Although NF-E2 related factor 2 (Nrf2) was found to be a transcriptional regulator that controls an expression of the β-globin gene, the notion is now widely accepted that this transfactor serves as a master regulator for the gene expression of a battery of proteins acting on anti-oxidative stress and detoxification of electrophiles. The function of Nrf2 that bears transcriptional activation depends solely on its nuclear localization, which is regulated by interaction with the cytosolic anchor protein Keap1 and its own turnover rate. In the present mini-review, we focus on the regulation of Nrf2 function and discuss the physiological and toxicological aspects of this transcriptional factor.
Gene expression changes in the lungs induced by paraquat (PQ) administration were studied in rats using DNA microarrays that were detectable for 1,090 genes per DNA microarray. The rats were subjected to subacute PQ exposure (7 mg/kg, s.c., daily for eight administrations). Two days after the final administration, the rats were divided into two groups. Group 1 experienced significant body weight loss and displayed signs of subacute PQ toxicity, but Group 2 showed no significant effects due to the PQ treatment. A control group, Group 3, was also included. In the comparison of the gene expression levels in the animals from Group 1 or Group 2 to the control animals treated by vehicle, 48 genes in Group 1 and 29 genes from Group 2 were differentially expressed. The twenty-eight genes were common to these two groups. These differentially expressed genes following paraquat treatment were classified as follows: 5 neurotransmitter receptor genes; 4 transporter genes; 4 voltage-gated ion channel genes; 2 lipid metabolism enzyme genes; 2 G-proteins involved in endocytosis and exocytosis genes; 7 cytokine genes; 4 ADP ribosylation genes involved in cell death and regeneration; CFTR gene, which is the causal gene for cystic fibrosis; neurofibromatosis type 1 gene, which is the causal gene for the neurofibromatosis type 1 that is known to accompany pulmonary fibrosis; and the causal gene for spinocerebellar ataxia. These genes may prove to be the keys for the elucidation of the mechanism of PQ toxicity, e.g. PQ-induced pulmonary fibrosis.
Flow cytometry (FCM) analysis of CD45, CD45R, CD71 and CD90 expression on Crj:CD(SD)IGS rat bone marrow cells was done after 5-fluorouracil (5-FU) administration to examine whether these lineage-specific cell surface antigens could be myelotoxic biomarkers. The expression of CD45 (CD45Low and CD45 High: differing in expression intensity), CD45R, CD71 and CD90 on bone marrow cells coincided with previous reports. After repeated administration of 5-FU at 50 mg/kg/day for 1-5 days, a time-dependent decrease in cells expressing CD45Low, CD71 and CD90 was observed, whereas a decrease in the CD45High expressing cells was not observed. Furthermore, the decrease was dose-dependent in CD45Low, CD71 and CD90 expressing cells after administration of 5-FU between 2 and 50 mg/kg/day for 4 days. After 4-day repeated dose of 5-FU at 50 mg/kg/day followed by a recovery period, the change in number of CD45Low, CD45R, CD71 and CD90 cells to the bottom and in recovery showed different kinetics. In contrast, the change in number of CD45High cells was minimal, and relatively stable after 5-FU administration. The results suggest that CD45, CD45R and CD90 could each be potential myelotoxic biomarkers for a total proportion of common leukocytes including T- and B-lymphocytes, for a total proportion of B-lymphocytes, and for a total proportion of T-lymphocytes plus immature B-lymphocytes and common progenitor cells, respectively. CD71 could be a single myelotoxic biomarker for erythroid cells. Further study is required for isolation of each of the myelo-lymphocytic lineages. However, the present study showed that FCM analysis could be available to assess the lineage or differentiation stage-specific response, such as the different extent and time-course or the kinetics (the time to reach the bottom and to recover to the normal level) of myelotoxic effect in rat bone marrow.
