHP-treated rats compared with controls. DHP-treated rats exhibited decreases in blood triglyceride, total cholesterol, phospholipid and glucose levels, which were likely related to biological effects of the PPARα agonist. Absolute and relative organ weights of the livers with pale brown discoloration and dark brown spots significantly increased in DHP-treated rats. Histopathological examinations revealed remarkable diffuse hypertrophy of hepatocytes with ground-glass appearance, intracytoplasmic inclusion bodies and/or vacuolation in the DHP-treated groups. These findings were associated with increases in serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and γ-glutamyltranspeptidase. The number and area of glutathione S-transferase placental form positive foci, a marker of hepatocellular preneoplastic lesions in rats, significantly increased in DHP-treated groups. Additionally, proliferating cell nuclear antigen positive liver cell counts in DHP-treated groups were significantly higher than those of the controls. Testicular alterations were not detected histopathologically, whereas absolute and relative prostate weights significantly decreased at both doses. These results indicate that DHP induces liver pre-neoplastic foci, and suggest the possibility that DHP is a possible genotoxic carcinogen in the liver of rats.
Bisphenol A (BPA) is known to be an endocrine disruptor that affects the development of reproductive system. The aim of the present study was to investigate a group of testicular genes dysregulated by prenatal exposure to BPA. Pregnant ICR mice were treated with BPA by subcutaneous administration on days 7 and 14 of pregnancy. Tissue and blood samples were collected from 6-week-old male offspring. Testes were subjected to gene expression analysis using a testis-specific microarray (Testis2), consisting of 2,482 mouse cDNA clones annotated with Medical Subject Headings (MeSH) terms indicative of testicular components and functions. To interpret the microarray data, we used the MeSH terms significantly associated with the altered genes. As a result, MeSH terms related to androgens and Sertoli cells were extracted in BPA-treated groups. Among the genes related to Sertoli cells, downregulation of Msi1h, Ncoa1, Nid1, Hspb2, and Gata6 were detected in the testis of mice treated with BPA (twice administered 50 mg/kg). The MeSH terms associated with this group of genes may provide useful means to interpret the testicular toxicity of BPA. This article concludes that prenatal BPA exposure downregulates expression of genes associated with Sertoli cell function and affects the reproductive function of male offspring. Additionally, a method using MeSH to extract a group of genes was useful for predicting the testicular and reproductive toxicity of prenatal BPA exposure.
Whereas nausea and emesis are burdensome side effects that lead to poor treatment compliance especially in chemotherapy, it is difficult to predict the emetic potential of agents in rats and mice because rodents do not vomit. We examined the effect of emetics on gastric retention and role of serotonin (5-hydroxytryptamine, 5-HT)3 receptor in chemotherapeutic-induced enhancement of gastric retention in rats. The gastric retention of solid material was determined using resin beads, which were suitable to beads made with metals or glasses in size, hardness and weight. Each rat was orally given distilled water (0.5 ml/rat) containing 40 resin beads via a plastic feeding tube. The stomach was removed at 1 hr post-dose and cut along the greater curvature under carbon dioxide anesthesia. Beads were given immediately after administration of the drugs except with cisplatin, when there was a 1 hr delay. Cancer chemotherapeutics including cisplatin(0.1-3 mg/kg i.v.) and doxorubicin(0.3-10 mg/kg i.v.) and a nauseant, copper sulfate(1-30 mg/kg p.o.) enhanced gastric retention of beads. Ondansetron, a 5-HT3 receptor antagonist, dose-dependently antagonized the enhanced gastric retention by cisplatin and doxorubicin. The copper sulfate-induced enhancement was also reversed by ondansetron. Our results suggest that 5-HT3 receptors mediate the cancer chemotherapeutic-enhanced gastric retention of solid material in rats. This implicates that the gastric retention of solid material is a useful marker to predict the potential of compounds to induce nausea and/or emesis in non-vomiting rodents.
