2-mercaptobenzothiazole generates γ-H2AX via CYP2E1-dependent production of reactive oxygen species in urothelial cells.

Ortho (o)-toluidine is a widely known carcinogenic substance associated with cancers of the human bladder. A study on British chemical factory workers exposed to 2-mercaptobenzothiazole, phenyl-ß-naphthylamine, aniline, and o-toluidine demonstrated the crucial roles of o-toluidine, 2-mercaptobenzothiazole, and phenyl-ß-naphthylamine in the development of bladder cancer. As genotoxic events are crucial steps in the initiation of cancer, in the present study, we aimed to examine the genotoxic potential of the four chemicals using phosphorylated histone H2AX (γ-H2AX), which is a sensitive and reliable marker of DNA damage, in cultured human urothelial cells. Of the four chemicals, 2-mercaptobenzothiazole was a particularly potent DNA-damaging agent. Moreover, mechanistic studies revealed that γ-H2AX generation by 2-mercaptobenzothiazole was mainly associated with the generation of reactive oxygen species via cytochrome P450 2E1-mediated metabolism. The findings of this study may provide information that is important for the assessment of risks associated with chemicals as well as the interpretation of epidemiological studies investigating occupational bladder cancer.

Hospital-based screening to detect patients with cadmium nephropathy in cadmium-polluted areas in Japan.

BACKGROUND: In health examinations for local inhabitants in cadmium-polluted areas, only healthy people are investigated, suggesting that patients with severe cadmium nephropathy or itai-itai disease may be overlooked. Therefore, we performed hospital-based screening to detect patients with cadmium nephropathy in two core medical institutes in cadmium-polluted areas in Akita prefecture, Japan. METHODS: Subjects for this screening were selected from patients aged 60 years or older with elevated serum creatinine levels and no definite renal diseases. We enrolled 35 subjects from a hospital in Odate city and 22 from a clinic in Kosaka town. Urinary ß2-microglobulin and blood and urinary cadmium levels were measured. RESULTS: The criteria for renal tubular dysfunction and the over-accumulation of cadmium were set as a urinary ß2-microglobulin level higher than 10,000 µg/g cr. and a blood cadmium level higher than 6 µg/L or urinary cadmium level higher than 10 µg/g cr., respectively. Subjects who fulfilled both criteria were diagnosed with cadmium nephropathy. Six out of 57 patients (10.5% of all subjects) had cadmium nephropathy. CONCLUSIONS: This hospital-based screening is a very effective strategy for detecting patients with cadmium nephropathy in cadmium-polluted areas, playing a complementary role in health examinations for local inhabitants. REGISTRATION NUMBER: No. 6, date of registration: 6 June, 2010 (Akita Rosai Hospital), and No. 1117, date of registration: 26 December, 2013 (Akita University).

Acute exposure to cadmium induces prolonged neutrophilia along with delayed induction of granulocyte colony-stimulating factor in the livers of mice.

Acute exposure to cadmium (Cd), a toxic heavy metal, causes systemic inflammation characterized by neutrophilia. To elucidate the mechanism of neutrophilia induced by Cd, we investigated the induction of granulocyte colony-stimulating factor (G-CSF), which regulates neutrophil production, in mice with acute Cd toxicity, and compared it with mice injected with lipopolysaccharide (LPS) as an inducer of general inflammatory responses. We injected BALB/c mice with Cd at 2.5 mg/kg i.p. or LPS at 0.5 mg/kg i.p. and sampled the peripheral blood and organs at time points up to 24 h. In Cd-treated mice, the peripheral neutrophil count increased steadily up to 24 h, whereas LPS-treated mice showed a more rapid increase with a peak at 12 h. The serum G-CSF level increased gradually to reach a plateau at 12-18 h in Cd-treated mice, but LPS-treated mice showed a marked increase, reaching a peak at 2-3 h. A gradual elevation of G-CSF mRNA expression up to 24 h was detected by real-time PCR in the livers of Cd-treated mice, but in LPS-treated mice its highest expression was observed in the liver with a rapid increase at 2 h. By in situ hybridization using G-CSF RNA probes, hepatic Kupffer cells were identified as G-CSF-producing cells in the liver. These results indicated that Cd has a characteristic effect of delayed induction of G-CSF in the liver, causing systemic inflammation accompanied by prolonged neutrophilia.