- Subcellular distribution of di-(2-ethylhexyl)phthalate (DEHP) in the testis was studied by single oral administration of [3,4,5,6-3H]-phthalic acid di-(2-ethylhexyl) ester (DEHP-3H) or phthalic acid di-(2-ethyl[1-3H]hexyl) ester (3H-DEHP) to 8-week-old male rats. Autoradiographs and electron microscopic autoradiographs were prepared from the testis, liver and kidney at 6 and 24 hr after administration and distribution of radioactive materials in the tissues were observed. In the autoradiographic specimen at 6 hr after administration of DEHP 3H-labeled at phthalic acid moiety (DEHP-3H), many grains were observed in the testis, mainly at the basal area of seminiferous tubules at the stages IX to I of the spermatogenic cycle. Electron microscopic autoradiographs taken at the same time revealed that localization of grains were in the smooth-surfaced endoplasmic reticulum and mitochondria of Sertoli cells. A few grains were also present at the Golgi apparatus and lysosome of Sertoli cells, and at the interfaces between the Sertoli cells or between Sertoli cells and spermatocytes, and in the cytoplasm of spermatocytes. Autoradiographs of the liver revealed grains in the centrilobular hepatocytes, localized at mitochondria, rough-surfaced endoplasmic reticulum and peroxisomes. In the kidney, the radioactivity was localized at the brush border of the tubular cells in the pars recta of proximal tubules. In the 24-hr specimen, the grain density in the seminiferous tubules obviously decreased. On the other hand, by autoradiography with DEHP 3H-labeled at the alcohol (3H-DEHP), only a few grains were observed in autoradiographs of the testes at 6 hr after administration. No grains were noted in autoradiographs of the liver and kidney with 3H-DEHP. The results showed that the phthalic acid ester was splitted rapidly in the body and only the phthalic acid moiety distributed into the cells.
Determination of `no observed adverse effect level' (NOAEL) is one of the prime objectives of a repeated subchronic experiment. The data collected in such experiments are huge, and it is a difficult and time-consuming task to determine NOAEL. In such situations, cluster analysis could be used as a valuable tool. In the present paper the determination of NOAEL is demonstrated using the data of a 28-day repeated oral toxicity study carried out in rats. Groups (10/sex/group) of Crj: CD rats were administered the test substance at low, middle, high and top dose levels by gastric intubation daily for 28 days. A concurrent control group was also maintained. In-life measurements included general behavior, body weight, food and water consumption. At termination various hematological and biochemical parameters were determined in the blood of individual animals in all groups. Urinalysis was also carried out at termination. Animals were sacrificed for microscopic and macroscopic findings. Analysis of data showed that 40 measurements (data 1) in the treatment groups were different from the control and 48 (data 2) were not treatment-related. Cluster analysis was carried out separately on data 1 and combined data 1 and data 2. This study revealed that for better judgment of NOAEL performing a cluster analysis on the data which alone showed a significant difference compared to control is more advisable than performing the cluster analysis on all data collected in the study. The advantage of cluster analysis is that quantitative data and qualitative data can be analyzed in annexation. In addition, cluster analysis assists in the result of the univariate analysis and displays NOAEL at once quite obviously.
It has generally been thought that iodine allergy is cross-sensitive to various iodine-containing chemicals. However, this concept seems to deviate from the immunological principle that immune recognition is specific. To solve this contradiction, we hypothesize that iodine allergy is an immunological reaction to iodinated autologous proteins produced in vivo by iodination reaction from various iodine-containing chemicals. Antisera to iodine were obtained from guinea pigs immunized subcutaneously with iodine-potassium iodide solution emulsified in complete Freund's adjuvant (CFA). The specificity of guinea pig anti-iodine antiserum was determined by enzyme-linked immunosorbent assay (ELISA) inhibition experiments using microplates coated with iodinated guinea pig serum albumin (I-GSA). Antibody activities were inhibited by I-GSA, diiodo-L-tyrosine, and thyroxine, but not by potassium iodide, monoiodo-L-tyrosine, 3,5,3'-triiodothyronine, monoiodo-L-histidine, or diiodo-L-histidine, or by ionic or non-ionic iodinated contrast media. The results that antigen recognition of anti-iodine antibody is specific to iodinated protein support our hypothesis. While protein iodination usually takes place both at histidine residues as well as at tyrosine residues, only iodinated tyrosine acted as an antigenic determinant and no antibody activities to iodinated histidine were detected in our experimental iodine allergy model.