We previously found that genetic polymorphism in cytochrome P450 2A6 (CYP2A6) is one of the potential determinants of tobacco-related lung cancer risk. It has been reported that the plasma concentration of cotinine, a major metabolite of nicotine, in carriers of wild-type alleles of CYP2A6 is considerably higher than that in carriers of null or reduced-function alleles of CYP2A6, raising the possibility that cotinine plays an important role in the development of lung cancer. As a novel mechanism of lung tumorigenesis mediated by CYP2A6, we investigated the effects of cotinine on the suppression of apoptosis and promotion of lung tumor growth. In human lung adenocarcinoma A549 cells, cotinine inhibited doxorubicin-induced cell death by suppressing caspase-mediated apoptosis. Enhanced phosphorylation of Akt, a key factor responsible for cell survival and inhibition of apoptosis, was detected after cotinine treatment. These data suggest that cotinine suppresses caspase-mediated apoptosis induced by doxorubicin through activation of the PI3K/Akt pathway. Furthermore, we clarified that cotinine significantly facilitated tumor growth in the Lewis lung cancer model and accelerated development of lung adenomas induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mice. We herein propose that cotinine induces tumor promotion by inhibiting apoptosis and enhancing cellular proliferation, thus underlining the importance of CYP2A6 in tobacco-related lung tumorigenesis.
The potential for health effects on humans with exposure to bisphenol A (BPA) has raised concerns, and the adverse effects of low-dose exposure to BPA on reproduction have been controversial. The purpose of the present study was to investigate the effects of low-dose exposure to BPA on reproductive development in F1 rat offspring. Pregnant female Sprague-Dawley rats (F0) were fed a diet containing low doses of BPA (0, 0.33, 3.3, or 33 ppm) from gestational day (GD) 6 through postnatal day (PND) 21. The weanlings (F1) from all dose groups were fed a normal diet ad libitum after weaning and then were subjected to necropsy at 5 weeks or 3 months of age. No BPA-related changes were observed in body weight or weight of any of the major reproductive organs in F1 males and females. Epididymis weight was significantly lower only in 3-month-old F1 males exposed to 33 ppm BPA. Anogenital distance (AGD), the ratio of AGD to the cube root of body weight, and relative ovary weight were significantly lower in 5-week-old F1 females exposed to 3.3 and 33 ppm BPA, but significant differences were not observed in 3-month-old females. There were no BPA-related effects on cauda epididymal sperm motility in 3-month-old F1 males. Plasma reproductive steroid hormone concentrations were not altered among groups in either sex. These outcomes indicate that low-dose exposure to BPA in the diet does not adversely affect reproductive development in F1 rat offspring.
Dietary fiber, maintaining the gut environment, contributes to better lung function among smokers. This study was aimed to investigate the role of dietary fiber on the anti-oxidant capacity and gut environment during exposure to cigarette smoke. The anti-oxidant capacity as well as caecal levels of organic acids and population of micro-flora in the gut was measured after 4 months’ exposure to cigarette smoke in mice (C57BL/6NcrSlc) fed with a cellulose-free diet. Animals were divided into control diet (AIN-93G)/non-smoke, cellulose-free diet/non-smoke, control diet/smoke, and cellulose-free diet/smoke groups. The anti-oxidant capacity in plasma was significantly suppressed by the cellulose-free diet in the non-smoke exposed mice. The suppression in the anti-oxidant capacity further declined following exposure to cigarette smoke. Both these changes in the anti-oxidant capacity were accompanied with changes in some organic acids levels in caecal contents. The anti-oxidant activity was significantly inversely correlated to succinic acid / acetic acid levels balance in caecal contents. In conclusion the cellulose-free diet suppressed the anti-oxidant capacity in mice, and the suppression further decreased by exposure to cigarette smoke. These changes in the anti-oxidant capacity may be related with changes in the gut environment.