Suppression of erythropoietin induction by diethylstilbestrol in rats.

Diethylstilbestrol is an estrogenic endocrine disrupter that has diverse health effects in humans. Bisphenol A is another estrogen-like chemical with possible similar effects to diethylstilbestrol, which has been increasingly used for industry to lead to globally widespread human exposure to it. Hematopoiesis is another of their possible targets, since estrogen suppresses erythropoietin induction to induce anemia. The aim of this study was to clarify the effects of diethylstilbestrol and bisphenol A on erythropoietin induction in rats. We observed the effects of one-shot subcutaneous injection of diethylstilbestrol or bisphenol A on hypoxia-, bleeding-, and cobalt-stimulated erythropoietin induction within 24 h and the hematological outcomes after repeated subcutaneous injection of diethylstilbestrol three times a week for 1 month in rats. Diethylstilbestrol at 10-1,000 µg/kg suppressed stimulus-elevated levels of plasma erythropoietin and its renal mRNA induction. In contrast, bisphenol A at 1,000 µg/kg did not suppress plasma erythropoietin elevated by any stimuli. Repeated injection of diethylstilbestrol at 1,000 µg/kg to rats for 1 month induced an anemic trend due to decelerated erythropoiesis through the insufficient production of erythropoietin, mimicking the effects of estradiol. In conclusion, diethylstilbestrol has a suppressive effect on erythropoietin induction, leading to deceleration of erythropoiesis and the development of anemia.

Relationship between personal-sampled air lead and blood lead in low-lead-exposure workers in Japan to apply multiple regression models determining permissible air lead concentration.

OBJECTIVES: We investigated the relationship between lead in air (Pb-A) measured by personal sampling and blood lead (Pb-B) in workers with relatively low lead exposure to estimate the permissible air concentration of lead corresponding to the biological tolerance value of Pb-B of 15 µg/dL. METHODS: We collected air samples at a lead-acid battery factory in Japan by personal sampling devices attached to 32 workers (19 males and 13 females) and measured Pb-A by a graphite furnace atomic absorption spectrophotometer in 2017-2020. In addition, we collected information on age, smoking habits, Pb-B, and urinary δ-aminolevulinic acid from the records of medical examinations for lead poisoning. Samples were collected two times from four workers, resulting in 36 data sets. RESULTS: Before analyses, we excluded four inappropriate data sets. The levels of Pb-A in the factory and Pb-B in the workers were almost under the current permissible limits. Multiple regression models showed significant correlations between Pb-B and Pb-A, and sex, and borderline significance between Pb-B and age. Based on them, we calculated Pb-A corresponding to Pb-B 15 µg/dL, and obtained similar values to the current occupational exposure limit (OEL) of 30 µg/m3 , with slight variation between sex and age. CONCLUSION: These results validate OEL, although supplementary conditions in terms of sex and age may be necessary.

Reassessment of the threshold of the blood lead level to increase urinary δ-aminolevulinic acid based on their relationship in recent lead workers in Japan.

OBJECTIVES: The present study investigated the quantitative relationship between blood lead (Pb-B) and urinary δ⁻aminolevulinic acid (ALA-U) in lead workers, and examined the Pb-B level that induces increases in ALA-U and the corresponding ALA-U. METHODS: We collected 10 562 data sets on Pb-B, ALA-U, age, and smoking habits from 808 workers (771 males and 37 females) who underwent multiple lead poisoning medical examinations at a lead-acid battery and lead smelting plant in Japan between 1995 and 2018. Females were excluded, and data collected in 169 subjects prior to engaging in lead work were used as the control. Pb-B and ALA-U levels were measured by graphite furnace atomic absorption spectrophotometry and high-performance liquid chromatography respectively. RESULTS: A significant dose-response relationship was observed between Pb-B and ALA-U based on Pb-B-classified observations of increases in ALA-U values and the prevalence of over-reference ALA-U as well as regression analyses independent of smoking habits. The results obtained revealed that the threshold of Pb-B to increase ALA-U was 25.1-35.0 µg/dL based on the significant elevation point of the prevalence of over-reference ALA-U and 16.2-22.3 µg/dL from a 3rd degree regression equation. CONCLUSIONS: We proposed a threshold of Pb-B to increase ALA-U of 20 µg/dL and a biologically acceptable value of ALA-U of 1 mg/L, corresponding to the threshold.

