An abbreviated history of the dose-response curve or chemical concentration-effect relationship is presented in this article. No attempt has been made to include all references on the subject. Just an outline, overview, and discussion of the most important eras are presented. The history of dose response may be divided roughly into three Eras, Preclassical, Classical, and Current. Paracelsus, who lived from 1493 to 1541, must be recognized as the first one to realize that dose was the most important issue to deal with in determining whether a chemical was toxic or not. However, that issue seems to have been forgotten and is still ignored by some. The Classical Era began about 1900 and ended about t; he time of the death of Gaddum in 1965, during which almost all of the progress had been made in solving the parameters to use in analyzing data. The Current Era began with the acceptance of the linearized multistage model in the mid-1970’s. In this author’s opinion, the biggest mistake in all of toxicology occurred with the acceptance and use of the linearized multistage for DNA-reactive carcinogens with dose on a linear scale. It had been clearly established in the Classical Era that dose should be plotted on a logarithmic scale. Plotting dose on a linear scale distorts the curve for low doses so that any attempt to detect the relationship of dose of carcinogen to carcinogenesis is impossible.
Botulinum neurotoxins (BoNTs) are potent toxicant proteins composed of a heavy chain (100 kDa) and a light chain (50 kDa) of seven (A-G) serotypes that is responsible for botulism syndrome. In this study, polypeptidesfrom C-terminal heavy chain of BoNTs serotypesA, B and Eto the length of 54, 45 and 48 amino acidrespectively wereselected, linked together usinga hydrophobic linker and expressed in E. coli. The expression efficiency of the chimeric protein was found to be 51%. The chimeric protein was produced in the form of inclusion body (IB) both at two studied temperatures, 30°C and 37°C. This IB was extracted by ultracentrifugation and followed for chimeric protein solubilization and purification using of ultrafiltration and preparative electrophoresis. The purified chimeric protein was characterized using blotting and ELISA. To evaluate the protection ability of this chimeric antigen against their active toxins, it was injected to mice and the antibody titer as well as the extent of protectivity were determined. Mice given three injections (10 µg/mice) of the antigen were protected against an intra-peritoneal administration of 10 LD50 of serotypes A and E, but 100 LD50 of serotype B. We conclude that a significant correlation exists between the antigenic characteristics and protection capability of the chimeric protein prepared in this study.
To explore the predictivity of dose range-finding (DRF) studies, we conducted asurvey by sending out questionnaires to 72 Japanese pharmaceutical companies.The survey yielded data for 108 and 85 compounds for which any embryo-fetaldevelopment (EFD) toxicities were observed in the definitive studies in rodentsand non-rodents, respectively. As a result of the analysis, 83% of studies inrodents and 80% in non-rodents showed EFD effects in the DRF studies. Whenfocusing on teratogenicity, 91% of studies in rodents and 100% in non-rodentswere judged “positive” in the DRF studies when all EFD toxicities were used asmarkers. When the effects of both the rodent and non-rodent studies wereevaluated together, the combination predictive value in the DRF studies was 96%for EFD toxicants and 100% for teratogens. To evaluate the influence of theexamination items, the predictive value was analyzed using 54 compounds forwhich full examinations (external, visceral and skeletal examination) wereconducted in both rodent and non-rodent DRF studies. When the results werejudged by including or excluding skeletal and visceral examinations results,the predictive values were not significantly different. In conclusion, theresults of this survey showed that a pair of the DRF studies in the rodents andnon-rodents is useful to increase the predictivity of DRF studies. In additionthe inclusion of observations such as fetal survival, body weight and externalexamination into the DRF studies are important to predict effects in thedefinitive studies.