Sodium valproate (VPA) is a major antiepileptic drug that is widely used for the treatment of epilepsy as well as other neuropsychiatric diseases. The present study was conducted to evaluate the ovarian toxicity of VPA using cultured rat ovarian follicles. Secondary follicles were isolated from the ovaries of 14-day-old female rats and cultured for 48 hr with VPA (0, 0.2, 1.0, and 5.0 mM). At 0, 24, and 48 hr of VPA treatment, follicular diameters were measured. After the culture, viability of follicles and expression of aromatase in the follicles were assessed, and progesterone, androstenedione, testosterone, and estradiol levels in culture media were measured. At all concentrations of VPA, follicular development was suppressed, and androstenedione, testosterone, estradiol, and combined levels of all steroid hormones tended to decrease in association with suppression of aromatase expression in granulosa cells. Additionally, the suppression of follicular development was associated with decreased viability of follicles and an increased progesterone level at 5.0 mM of VPA. The decrease in the combined levels of all steroid hormones implies that VPA suppresses the synthetic pathway from cholesterol to estradiol including de novo synthesis of cholesterol. In conclusion, VPA induces ovarian toxicity via suppression of development and abnormal steroid hormone synthesis in cultured rat ovarian follicles.
We investigated the role of glutathione S-transferases Mu 1 (GSTM1) in acetaminophen (APAP)-induced hepatotoxicity using Gstm1-null mice. A single oral administration of APAP resulted in a marked increase in plasma alanine aminotransferase accompanied by hepatocyte necrosis 24 hr after administration in wild-type mice, but its magnitude was unexpectedly attenuated in Gstm1-null mice. Therefore, it is suggested that Gstm1-null mice are resistant to APAP-induced hepatotoxicity. To examine the mechanism of this resistance in Gstm1-null mice, we measured phosphorylation of c-jun N-terminal kinase (JNK), which mediates the signal of APAP-induced hepatocyte necrosis, by Western blot analysis 2 and 6 hr after APAP administration. A marked increase in phosphorylated JNK was observed in wild-type mice, but the increase was markedly suppressed in Gstm1-null mice. Therefore, it is suggested that suppressed phosphorylation of JNK may be a main mechanism of the resistance to APAP-induced hepatotoxicity in Gstm1-null mice, although other possibilities of the mechanism cannot be eliminated. Additionally, phosphorylation of glycogen synthase kinase-3β and mitogen-activated protein kinase kinase 4, which are upstream kinases of JNK in APAP-induced hepatotoxicity, were also suppressed in Gstm1-null mice. A decrease in liver total glutathione 2 hr after APAP administration, which is an indicator for exposure to N-acetyl-p-benzoquinoneimine, the reactive metabolite of APAP, were similar in wild-type and Gstm1-null mice. In conclusion, Gstm1-null mice are considered to be resistant to APAP-induced hepatotoxicity perhaps by the suppression of JNK phosphorylation. This study indicates the novel role of GSTM1 as a factor mediating the cellular signal for APAP-induced hepatotoxicity.
The placenta secures the embryo and fetus to the endometrium and releases a variety of steroid and peptide hormones that convert the physiology of a female to that of a pregnant female. Chemical-induced alteration or deviation of placental function in the maternal and extraembryonic tissue can ultimately lead to pregnancy loss, congenital malformation and fetal death. The 6-mercaptopurine (6-MP), an anti-leukemic drug, is known to produce undesired effects on some organs, then the placenta/embryo toxicity of 6-MP was investigated in pregnant rats given 60 mg/kg with two intraperitoneal injections on gestation days (GD) 11 and 12. The rats were sacrificed and their placentas were collected on GD13 or 15. On GD15 small and limb-defected embryos were found in the 6-MP-treated rats. Placental weights were significantly reduced on GD15, as well as a reduced number of cells was detected in the labyrinth zone with both the labyrinth and basal zones having thinned. Cleaved caspase-3-positive cells increased in number in the labyrinth zone, while in the basal zone, glycogen cells reduced with cytolysis. The number of spongiotrophoblasts and trophoblastic giant cells also increased by 6-MP treatment. The 6-MP-treatment resulted in the increased xanthine oxidase (Xdh) expression in the placenta, which gene is related to the ischemic condition of tissues. These data suggest that apoptosis of the labyrinth zone cells may lead to decreased materno-fetal exchange. Moreover, subsequent ischemia in the placental tissue may occur and induce Xdh expression.