Latest status of cadmium accumulation and its effects on kidneys, bone, and erythropoiesis in inhabitants of the formerly cadmium-polluted Jinzu River Basin in Toyama, Japan, after restoration of rice paddies.

PURPOSE: The cadmium-polluted Jinzu River Basin in Toyama, Japan, where nephropathy and itai-itai disease were endemic among resident farmers decades ago, has been almost completely restored. The aim of this study is to investigate whether inhabitants there would still exhibit cadmium accumulation and its effects on kidneys, bones, and erythropoiesis. METHODS: We performed a cross-sectional study of 150 subjects from the polluted area and 144 controls from the same prefecture. Participants included female inhabitants from 34 to 74 years of age who underwent examinations to gather anthropometrical and medical information, obtain rice, blood and urine samples, and measure bone mineral density. RESULTS: Cadmium concentration in rice from the polluted area was lower than the level in the control area. Blood and urinary cadmium and urinary ß(2)-microglobulin levels were higher in subjects from the polluted area than controls, and the urinary ß(2)-microglobulin was independently affected by urinary cadmium. Bone mineral density did not differ between the two areas, but it was affected by renal tubular function in subjects from the polluted area. Serum bone alkaline phosphatase was lower in subjects from the polluted area compared to controls. We detected three cases of cadmium nephropathy among the subjects. One of them suffered from a renal anemia type of itai-itai disease. CONCLUSION: Inhabitants in the formerly polluted area still had high cadmium accumulations and showed a characteristic natural history of chronic cadmium toxicity, indicating that the risk remains for developing nephropathy or itai-itai disease in the future.

Exposure Assessment of Cadmium in Female Farmers in Cadmium-Polluted Areas in Northern Japan.

Akita prefecture is located in the northern part of Japan and has many cadmium-polluted areas. We herein performed an exposure assessment of cadmium in 712 and 432 female farmers in two adjacent cadmium-polluted areas (A and B, respectively), who underwent local health examinations from 2001-2004. We measured cadmium concentrations in 100 food items collected from local markets in 2003. We then multiplied the intake of each food item by its cadmium concentration in each subject to assess cadmium intake from food and summed cadmium intake from all food items to obtain the total cadmium intake. Median cadmium intake levels in areas A and B were 55.7 and 47.8 µg/day, respectively, which were both higher than that of the general population and were attributed to local agricultural products, particularly rice. We also calculated weekly cadmium intake per body weight and compared it to the previous provisional tolerable weekly intake reported by the Joint FAO (Food and Agriculture Organization)/WHO (World Health Organization) expert committee on food additives or current tolerable weekly intake in Japan of 7 µg/kg BW/week. Medians in areas A and B were 7.2 and 6.0 µg/kg BW/week, respectively. Similar estimated values were also obtained by the Monte Carlo simulation. These results demonstrated that the cadmium exposure levels among the farmers were high enough to be approximately the tolerable weekly intake.

Gene expression signatures in peripheral blood cells from Japanese women exposed to environmental cadmium.