Cigarette smoke (CS), a major risk factor in emphysema, causes cell death by incompletely understood mechanisms. Death-inducing signaling complex (DISC) formation is an initial event in Fas-mediated apoptosis. We demonstrated cigarette smoke extract (CSE) induced DISC formation in human lung fibroblasts (MRC-5). The aim of this study was to investigate the involvement of extracellular signal-regulated kinase (ERK) MAPK activation in CSE induced DISC formation. Immunoprecipitaion (IP) for Fas and Western Immunoblot (IB) analysis for caspase 8 were then performed to show DISC. Lactate dehydrogenase (LDH) release was measured using a cytotoxicity detection kit. MTT assay was used as a measure of cell viability. We demonstrated that CSE induces DISC formation in MRC-5 using IP for Fas and IB for caspase 8. ERK was expressed in MRC-5 exposed to CSE. MEK-1 inhibitor (PD98059) decreased DISC formation in MRC-5 exposed to 20% CSE at 1 hr, and cell viability, as assessed by colorimetric MTT assay, was increased in MEK-1 inhibitor treated MRC-5 cells after 24 hr CSE exposure compared to the control. Inhibiting ERK significantly decreased the caspase-3,-8 activity in MEK-1 inhibitor treated MRC-5 cells compared to the control.The DISC formation, initial event of extrinsic apoptotic pathway, is a primary component of CSE- induced death in MRC-5, and ERK activation plays an active role in the DISC formation and downstream pathway. These results suggest that modulation of ERK may have therapeutic potential in the prevention of smoke-related lung injury.
Reactive oxygen species (ROS) play an important role in ageing and age-related neurodegenerative changes including Parkinson’s disease (PD). PD is characterized by signs of major oxidative stress and mitochondrial damage in the pars compacta of the substantia nigra. Present study was designed to investigate whether the Centella asiatica extract (CAE) would prevent 1-methyl-4-phenyl-1,2,3,6 - tetrahydropyridine (MPTP) - induced neurotoxicity in aged Sprague-Dawley rats. Adult, male Sprague-dawley rats of 300-350 g were divided into control, C. asiatica alone, MPTP alone (20 mg/kg, for 21 days) and MPTP with C. asiatica (300 mg/kg for 21 days) groups. Effect of aqueous extract of C. asiatica on oxidative biomarker levels in corpus striatum and hippocampus homogenate was examined. MPTP-challenged rats elicited a significant increase in lipid hydroperoxides (LPO) (p < 0.01), protein-carbonyl-content (PCC) (p < 0.01) and xanthine oxidase (XO) (p < 0.01) when compared with control rats. There was a significant decrease in total antioxidants (TA) (p < 0.001), superoxide dismutase (SOD) (p < 0.001), glutathione peroxidase (GPx) (p < 0.01) and catalase (CAT) (p < 0.001) levels with MPTP treatment. Supplementation of CAE reduced LPO and PCC and significantly increased (p < 0.01) TA and antioxidant enzyme levels (p < 0.01) in corpus striatum and hippocampus. These results show that administration of C. asiatica was effective in protecting the brain against neurodegenerative disorders such as Parkinsonism.
We observed that first delivery was delayed when a group of paired mice fed with non-organic common rice compared to a group fed with organic rice. This led us to hypothesize that pesticides or other soil contaminants may be responsible for the effect on mice reproduction. We then found that the non-organic rice was contaminated with a pesticide etofenprox and nonylphenol, butylphenol and diethylphthalate which are used as agricultural detergents or plasticizers of agricultural film, that are all suspected to be estrogenic. Therefore, the chemicals were administered to mice at the levels detected in rice, and we subsequently observed that first delivery and sperm count of the animals were significantly impaired. This study is the first to show that rice contaminated with agricultural chemicals affects reproduction in the mammal.
Acrylamide (AA) has been reported to be formed in fried and baked foods with various concentrations, and exposure levels to AA from cooked foods in children are estimated to be higher than those in adults. In order to evaluate the carcinogenicity of AA exposure during childhood, we conducted a medium-term carcinogenicity study with prepubertal administration of AA followed by treatments of a multi-organ-targeted genotoxic carcinogen and a promoting agent for thyroid carcinogenesis in rats. A total of 36 postpartum F344 rats were given drinking water containing AA at 0, 20, 40 or 80 ppm for 3 weeks during the lactation period, and their weaned offspring received the same AA-containing water for 3 more weeks. Offspring were then injected with N-methyl-N-nitrosourea (MNU; 40 mg/kg body weight, i.p.) once at week 7 after birth. Half the animals of the 0 and 40 ppm groups were additionally treated with the anti-thyroid agent sulfadimethoxine (SDM; 125 ppm) in the drinking water thereafter. Offspring were subjected to complete necropsy at week 50. All the major organs and macroscopic abnormalities were excised and examined histopathologically. There was no significant difference in the incidences of hyperplastic and neoplastic lesions in the target organs of AA and/or MNU, such as the brain, spinal cord, pituitary gland, thyroid, adrenal glands, uterus, mammary glands, clitoral gland and tunica vaginalis. In conclusion, no significant modifying actions of AA on MNU-induced multi-organ carcinogenesis were exhibited in any organs of rats when exposed prepubertally under the present experimental conditions.
