Increased air pollution, containing carcinogenic particulate matter smaller than 2.5 μm (PM_2.5), has gained particular attention in recent years as a causative factor in the increased incidence of respiratory diseases, including lung cancer. Extensive carcinogenicity studies conducted recently under Good Laboratory Practice conditions by National Toxicology Program in the USA, Ramazzini Foundation in Italy or Contract Research Organizations on numerous chemical compounds have demonstrated the importance of considering dose levels, times and duration of exposure in the safety evaluation of carcinogenic as well as classical toxic agents. Data on exposure levels to chemical carcinogens that produce tumor development have contributed to the evaluation of human carcinogens from extrapolation of animal data. A popular held misconception is that the risk from smoking is the result of inhaling assorted particulate matter and by products from burning tobacco rather than the very low ng levels of carcinogens present in smoke. Consider the fact that a piece of toasted bread contains ng levels of the carcinogen urethane (ethyl carbamate). Yet, no one has considered toast to be a human carcinogen. Future human carcinogenic risk assessment should emphasize consideration of inhalation exposure to higher levels of benzo (a) pyrene and other possible carcinogens and particulate matter present in polluted air derived from automobile exhaust, pitch and coal tar on paved roads and asbestos, in addition to other environmental contaminant exposure via the food and drinking water.

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Organotin compounds have been widely used as antifouling biocides for ships and fishing nets, agricultural fungicides and rodent repellents. These widespread uses have resulted in the release of increasing amounts of organotins into the environment. In aquatic invertebrates, particularly marine gastropods, organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), induce irreversible sexual abnormality in females which is termed “imposex” at very low concentrations. Although it has been theorized that these compounds act as potential competitive inhibitors of aromatase, which converts androgen to estrogen, and then increase levels of unconverted androgens in gastropods, their effective concentrations for aromatase inhibition are high. In addition to wildlife, organotins may have various undesirable effects on human health. Contrary to the theory of organotin-induced aromatase inhibition in gastropods, in human choriocarcinoma cells, these compounds markedly enhance estradiol biosynthesis along with the increase of both aromatase activity and 17β-hydroxysteroid dehydrogenase type I (17β-HSD I) activity, which converts low-activity estrogen estrone to the biologically more active form estradiol, at the same low concentrations. Although there are many reports describing the potential toxicity of organotins in human and mammals, the critical target molecules for the toxicity of organotin compounds remain unclear. Recently, organotin compounds including TBT and TPT were identified as nanomolar agonists for retinoid X receptor (RXR) and peroxisome proliferator-activated receptor (PPAR) γ, which are members of the nuclear receptor superfamily. Here, we review the potential genetics action and subsequent toxicity induced by organotins via these nuclear receptors.

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Safety assessment of biopharmaceuticals in preclinical studies is guided by the ICH S6 guideline issued in 1997. Along with enormous experiences and knowledge on safety assessment of some classes of biopharmaceuticals over the last decade, the necessity and feasibility of updating the guideline has been discussed. According to a recommendation by safety experts at the ICH meeting in Chicago in 2006, regional discussions of ICH S6 were held in the USA, EU and Japan. The meeting to clarify the values, challenges and recommendations for ICH S6 from Japanese perspective was held as a part of the first Drug Evaluation Forum in Tokyo on August 10, 2007. Of utmost importance, the “case-by-case” approach must be preserved as the basic principle of the ICH S6 guideline. It is our opinion that oligonucleotides, siRNA, aptamers and related molecules should be excluded from ICH S6 and may be more appropriate for separate guidance. However, based on experiences and accumulated knowledge, there are a number of issues that can be updated including new types of biopharmaceuticals such as bioconjugates, use of homologous proteins and transgenic animals, reproductive/developmental toxicity studies in non-human primates, in vitro cardiac ion channel assay and alternative approaches for carcinogenicity assessment. Preliminary recommendations for some of these topics were outlined at the meeting. The overall Japanese recommendation is that the ICH S6 guideline should be updated to address these topics.