The objective of this study was to examine the effects of environmental cadmium (Cd) exposure on the gene expression profile of peripheral blood cells, using an original oligoDNA microarray. The study population consisted of 20 female residents in a Cd-polluted area (Cd-exposed group) and 20 female residents in a non-Cd-polluted area individually matched for age (control group). The mRNA levels in Cd-exposed subjects were compared with those in respective controls, using a microarray containing oligoDNA probes for 1867 genes. Median Cd concentrations in blood (3.55 microg/l) and urine (8.25 microg/g creatinine) from the Cd-exposed group were 2.4- and 1.9-times higher than those of the control group, respectively. Microarray analysis revealed that the Cd-exposed group significantly up-regulated 137 genes and down-regulated 80 genes, compared with the control group. The Ingenuity Pathway Analysis Application (IPA) revealed that differentially expressed genes were likely to modify oxidative stress and mitochondria-dependent apoptosis pathways. Among differentially expressed genes, the expression of five genes was positively correlated with Cd concentrations in blood or urine. Quantitative real-time PCR (RT-PCR) analysis validated the significant up-regulation of CASP9, TNFRSF1B, GPX3, HYOU1, SLC3A2, SLC19A1, SLC35A4 and ITGAL, and down-regulation of BCL2A1 and COX7B. After adjustment for differences in the background characteristics of the two groups, we finally identified seven Cd-responsive genes (CASP9, TNFRSF1B, GPX3, SLC3A2, ITGAL, BCL2A1, and COX7B), all of which constituted a network that controls oxidative stress response by IPA. These seven genes may be marker genes useful for the health risk assessment of chronic low level exposure to Cd.

[Anemia induced by cadmium intoxication].

Anemia is commonly induced by chronic cadmium (Cd) intoxication. Three main factors are involved in the development of Cd-induced anemia: hemolytic, iron-deficiency, and renal. Intravascular hemolysis can occur at the early stage of Cd exposure owing to the direct damaging effect on erythrocytes. In addition, Cd that accumulates in erythrocytes affects membrane cytoskeletons and decreases cell deformability, and these cells are then trapped and destroyed in the spleen. Iron deficiency can be detected in animals after an oral exposure to Cd, which competes with iron for absorption in the intestines, leading to anemia. However, an increase in body iron content along with anemia is often observed in cases of parenteral exposure or itai-itai disease. Therefore, it is estimated that Cd disrupts the efficient usage of iron in hemoglobin synthesis in the body. Renal anemia is observed during the very last phase of chronic, severe Cd intoxication, such as itai-itai disease, showing a decrease in the production of erythropoietin from renal tubular cells. Because the renal anemia is based on the same pathophysiology as Cd-induced osteomalacia, which is derived from the disturbance of mineral metabolism due to renal tubular dysfunction, it is reasonable to include renal anemia in the criteria for the diagnosis of itai-itai disease. Hemodilution could also contribute to the development of Cd-induced anemia. Bone marrow hypoplasia or the inhibition of heme synthesis might only be involved in Cd-induced anemia in severe cases of Cd intoxication.

Dietary glycemic index and load in relation to metabolic risk factors in Japanese female farmers with traditional dietary habits.

BACKGROUND: Little is known about the relation of dietary glycemic index (GI) and glycemic load (GL) to metabolic risk factors, particularly in non-Western populations. OBJECTIVE: We examined the cross-sectional associations between dietary GI and GL and several metabolic risk factors in healthy Japanese women with traditional dietary habits. DESIGN: The subjects were 1354 Japanese female farmers aged 20-78 y from 5 regions of Japan. Dietary GI and GL were assessed with a self-administered diet-history questionnaire. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Fasting blood samples were collected for biochemical measurements. RESULTS: The mean dietary GI was 67, and the mean dietary GL (/1000 kcal) was 88 (GI for glucose = 100). White rice (GI = 77) was the major contributor to dietary GI and GL (58.5%). After adjustment for potential dietary and nondietary confounding factors, dietary GI was positively correlated with BMI (n = 1354; P for trend = 0.017), fasting triacylglycerol (n = 1349; P for trend = 0.001), fasting glucose (n = 764; P for trend = 0.022), and glycated hemoglobin (n = 845; P for trend = 0.038). Dietary GL was independently negatively correlated with HDL cholesterol (n = 1354; P for trend = 0.004) and positively correlated with fasting triacylglycerol (P for trend = 0.047) and fasting glucose (P for trend = 0.012). CONCLUSIONS: Both dietary GI and GL are independently correlated with several metabolic risk factors in subjects whose dietary GI and GL were primarily determined on the basis of the GI of white rice.