We hypothesize that iodine allergy is an immune response to iodinated autologous proteins generated in vivo from iodine-containing organic and inorganic chemicals. In this report, effects of protein iodination on elicitogenic activity in guinea pig iodine allergy model and iodinated protein antigen generation in vitro from iodine-containing chemicals were investigated. Active cutaneous anaphylaxis (ACA) and delayed-type hypersensitivity (DTH) tests were performed in guinea pigs immunized with iodine. The amount of iodine (I2) reacted to proteins for giving them an eliciting activity of ACA was ≥0.15 μmol for 1 mg of albumin. DTH reactions were provoked by intradermal injection of 106 PECs reacted with ≥0.075 μmol of I2. I2 was generated from a potassium iodide (KI) solution or iodinated contrast media by UV light irradiation. X-ray irradiation of KI and iodinated contrast media in the presence of protein resulted in the generation of iodinated protein antigens. The generation of iodinated protein antigens was inhibited in the presence of reducing agents. Therefore, it is noteworthy that iodine allergy of the present hypothesis is dependent on reactive oxygens. By presenting these ex vivo and in vitro data, we discuss the possibilities for the generation of iodinated protein antigens in vivo.
We hypothesize that iodine allergy is an immune response to iodinated self proteins produced in vivo from various iodine-containing chemicals. Since an antigenic determinant of experimental iodine allergy is diiodotyrosine (DIT), we designed low molecular weight DIT derivatives having provocative antigenicity without sensitizing immunogenicity. Tetraiododityrosine and hexaiodotrityrosine provoked dose-dependent skin reactions in guinea pigs previously immunized with iodine. No guinea pigs immunized with hexaiodotrityrosine showed anaphylactic reaction by i.v. challenge with hexaiodotrityrosine and none of their antisera showed positive passive cutaneous anaphylaxis (PCA) reaction in guinea pigs, indicating the non-immunogenic nature of the compound. Erythrosine, one of the color additives having a structure common with DIT, was assessed for its immunological property. Enzyme-linked immunosorbent assay (ELISA) inhibition studies on erythrosine revealed that the inhibitory activity of erythrosine was stronger than that of DIT. Furthermore, erythrosine provoked a PCA reaction in animals sensitized with anti-iodine antisera. In conclusion, hexaiodotrityrosine is thought to be useful for skin testing of iodine allergy without any fear of sensitization to the allergen. Erythrosine was shown to provoke an experimental iodine allergy and, also, the relationships between the new concept of iodine allergy and features of clinical findings of adverse effects by iodocontrast media are discussed.
In order to clarify the mechanism of the neurotoxicity of 5-FU and/or its masked compounds, we studied the effects of α-fluoro-β-alanine (FBAL) and fluoroacetic acid (FA) on the formation of vacuolar changes in the dog cerebrum, using the dosage of 3.0 mg/kg/day of FBAL-HCl (FBAL·HCl) and 0.03 mg/kg/day of FA-Na (FA·Na), respectively. These 2 compounds were selected because they are the metabolites of 5-FU claimed to be responsible for the neurotoxic effects of 5-FU and/or its masked compounds, and we wanted to confirm their effects. Tegafur-uracil mixture (UFT) was used as a positive control drug for the formation of vacuolar changes in the dog cerebrum. All compounds were orally administered daily for 3 months to beagle dogs. Each study group consisted of 3 males. Neurotoxic signs such as hyperesthesia and/or excitement, as well as convulsions, were observed in both FBAL·HCl and FA·Na groups; these toxic signs were also found in the UFT group. Slight loss of body weight gain and of food consumption was observed in the FBAL·HCl and UFT groups. Neuropathologically, vacuolar changes were detected in several areas of the dog cerebrum following administration of FBAL·HCl, FA·Na or UFT. In terms of morphology, the neuropathological effects of these 2 drugs were very similar to those induced by UFT. In conclusion, we clearly showed that FBAL is one of the main substances that cause neurotoxic signs and neuropathological changes in dogs intoxicated by 5-FU or its masked compounds. Moreover, FA might be considered to be a causative factor in addition to FBAL.