Teriparatide, a therapeutic agent for osteoporosis, has been reported to increase the incidences of bone neoplasms such as osteosarcoma when administered subcutaneously to Fischer 344 (F344) rats for a long term, but its non-carcinogenic dose level following 2-year daily administration has not been established. Here we report detailed studies on the carcinogenicity of teriparatide following long-term administration. When teriparatide was administered subcutaneously to male and female Sprague-Dawley (SD) rats daily for 2 years, the incidence of osteosarcoma was increased at 13.6 μg/kg/day. The non-carcinogenic dose level was 4.5 μg/kg/day for both males and females. The development of osteosarcoma in SD rats depends on the dose level of, and treatment duration with, teriparatide. Responses of the bones to teriparatide were similar between F344 and SD rats in many aspects. These results suggested that the carcinogenic potential of teriparatide in SD rats is essentially the same as in F344 rats.
We investigated the age-related changes in reproductive function in female rats of the Crl:CD(SD) strain. The estrous cycles were monitored from 6 to 42 weeks of age. Other females were mated with males at 6, 8, 10, 15, 19, 23, 27, 31, 35 and 40 weeks of age and caesarean section was performed on day 20 of gestation to examine their fetuses, and then reproductive parameters were calculated and fetal growth was observed. The percentage of rats exhibiting irregular estrous cycles increased from 23 weeks of age. The copulatory and fertility indices decreased from 35 and 27 weeks of age, respectively. Dams at 6 weeks of age showed low numbers of corpora lutea and implantation sites. Although the number of corpora lutea was not affected by advanced maternal age, number of implantation sites decreased from 27 weeks of age. The pre- and post-implantation loss rates increased from 23 and 31 weeks of age, respectively, and the number of live fetuses decreased from 27 weeks of age. Fetal growth retardation was observed in maternal rats older than 31 weeks of age. The external observations on the fetuses revealed umbilical hernia at 35 to 40 weeks of age. These data indicated that maternal aging affected reproductive function and fetal development from 23 and 31 weeks of age, respectively. It was considered that the appropriate age of Crl:CD(SD) female rats for the assessment of reproductive toxicity were 8 to 22 weeks.
Thin, gel-like, hydrated soft contact lenses are directly placed on human eyes and can be used with a variety of eye drops containing over-the-counter drugs, even though some labels suggest caution. In this study, evaluation was carried out of the cytotoxic potential of soft contact lenses that had adsorbed the active ingredients of over-the-counter eye drops. Although there are some guidelines for in vitro cytotoxicity tests for preclinical biological evaluation of medical materials and devices, the shape of soft contact lenses makes these tests difficult to apply. We developed a new cytotoxicity test in which cored lenses 6 mm in diameter were pretreated with active ingredients and placed in direct contact with Chinese hamster lung V79 cells. Among the 11 chemicals tested, anionic and hydrated type-IV lenses that had been soaked in benzalkonium chloride, berberine chloride, and chlorhexidine indicated cytotoxic potential toward the cultured cells. Solutions of 0.002%-0.004% benzalkonium chloride, 0.025% berberine chloride, and 0.008% chlorhexidine were not cytotoxic to the cultured cells under the experimental conditions; however, type-IV lenses that had been immersed in the same concentrations of these active ingredients showed significant cytotoxicity to the cultured cells. These results suggest that cytotoxicity to the ocular surface may be caused by soft contact lenses that have been soaked in the usual ranges of nontoxic eye drops containing benzalkonium chloride, berberine chloride, and chlorhexidine. This modified in vitro safety test system for cored soft contact lenses is of use for evaluating the cytotoxic potential of ophthalmological drugs adsorbed on soft contact lens surfaces.