Dietary patterns associated with bone mineral density in premenopausal Japanese farmwomen.

BACKGROUND: Because several nutrients are known to affect bone mineral density (BMD), the analysis of dietary patterns or combinations of foods may provide insights into the influence of diet on bone health. OBJECTIVE: We evaluated associations between dietary patterns and BMD in Japanese farmwomen. DESIGN: The study included 291 premenopausal farmwomen (aged 40-55 y) who participated in the Japanese Multi-centered Environmental Toxicant Study (JMETS; n = 1407). Forearm BMD was measured by using dual-energy X-ray absorptiometry. Diet was assessed by using a validated self-administered diet history questionnaire comprising 147 food items, from which 30 food groups were created and entered into a factor analysis. RESULTS: Four dietary patterns were identified. The "Healthy" pattern, characterized by high intakes of green and dark yellow vegetables, mushrooms, fish and shellfish, fruit, and processed fish, was positively correlated with BMD after adjustment for several confounding factors (P = 0.048). In contrast, the "Western" pattern, characterized by high intakes of fats and oils, meat, and processed meat, tended to be inversely associated with BMD; however, the association was not significant (P = 0.08). CONCLUSION: A dietary pattern with high intakes of fish, fruit, and vegetables and low intakes of meat and processed meat may have a beneficial effect on BMD in premenopausal women.

Dioxin concentration in human milk in Hebei province in China and Tokyo, Japan: potential dietary risk factors and determination of possible sources.

Very limited information is available on body burdens and environmental levels of dioxins and dioxin-like PCBs (dl-PCBs) in mainland China. In the current studies, human milk samples were collected from 30 breastfeeding mothers in Shijiazhuang city (industrialized) and 11 in the Tanshan countryside (agricultural) of Hebei Province in northern China. An additional 20 samples were obtained from mothers in Tokyo, Japan. PCDDs, PCDFs, and dl-PCBs in human milk were analyzed by high-resolution gas chromatography/high-resolution mass spectrometry. Our results show that arithmetic means for body burdens of PCDDs/Fs and dl-PCBs in Hebei were 3.6 and 1.9 pg TEQg(-1) fat, respectively, which were only about one fourth of the levels in Japan. In addition, no difference was found in the chemical levels except dl-PCBs between the urban and rural areas. Based on the results of an in-person interview of the Chinese mothers using a 59-item questionnaire, freshwater fish consumption was found to correlate with the body burden of dioxins. Principal component analysis of dioxin congeners revealed that the patterns of dioxins in the Hebei urban and rural areas are quite similar; however, they are clearly different from those in Japan. Collectively, our results suggest that the lower body burdens of dioxin in Hebei may be due in part to the relatively slow industrialization and a lower consumption of marine foods. Finally, the results indicate that comprehensive monitoring of dioxins and dl-PCBs in humans as well as in the environment and foods is necessary in China.

A soy diet accelerates renal damage in autoimmune MRL/Mp-lpr/lpr mice.

Isoflavones, which are phytoestrogens present in large quantities in soy and soy-derived products, have estrogenic activity, inhibit protein tyrosine kinase, and exert other effects in the human body. Thus, the recent spread of soy consumption in Western populations emphasizes the need to more fully understand the potential effects in the body, especially in abnormal immune conditions. In the present study, the influence of a soy diet on lupus disease in MRL/Mp-lpr/lpr (MRL/lpr) mice was investigated. Weanling female MRL/lpr mice (4 weeks) were fed a soy diet (20% soybean protein and 5% soybean oil). The soy diet exacerbated renal damage; findings in this mouse strain included accelerated proteinuria, elevated serum creatinine concentrations, and reduced creatinine clearance. No effects were detected, however, in C3H/HeN mice, which have the same H-2(k) genetic background as MRL/lpr mice do. A tendency toward an increase in thymus weight and proliferation of T cells in spleen and B cells in lymph nodes were found at the age of 16 weeks. These findings indicate that a soy diet, in comparison with a casein diet, significantly exacerbates the clinical course of this autoimmune disease. Further research on the mechanism of this effect of soy-rich diets is needed, and isoflavone supplementation for systemic lupus erythematosus patients should be carefully reevaluated.