To determine the threshold dose of dicyclanil (DC) that induces hepatocellular tumor-promoting effects associated with reactive oxygen species (ROS) generation via their metabolic pathways, partial hepatectomized ICR male mice were fed diets containing 0, 187.5, 375 or 750 ppm DC after an intraperitoneal injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Immunohistochemically, the proliferating cell nuclear antigen (PCNA)-positive cell ratio was significantly increased in the DEN + 750 ppm DC group compared with the DEN alone group. However, significant increases in the number of γ-glutamyltranspeptidase (GGT)-positive cells and formation of microsomal ROS were not observed in the DEN + DC groups compared with the DEN alone group. Real-time polymerase chain reaction (RT-PCR) showed that the expression of Cyp1a1, Cyp1a2, and OGG1genes was significantly up-regulated in mice given diets containing 375 ppm DC or more, 187.5 ppm DC or more, and 750 ppm DC, respectively. These results suggest that the threshold dose of DC that induces ROS-mediated liver tumor promotion in mice is more than 750 ppm, although expression of the Cyp1a2 gene, which is related to ROS generation, was up-regulated in the liver of mice, even at a DC dose of 187.5 ppm.
The purpose of a toxicity test is to determine the no-observed-effect level (NOEL) of test substance through biological and pharmacological techniques. If the low dose not does show statistically significant and biologically relevant changes in the data evaluated in a study, the usual practice is to consider this dose as the NOEL. To overcome this, 6 types of techniques that seemed to be appropriate are presented in this paper by investigating the results of several domestic and foreign theses on toxicology. The most appropriate techniques appear to be the trend test, comparison between treatment group and historical control by t-test, and confirmation that all individual values lie within the 95% confidence interval (2 SD) of the historical control value, if a significant difference is admitted in the low dose.
This study was designed to evaluate any adverse effect of a hot water extract of black soybeans (Glycine max (L.) Merr.), when administered to both sexes of Crj:CD(SD)IGS rats at dietary levels of 0 (control), 0.5, 1.5 and 5.0% (6 rats/sex/group). During the study, the treatment had no adverse effects on clinical signs, survival, body weights, and food and water consumption, or on findings of ophthalmology, urinalysis, hematology, or blood biochemistry. Organ weights, gross pathology and histopathology exhibited no differences of toxicological significance between control and treated rats. Thus, the no-observed-adverse-effect level (NOAEL) of black soybean extract was concluded to be 5.0% (3,618 mg/kg body weight/day for males and 4,066 mg/kg body weight/day for females) from the present study.
Photosafety evaluation is becoming important during the drug development process in pharmaceutical companies. Both in vitro and in vivo test systems have been developed for the evaluation of phototoxic potential of chemicals. In the present study, we conducted an in vivo phototoxicity test using BALB/c mice. The mice were treated with sparfloxacin, lomefloxacin, or a quinoline derivative orally followed by the irradiation of simulated sunlight, and resulting phototoxic reactions of the ears were assessed. Sparfloxacin and lomefloxacin, but not the quinoline derivative, are well known to cause photoirritation in humans. All three drugs exhibited positive reaction in the 3T3 neutral red uptake phototoxicity test (3T3 NRU PT). In the in vivo test, sparfloxacin and lomefloxacin exhibited positive skin reaction in mice, but the quinoline derivative did not. The results of in vivo phototoxicity test in the mice coincided with phototoxic potential of these drugs in humans. The exposure levels of sparfloxacin or lomefloxacin at the minimum effective dose that exhibited phototoxic reaction in the mice were comparable with those in humans treated with the recommended therapeutic dose.