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Warfarin is commonly used worldwide as a rodenticide. It inhibits coagulation of blood by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity. An inadequate supply of vitamin K blocks the production of prothrombin and causes hemorrhage. It has been reported that repeated or long-term treatments with this drug cause resistance in wild rodents. However, the mechanism of warfarin resistance in rodents is still not known precisely. Recent studies reported and identified the function of the molecule, vitamin K epoxide reductase complex subunit 1 (VKORC1), which is the main unit of VKOR. An amino acid substitution in VKORC1 is one of the supposed mechanisms of warfarin resistance. An accelerated detoxification system involving cytochrome P450 (CYP) could also cause the rodenticide resistance. Administration of SKF-525A, a potent inhibitor for P450, increased the mortality due to reduction of warfarin metabolism in warfarin-resistant rats. Meanwhile, the appearance of warfarin-resistant rodents has led to the development of the more effective and toxic rodenticide superwarfarin, which is widely used in Europe and the USA. However, animals resistant to this second-generation rodenticide have already been reported in Europe. In this review, we focus on the mechanism and the pleiotropic effects of pesticide resistance in wild rodents.

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If titanium dioxide nanoparticles are inert and non-biodegradable, they must be evaluated similarly to fullerenes, carbon nanotubes and asbestos. We surveyed the titanium level in typical raw food materials, and then intravenously injected titanium dioxide nanoparticles (primary particle diameter: 15 nm; secondary particle size: 220 nm) in mice and determined their tissue distribution and elimination. As a result, an unexpectedly high titanium concentration was observed in several foods. It was also detected in blood and tissues of healthy mice without administration of titanium dioxide nanoparticles. Then, forced i.v. injection of the nanoparticles was performed in mice. The titanim level was significantly increased in blood and tissues, but no increase was found in the brain after i.v. injection. Most titanium was concentrated in the liver after injection, but the liver level decreased over time (ca. 30% decrease in 1 month). These data show that titanium must be eliminated from the body, and suggest that we should reconsider an evaluation method for toxicity of titanium dioxide nanoparticles.

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This study examined the deleterious effects of di(n-butyl) phthalate (DBP) on the male reproductive organs in hypothyroid rats. Hypothyroidism was induced in prepubertal male rats (28 days of age) by an intraperitonial (i.p.) injection of 10 mg/kg/day propylthiouracil (PTU) for 30 days. DBP (100 and 500 mg/kg/day) was administered by oral gavages to the intact or hypothyroid rats for 30 days. The body weight of the PTU-treated rats was significantly lower than the control group. The total triiodothyronine (T3) and thyroxine (T4) serum level was lower, and the thyroid-stimulating hormone (TSH) level was higher in the hypothyroid rats than in the control rats. The DBP treatment rats showed significantly lower testes, epididymides, seminal vesicles, and ventral prostate weights than the untreated rats. The hypothyroid rats had significantly higher thyroid weights and lower adrenal glands weights than the control rats. The histomorphological examination showed diffused Leydig cells hyperplasias and germ cells loss in the DBP (500 mg/kg)-treated rats, whereas these effects were mild in the DBP-treated hypothyroid rats. The serum levels of monobutyl phthalate (MBP) were significantly lower in PTU-induced hypothyroid rats than in the DBP-treated rats. This data suggests that the hypothyroid status might offer some protection from male reproductive organ toxicity caused by a disturbance in the metabolic activation of the parent compound, DBP.

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Effects of dose and duration of phenobarbital (PB) administration and those of co-administration of PB and vitamin K on blood coagulation-related parameters were examined in specific pathogen-free (SPF) rats of Sprague-Dawley strain kept on an ordinary diet. In Experiment 1, oral administration of PB (0, 25, 50, 100 or 150 mg/kg/day) for 2 weeks induced increases in hepatic cytochrome P450 content and CYP2B expression, prolongation of coagulation time (activated partial thromboplastin time (APTT) and Thrombotest^® (TBT)) and an increase in anti-thrombin III (AT III) concentration in a dose-dependent manner. In Experiment 2, PB administration (100 mg/kg/day) for up to 14 days produced time-dependent increases in hepatic cytochrome P450 content and CYP2B (CYP2B1 and CYP2B2) expression. APTT was prolonged from day 1 and AT III concentration was increased from day 2, whereas the coagulation time (TBT) was prolonged from day 7. In Experiment 3, APTT prolonged by PB (100 mg/kg/day) was shortened after vitamin K₂ (30 mg/kg/day) co-administration, although AT III concentration was still increased. This suggests that not AT III but PB-induced vitamin K deficiency may play an important role in PB-induced prolongation of coagulation time in SPF rats kept on an ordinary diet.