[Effects of volcanic sulfur dioxide on reconstruction workers and residents returning to Miyake Island].

Four and half years have passed since Mt. Oyama in Miyake Island erupted. Ambient sulfur dioxide (SO2) is still above the environmental air standard in parts of the island, even though emission of the volcanic gas has diminished. Reconstruction of life-support infrastructure in the island and safety measures started in July 2002, and a short-term trial stay project for former residents was completed in April 2003 for the total rehabilitation of the island. We conducted health examinations, and questionnaires on clinical symptoms among the reconstruction workers in January 2003, as well as questionnaires on respiratory symptoms among former residents who joined the trial stay, in late fall of 2003. The peak expiratory flow rate and symptoms of the workers and the symptoms of the short-term residents were not correlated to SO2 concentrations. In addition, we investigated the medical histories of outpatients at Miyake-mura National Insurance Center Clinic. We could not recognize any cases directly connected with higher ambient SO2 concentration in the area of the island, but there was a male worker, in his thirties, who suffered an initial attack of bronchial asthma, which required intensive care and treatment. As a general rule, all the workers and the residents are asked to wear gas masks for SO2 when its concentration is above 2.0 ppm and to stay in the house with desulfuration facilities as a precautionary measure at night. The residents are now well informed about the risks of volcanic gas and preventive measures for adverse health effects. We could not evaluate correlations between SO2 exposure indices and health effects sufficiently due to the limitation of the field study, but this study presents useful pieces of information as a risk communication for reconstruction workers and former inhabitants to the island.

Oestrogen causes G2/M arrest and apoptosis in breast cancer cells MDA-MB-231.

Flavonoids have been shown to exert many biological activities within cancer cells, and oestrogen is known to be structurally related to flavonoids. We investigated the effects of oestrogen in cancer cells to determine if its activities would be similar to those of flavonoids. When 50 microM 17 beta-oestradiol (oestradiol) was added to the oestrogen receptor (ER) alpha-negative breast cancer cell line MDA-MB-231, growth arrest was apparent, similar to that observed with genistein and daidzein. Oestradiol exhibited a dose response curve for the growth arrest similar to those of genistein and daidzein. Apoptosis occurred in the breast cancer cells after treatment with 50 microM oestradiol, genistein, or daidzein, with similar profiles. Flow cytometry analysis revealed that oestradiol treatment caused G2/M arrest and apoptosis. Cell-cycle arrest at G2/M began at 6 h after treatment, and apoptosis began within 24 h. Because MDA-MB-231 cells are ER alpha negative, these results suggest that oestradiol induces cell-cycle arrest and apoptosis through an ER alpha-independent pathway.

Acute exposure to cobalt induces transient methemoglobinuria in rats.

We observed transient excretion of dark-brown urine after acute exposure to cobalt in rats and investigated the mechanism of it. We injected cobalt into rats s.c. at a dose of 15 mg/kg and collected urine, peripheral blood, and organ samples at the indicated times after injection. Biochemical and histopathological examinations of these samples were conducted. Obvious macroscopic and biochemical methemoglobinuria was observed just after injection of cobalt, but the level of urinary methemoglobin decreased gradually, almost disappearing by 24 h. The levels of cobalt in peripheral blood and urine showed a very similar pattern to that of methemoglobinuria. Neither anemia nor bilirubinemia was observed, indicating no extrarenal intravascular hemolysis. Pathological examination of the kidneys revealed that the glomerular capillaries were filled with red blood cells at 1 h after injection. Electron microscopy showed deformed red blood cells in the glomerular capillaries and condensed hemoglobin in Bowman's capsule that passed through the basement membrane. There were no trends toward increases in plasma levels of creatinine or blood urea nitrogen. These results indicate that exposure to cobalt induces transient methemoglobinuria through the lysis of red blood cells and oxidation of iron in hemoglobin at the glomerular capillaries without causing renal dysfunction.