Spontaneous clonic convulsions in Sprague-Dawley (SD) rats are occasionally observed in chronic toxicity studies and carcinogenicity studies. To estimate the incidence and features of the spontaneous convulsions, data from 11 oral gavage carcinogenicity studies using Charles River SD rats, which were conducted at our laboratory between 2003 and 2010, were collected (N = 2990 for each sex). The total incidence of animals which showed spontaneous clonic convulsions at least once during the 2-year study period was 2.4% (2.9% in males and 1.9% in females). In those carcinogenicity studies, the earliest observation of convulsions was 25 weeks of age in males and 20 weeks of age in females, and the average age at the first occurrence of convulsions in all animals was approximately 66 weeks of age. Some animals showed convulsions only once, but others on several or numerous occasions. Most convulsions were observed during the oral gavage procedure, especially when holding the animals. Approximately 0.3% of animals died immediately after the seizure. No related histopathological abnormalities in the brain have been recorded for such dead animals following routine examination.
Endotoxin-induced shock, which is a common problem in the intensive care unit, has a high mortality rate. Cytokines produced from leukocytes that are activated by endotoxins have been implicated in the pathophysiology of endotoxin-induced shock. The present study was conducted in order to clarify the effects of acute low-dose ethanol administration on endotoxin-induced shock and cytokine responses in rats. Twenty rats were randomly assigned to 4 groups: control group received saline, ethanol group received acute low-dose ethanol (100 mg•kg-1•hr-1 for 2 hr, i.v.), endotoxin group received endotoxin (Escherichia coli lipopolysaccharide; 15 mg/kg, i.v.), and endotoxemia-ethanol group received acute low-dose ethanol after the injection of endotoxin. After the endotoxin injection, hemodynamics, acid-base statuses, and the plasma concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were assessed in each of the 4 groups. The systolic arterial pressure was significantly greater in endotoxemia-ethanol group than in endotoxemia group from 0.5 to 1.0 hr after injection. The base excess was significantly greater in endotoxemia-ethanol group than in endotoxemia group from 3 to 4.5 hr after injection. Plasma TNF-α concentration in endotoxemia-ethanol group was significantly lower than that in endotoxemia group 3 hr after injection, and plasma IL-10 concentration in endotoxemia-ethanol group was significantly higher than that in endotoxemia group from 3 to 4.5 hr after injection. Acute low-dose ethanol administration inhibited endotoxin-induced shock, metabolic acidosis, inflammatory cytokine responses, and activated anti-inflammatory cytokine responses in rats.
Human exposure to inorganic arsenic (iAsIII), a potent oxidative stressor, causes liver injuries, which can lead to cancer. It is known that induction of haem oxygenase-1 (HMOX1) reduces arsenite-induced cytotoxicity in the liver. In a two-step reaction HMOX1 generates BR from haem and BR, in turn is oxidised to its oxidative metabolites by radical oxygen species (ROS). Bilirubin oxidative metabolites (BOMs), such as tripyrroles and propentdyopent have been identified and measured in rat and human urine. Additionally, two novel dipyrroles (with m/z values 333 and 315) have recently been identified in vitro (Abu-Bakar et al. (2011). Toxicol. Appl. Pharmacol., 257, 14-22) but their presence in biological samples has yet to be demonstrated.The aim of the present study was to identify the novel dipyrroles in urine of control and iAsIII treated mice. Acute iAsIII exposure generated transient oxidative stress in the liver, which associated with temporal induction of hepatic HMOX1, the rate-limiting enzyme of BR biosynthesis. The increase in the total BR levels was modest compared to the strong induction of HMOX1. Additionally, urinary dipyrroles (m/z values 333 and 315) escalated after iAsIII exposure. The results suggest that dipyrrolic BOMs can be used as marker for oxidative stress.
Dental amalgam is a source of exposure to elemental mercury vapor in the general population. The aim of this study was to elucidate the effect of elemental mercury vapor exposure from dental amalgam restorations on gene expression profiles. Out of 26,962 rat genes, mercury vapor was found to increase the expression of 1 gene (Atp1b3) and decrease the expression of 1 gene (Tap1) in the cerebrum, increase the expression of 1 gene (Dnaja2) in the cerebellum, increase the expression of 2 genes (Actb and Timm23) and decrease the expression of 1 gene (Spink3) in the liver, increase the expression of 2 genes (RT1-Bb and Mgat5) and decrease the expression of 6 genes (Tnfaip8, Rara, Slc2a4, Wdr12, Pias4 and Timm13) in the kidney.