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Drug-induced QT interval prolongation is a critical issue in development of new chemical entities, so the pharmaceutical industry needs to evaluate risk as early as possible. Common marmosets have been in the limelight in early-stage development due to their small size, which requires only a small amount of test drug. The purpose of this study was to determine the utility of telemetered common marmosets for predicting drug-induced QT interval prolongation. Telemetry transmitters were implanted in common marmosets (male and female), and QT and RR intervals were measured. The QT interval was corrected for the RR interval by applying Bazett’s and Fridericia’s correction formulas and individual rate correction. Individual correction showed the least slope for the linear regression of corrected QT (QTc) intervals against RR intervals, indicating that it dissociated changes in heart rate most effectively. With the individual correction method, the QT-prolonging drugs (astemizole, dl-sotalol) showed QTc interval prolongations and the non-QT-prolonging drugs (dl-propranolol, nifedipine) did not show QTc interval prolongations. The plasma concentrations of astemizole and dl-sotalol associated with QTc interval prolongations in common marmosets were similar to those in humans, suggesting that the sensitivity of common marmosets would be appropriate for evaluating risk of drug-induced QT interval prolongation. In conclusion, telemetry studies in common marmosets are useful for predicting clinical QT prolonging potential of drugs in early stage development and require only a small amount of test drug.

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Male Wistar rats were exposed by inhalation to N,N-dimethylformamide (DMF) at 0 (control), 200 or 400 ppm (v/v) for 6 hr/day, 5 days/week and 4 weeks, and each inhalation group received DMF-formulated drinking water at 0, 800, 1,600 or 3,200 ppm (w/w) for 24 hr/day, 7 days/week and 4 weeks. Both the combined inhalation and oral exposures and the single-route exposure through inhalation or ingestion induced centrilobular hypertrophy and single-cell necrosis of hepatocytes, increased plasma levels of alanine aminotransferase (ALT), increased percentage of proliferating cell nuclear antigen (PCNA)-positive hepatocytes without glutathione-S-transferase placental form (GST-P)-positive liver foci, and increased relative liver weight. Those hepatic parameters of the DMF-induced effects were classified into hypertrophic, necrotic and proliferative responses according to the pathological characteristics of affected liver. While magnitudes of the hypertrophic and necrotic responses were linearly increased with an increase in amounts of DMF uptake in the single-route exposure groups, those dose-response relationships tended to level off in the combined-exposure groups. Saturation of the hypertrophic and necrotic responses at high dose levels might be attributed to suppression of the metabolic conversion of DMF to its toxic metabolites. Percentage of PCNA-stained hepatocytes classified as the proliferative response was increased more steeply in the combined-exposure groups than in the single-route exposure groups. It was suggested that the proliferative response of hepatocytes to the combined exposures would be greater than that which would be expected under an assumption of additivity for the component proliferative responses to the single-route exposures through inhalation and ingestion.

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Large-scale clinical studies have shown that the biguanide drug metformin, widely used for type 2 diabetes, to be very safe. By contrast, another biguanide, phenformin, has been withdrawn from major markets because of a high incidence of serious adverse effects. The difference in mode of action between the two biguanides remains unclear. To gain insight into the different modes of action of the two drugs, we performed global gene expression profiling using the livers of obese diabetic db/db mice after a single administration of phenformin or metformin at levels sufficient to cause a significant reduction in blood glucose level. Metformin induced modest expression changes, including G6pc in the liver as previously reported. By contrast, phenformin caused changes in expression level of many additional genes. We used a knowledge-based bioinformatic analysis to study the effects of phenformin. Differentially expressed genes identified in this study constitute a large gene network, which may be related to cell death, inflammation or wound response. Our results suggest that the two biguanides show a similar hypoglycemic effect in db/db mice, but phenformin induces a greater stress on the liver even a short time after a single administration. These findings provide a novel insight into the cause of the relatively high occurrence of serious adverse effect after phenformin treatment.