Chlorella (Parachlorella beyerinckii CK-5), previously identified as Chlorella vulgaris CK-5, is a unicellular green algae that has for many years been used as a nutritional supplement. In order to investigate the effects of methylmercury (MeHg) detoxification by Chlorella, we examined the absorption and excretion of MeHg in mice. Female C57BL/6N mice were randomly divided into three groups of five, and were housed in metabolism cages. Mice were orally administered MeHg chloride at doses of 5 mg (4 mg Hg)/kg body weight with or without 100 mg/mouse of P. beyerinckii powder (BP), and were assigned to either a MeHg group or MeHg + BP group, accordingly. Twenty-four hr after oral administration, feces and urine were collected, and blood, liver, and kidney samples were obtained. Total mercury contents in the samples obtained were determined using an atomic absorption method. The amounts of Hg excreted in feces and urine of the MeHg + BP group were increased nearly 1.9 and 2.2-fold compared with those of the MeHg group. On the other hand, blood and organ Hg levels were not significantly different between two groups. These results suggest that the intake of BP may induce the excretion of Hg both in feces and urine, although it does not affect MeHg absorption from the gastrointestinal tract. The effect of BP on the tissue mercury accumulation may become evident in a long-term experiment.
It is important for toxicological assessment of nanoparticles to determine the penetration of nanoparticle in skin qualitatively and quantitatively. Skin penetration of four different types of rutile titanium dioxide (TiO2) (T-35, 35 nm, non-coating; TC-35, 35 nm, with almina/silica/silicon coating; T-disp, 10 x 100 nm, mixture of almina coated and silicon coated particles, dispersed in cyclopentasiloxan; T-250, 250 nm, non-coating) was determined with in vitro intact, stripped, and hair-removed skin of Yucatan micropigs to study the effect of dispersion and skin conditions. The TiO2 was suspended in a volatile silicone fluid used for cosmetics, cyclopentasiloxane, at a concentration of 10%. The suspension was applied at a dose 2 µl/cm2 for 24 hr, followed by cyanoacrylate stripping. The Ti concentration in skin was determined by ICP-MS. T-35 and T-250 easily aggregated in suspension with a mean diameter greater than 1 µm. TC-35 and T-disp showed good dispersion properties with a mean diameter in suspension of approximately 100 nm. No penetration was observed regardless of TiO2 type in intact and stripped skin. The concentration of Ti in skin was significantly higher when TC-35 was applied on hair-removed skin. SEM-EDS observation showed that Ti penetrated into vacant hair follicles (greater than 1 mm below the skin surface), however, it did not penetrate into dermis or viable epidermis.
Profiles of Chemical Effects on Cells (pCEC) is a toxicogenomics database with a system of classifying chemicals that have effects on human health. This database stores and handles gene expression profiling information and categories of toxicity data. Chemicals are classified according to the specific tissues and cells they affect, the gene expression changes they induce, their toxicity and biological functions in this database system. The pCEC system also analyzes relationships between chemicals and the genes they affect in specific tissues and cells. The reason why we developed pCEC is to support decision-making within the context of environmental regulation. Especially, exposure to environmental chemicals during fetal and newborn development may result in a predisposition to various disorders such as cancer, learning disabilities and allergies later in life. The identification and prediction of hazardous chemicals using limited information are important issues in human health risk management. Therefore, various toxicity information including lethal dose 50 (LD50), toxicity pathways and pathological data were loaded into pCEC. pCEC is also a facility for query, analysis and prediction of unknown toxicochemical reaction pathways and biomarkers which are based on toxicoinformatical data mining approaches. This database is available online at http://project.nies.go.jp/eCA/cgi-bin/index.cgi. The current version of the database has information on the hepatotoxicity, reproductive toxicity and embryotoxicity of chemicals.
In order to investigate nickel toxicity against the yeast Saccharomyces cerevisiae, genomic responses to nickel chloride were examined using yeast DNA microarrays. Microarray analysis revealed that exposure to 25 mM nickel chloride for 2 hr induced changes in gene expression in S. cerevisiae. Nickel chloride increased expression levels in 601 genes and decreased expression levels in 696 genes in S. cerevisiae.
To investigate peroxisomal proliferator-activated receptor α (PPARα) signal responses in heart muscle, we performed LC-MS/MS-based proteomics analysis of heart muscle from rats given fenofibrate or clofibrate. Fenofibrate increased the expression of ACAA2, DECR1, and ECH1 consistent with activation of PPARα. Fenofibrate and clofibrate reduced the expression of 10 and 12 proteins, respectively with the expression of ACSL1, SLC25A4, A1BG, HADHA, ATP2A2, BDH1, ETFDH, HADHB, and CPT2 being reduced in common with both of fibrate-treated groups. The approach adopted in this study provides an efficient method for monitoring global changes in protein expression.