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.

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.

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.

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.

Age-relevant renal effects of cadmium exposure through consumption of home-harvested rice in female Japanese farmers.

There are cadmium-polluted areas in Japan, where farmers may be at risk of renal dysfunction due to cadmium exposure through consumption of home-harvested rice. The aims of this study were to investigate levels of cadmium exposure and accumulation and their renal effects in female farmers residing in cadmium-polluted areas, and to consider the relevance of age to the effects of cadmium. We conducted a cross-sectional study of 1200 women (40-79years old) without symptomatic disorders in two cadmium-polluted areas and one unpolluted area as a control. Rice, blood, and urine samples were collected to measure the cadmium levels, together with urinary levels of α1-microglobulin and ß2-microglobulin for renal tubular function. Cadmium levels in rice were significantly higher in the polluted areas than control area. Blood and urinary cadmium levels, along with urinary protein levels, were also significantly higher in the polluted areas, especially among the elder subjects. There was one case of cadmium nephropathy in the polluted areas. Age- and urinary cadmium-specific analysis for all the subjects showed a mild linear dose-response relationship between urinary cadmium and proteins in the younger women, and a steep progress of renal dysfunction over the threshold of urinary cadmium (10µg/g creatinine) in the older women. In conclusion, the aged women in the polluted areas showed high accumulation of cadmium and deterioration of renal function through consumption of rice. Also, the aging process itself appeared to contribute to the different renal effects of cadmium observed in the elderly population.

Relationships between QT interval and heart rate variability at rest and the covariates in healthy young adults.

To clarify the links between ECG QT-related parameters and heart rate variability (HRV) and the covariates possibly distorting them, the averaged RR and QT intervals in a single lead ECG were measured for 64 male and 86 female subjects aged 18-26. The QT index, defined by Rautaharju et al., in the young adults was not significantly related to any HRV parameters nor heart rate, but the Bazett's corrected QT (QTc) interval was associated negatively with the parasympathetic activity and positively with heart rate. No significant differences in the QTc interval, QT index or heart rate were seen between the men and women, but they significantly differed between both sexes after adjustment for possible covariates such as age and body mass index (BMI). Significant sex differences in parasympathetic parameters of the HRV were unchanged before and after the adjustment, but significant differences observed in the unadjusted sympathetic parameters disappeared after adjusting for covariates. Age, BMI and body fat percentage also were significant covariates affecting these ECG parameters. Consequently, QT index, unaffected by heart rate and HRV parameters, appears to be a more useful indicator than the QTc interval. Instead, the QT index and HRV parameters are recommended to be simultaneously measured in epidemiological research because they are probably complementary in assessing autonomic nervous function. Also, these parameters should be analyzed in men and women separately.

[Current status of cadmium exposure among Japanese, especially regarding the safety standard for cadmium concentration in rice and adverse effects on proximal renal tubular function observed in farmers exposed to cadmium through consumption of self-grown rice].

Because the staple food in Japan is rice, which absorbs cadmium (Cd) from the soil efficiently, rice is the main source of exposure to Cd in the Japanese population. In addition, there have been many Cd-contaminated farming areas in Japan. Therefore, a safety standard for the Cd concentration in rice was set as 0.4 ppm by the Japanese government. This safety standard has been followed for decades without any appropriate scientific or legal basis. However, recent epidemiological studies of female Japanese farmers exposed to Cd through self-grown rice, that is, a series of Japanese Multi-centered Environmental Toxicant Study (JMETS), showed evidence that the safety standard is appropriate. Therefore, general Japanese consumers are unlikely exposed to Cd excessively with the application of this safety standard, considering the trend of decreasing amount of rice consumed among the Japanese population. On the other hand, Japanese farmers were found to be at risk of Cd exposure through the consumption of self-grown rice with a high Cd concentration. Actually, the JMETS showed that female farmers at 70 years of age or older had a decreased proximal renal tubular function due to the high renal accumulation of Cd. On the basis of these findings, "medical examinations for Cd exposure" have recently been implemented for farmers residing in Cd-polluted areas in northern Japan. Because it has been estimated that such Cd-polluted areas are actually larger, it is necessary to implement medical examinations of more farmers there, particularly the elderly.

Cadmium induces anemia through interdependent progress of hemolysis, body iron accumulation, and insufficient erythropoietin production in rats.

Cadmium is a toxic heavy metal and distributed widely in the environment. In addition to damaging the liver, kidneys, and bone, cadmium causes anemia through hemolysis, iron deficiency, and insufficient erythropoietin (EPO) production (renal anemia) along with changes in iron metabolism. Here, we investigated the role of iron in the interdependent progress of three types of anemia in cadmium-injected rats fed iron-sufficient or iron-deficient diets for 1 or 3 months. Cadmium injections for 1 month induced renal anemia without renal injury. Injections for 3 months induced hemolysis, iron deficiency, and renal anemia, accompanied by hepatic and renal damage. Iron concentrations in the liver, kidney, and spleen were increased, derived from internally released iron from hemolyzed red blood cells, increased duodenal iron absorption, insufficient erythropoiesis, and hepatic ferritin overproduced by cadmium-induced interleukin-6. Therefore, the iron deficiency anemia was actually apparent. Cadmium suppressed renal EPO production through a direct effect, accumulated iron, and destruction of EPO-producing cells. Increased duodenal iron absorption could be attributed to hypertrophy of the duodenal mucosa derived from anemia. Thus, insufficient EPO production and iron accumulation are the central factors driving anemia in cadmium toxicity.

[Application of the benchmark dose approach to epidemiological endpoints with clinical standards].

OBJECTIVE: By publishing the scientific opinion entitled "Use of the benchmark dose (BMD) approach in risk assessment: Guidance of the Scientific Committee," the European Food Safety Authority (EFSA) recommended that the BMD approach, as an alternative to the traditionally used no-observed-adverse-effect level approach, be used as the method of choice for the determination of the reference point for deriving health-based guidance values and margins of exposure, and described the BMD approach as being extensively applicable to all chemicals, even to dose-effect assessment of epidemiological data. When the BMD approach was first proposed, the approach was expected to estimate the dose causing a low but measurable target organ effect. We examined whether the BMD approach can be applied to epidemiological endpoints with clinical standards. METHODS: Comparisons between the BMD approach recommended by the EFSA and classical BMD approach (hybrid method) were conducted using epidemiological data with clinical standards. RESULTS: The 95% lower confidence limit of the EFSA-based BMD tended to be considerably lower than that of the hybrid method. The former approach is easily applicable to dose-response data in published papers, though it is difficult to adjust for possible confounders. The cutoff values, calculated by the hybrid method, of epidemiological endpoints are nearly concordant with the clinical standards. CONCLUSIONS: By using the BMD approach recommended by the EFSA, human health safety may be better guaranteed because of the lower reference points for hazardous substances. However, its application to epidemiological data does not always reflect toxicological implications in light of the clinical standard.

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.