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Toxicokinetics (TK) is usually performed by measurement of the total drug concentrations in plasma. However, free drug concentrations in plasma are considered to correlate directly with toxicodynamics (TD). In the present study, to evaluate the applicability of TK/TD analysis based on free drug concentrations, we investigated the TK/TD of clofibrate, which binds to albumin with a higher ratio, using an albumin-deficient mutant strain, Nagase analbuminemia rats (NAR). TK, blood chemistry, histopathology, drug and fatty acid metabolizing enzymes and microarray analysis in the liver were examined after a 4-day oral administration of clofibrate. Compared to Sprague-Dawley (SD) rats, the parent strain of NAR, 4.1-fold higher AUC_0-24hr based on free drug concentrations (3445 versus 844 µg·hr/ml) was observed in NAR when both rats showed the same level of AUC_0-24hr based on the total drug concentrations (4436 versus 4237µg·hr/ml). Additionally, more severe hepatocellular hypertrophy, increase in aspartate transaminase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), decrease in total cholesterol (T.CHO), phospholipid (PL), triglyceride (TG), and non-esterified fatty acid (NEFA), and increase in the mRNA levels of fatty acid metabolizing enzymes (FAOS, CAT, and CPT) were observed in NAR at the same dose. These results demonstrated that NAR developed more severe toxicities and pharmacological effects than SD rats correlating with the higher AUC of the free drug concentrations. The results also suggested that TK/TD analysis based on the free drug concentration is appropriate to interpret the relationship between exposure and toxicity in cases of protein binding saturation including protein decrease or species differences on protein binding, especially when drugs showing a higher protein binding ratio are dosed.

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Olanzapine is a second-generation atypical antipsychotic that is increasingly used in preference to older antipsychotic agents. Limited data is available concerning the toxic effects of olanzapine after deliberate overdose. Two patients presented to our institution after massive olanzapine ingestion, and required prolonged ventilatory support due to the development of coma and respiratory depression. Serum olanzapine concentrations were orders of magnitude higher than those associated with therapeutic doses, and remained elevated for several days after ingestion. Both patients made a full recovery with only supportive care, despite having initial serum drug concentrations > 2500 µg/l. These reports indicate the potential for olanzapine ingestion to cause coma that may persist for several days after overdose.

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(-)-Epigallocatechin 3-O-gallate (EGCG), a major catechin in green tea, suppresses renal failure in animals, and inhibits the growth of mesangial cells and opossum kidney proximal tubular cells. In addition, gallic acid, a structural constituent of this catechin, induces apoptosis in tumor cell lines. However, the effects of catechins on renal fibroblastic cells have not been investigated. In this experiment, the growth of normal rat kidney interstitial fibroblast (NRK-49F) cells was significantly inhibited by EGCG at concentrations higher than 6.25 µM, and almost completely inhibited at concentrations over 200 µM. The numbers of in situ end-labeled (ISEL) cells in cultures treated with EGCG at 6.25 to 200 µM increased dose-dependently. Furthermore, exposure to 6.25 to 50 µM EGCG for 24 hr led to a significant increase in caspase-3 activity compared to the control. These results suggest that EGCG induces apoptosis in NRK-49F cells.

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The feasibility of transgenic tobacco, genetically engineered to express bacterial polyphosphate (polyP) for phytoremediation of cadmium pollution was examined. The transgenic tobacco showed more resistance to Cd²⁺ and accumulated more Cd²⁺ than its wild-type progenitors. These results suggest that polyP has abilities to reduce Cd²⁺ toxicity, probably via a chelation mechanism, and to accumulate cadmium in the transgenic tobacco. Based on the results obtained in this study, polyP-mediated Cd²⁺ accumulation may serve as a useful strategy for Cd²⁺ phytoremediation.

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The aim of this study is to report how pregnancy alters hematology and clinical chemistry values in rats. Female and male Sprague-Dawley rats were mated; the day of copulation was designated as Day 0. Hematology and clinical chemistry measurements were conducted on Days 7, 14, 17 and 21 in pregnant rats. Measurements were also conducted in non-pregnant rats. Red blood cells (RBC), hemoglobin (Hb), hematocrit (Ht), total protein and albumin decreased on Days 7, 14, 17 and 21; sodium, chloride and glucose decreased on Days 14, 17 and 21; iron decreased on Days 17 and 21; hemoglobin content of reticulocytes (CHr), calcium, inorganic phosphorus and the albumin/globulin ratio decreased on Day 21; and total cholesterol, phospholipid and high-density lipoprotein cholesterol decreased on Day 14 in pregnant rats compared with non-pregnant rats. Reticulocyte increased on Days 7, 14 and 17; mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, neutrophil count and rate increased on Days 14, 17 and 21; platelets, fibrinogen, triglyceride and free fatty acid increased on Days 17 and 21; and activated partial thromboplastin time was prolonged on Days 17 and 21 in pregnant rats compared with non-pregnant rats. The decreased RBC, Hb, Ht, CHr and iron in pregnant rats indicated that they suffered from iron deficiency anemia. These data can be used as background information for effective evaluation in reproductive toxicology studies.

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