Fujio Kayama

Arsenic can mediate skin neoplasia by chronic stimulation of keratinocyte-derived growth factors

Although numerous epidemiological studies have shown that inorganic arsenicals are human skin carcinogens, there is currently no accepted mechanism for its action or an established animal model for its study. We observed increased mRNA transcripts and secretion of keratinocyte growth factors, including granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-alpha (TGF-alpha) and the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) in primary human epidermal keratinocytes cultured in the presence of low micromolar concentrations of sodium arsenite. Total cell numbers, as well as c-myc expression and incorporation of [3H]thymidine, both indicators of cell proliferation, were also elevated in keratinocyte cultures treated with sodium arsenite. As an in vivo model, the influence of arsenic on mouse skin tumor development was studied in transgenic TG.AC mice which carry the v-Ha-ras oncogene, and can serve as a genetically initiated model for skin carcinogenesis. Following low-dose application of 12-O-tetradecanoyl phorbol-13-acetate (TPA), a marked increase in the number of skin papillomas occurred in transgenic mice receiving arsenic in the drinking water as compared to control drinking water. Papillomas did not develop in arsenic-treated transgenic mice that had not received TPA or arsenic-treated wild-type FVB/N mice, suggesting that arsenic is neither a tumor initiator or promoter but rather an enhancer. Injection of anti-GM-CSF antibodies following application of TPA in transgenic mice reduced the number of papillomas. Consistent with that observed in human keratinocyte cultures, increases in GM-CSF and TGF-alpha mRNA transcripts were found within the epidermis of arsenic-treated mice when compared to controls within 6 weeks of treatment. These results suggest that arsenic enhances papilloma development via the chronic stimulation of keratinocyte-derived growth factors and represents the first example of a chemical carcinogen that acts in this manner. These studies suggest that in vitro studies with human keratinocyte cultures examined in conjunction with TG.AC transgenic mice can provide a useful model for examining the tumor enhancing properties of environmental chemicals.

Effects of diverse dietary phytoestrogens on cell growth, cell cycle and apoptosis in estrogen-receptor-positive breast cancer cells

Phytoestrogens have attracted attention as being safer alternatives to hormone replacement therapy (HRT) and as chemopreventive reagents for breast cancer because dietary soy isoflavone intake has been correlated with reduction in risk. To identify safe and effective phytoestrogen candidates for HRT and breast cancer prevention, we investigated the effects of daidzein, genistein, coumestrol, resveratrol and glycitein on cell growth, cell cycle, cyclin D1 expression, apoptosis, Bcl-2/Bax expression ratio and p53-dependent or NF-kappaB-dependent transcriptional activity in MCF-7 breast cancer cells. Phytoestrogens, except for glycitein, significantly enhanced estrogen-response-element-dependent transcriptional activity up to a level similar to that of 17beta-estradiol (E(2)). E(2) increased cell growth significantly, coumestrol increased cell growth moderately, and resveratrol and glycitein reduced cell growth. Phytoestrogens, except for glycitein, stimulated the promotion of cells to G(1)/S transition in cell cycle analysis, similar to E(2). This stimulation was accompanied by transient up-regulation of cyclin D1. While genistein, resveratrol and glycitein all increased apoptosis and reduced the Bcl-2/Bax ratio, resveratrol reduced this ratio more than either genistein or glycitein. Moreover, resveratrol significantly enhanced p53-dependent transcriptional activity, but slightly reduced NF-kappaB-dependent transcriptional activity. On knockdown analysis, genistein, resveratrol and glycitein all reduced the Bcl-2/Bax ratio in the presence of apoptosis-inducing stimuli, and estrogen receptor (ER) alpha silencing had no effect on these reductions. In contrast, in the absence of apoptosis-inducing stimuli, only resveratrol reduced the ratio, and ERalpha silencing abolished this reduction. Thus, resveratrol might be the most promising candidate for HRT and chemoprevention of breast cancer due to its estrogenic activity and high antitumor activity.

Rationale and study design of the Japan environment and children’s study (JECS)

BackgroundThere is global concern over significant threats from a wide variety of environmental hazards to which children face. Large-scale and long-term birth cohort studies are needed for better environmental management based on sound science. The primary objective of the Japan Environment and Children’s Study (JECS), a nation-wide birth cohort study that started its recruitment in January 2011, is to elucidate environmental factors that affect children’s health and development.Methods/DesignApproximately 100,000 expecting mothers who live in designated study areas will be recruited over a 3-year period from January 2011. Participating children will be followed until they reach 13 years of age. Exposure to environmental factors will be assessed by chemical analyses of bio-specimens (blood, cord blood, urine, breast milk, and hair), household environment measurements, and computational simulations using monitoring data (e.g. ambient air quality monitoring) as well as questionnaires. JECS’ priority outcomes include reproduction/pregnancy complications, congenital anomalies, neuropsychiatric disorders, immune system disorders, and metabolic/endocrine system disorders. Genetic factors, socioeconomic status, and lifestyle factors will also be examined as covariates and potential confounders. To maximize representativeness, we adopted provider-mediated community-based recruitment.DiscussionThrough JECS, chemical substances to which children are exposed during the fetal stage or early childhood will be identified. The JECS results will be translated to better risk assessment and management to provide healthy environment for next generations.

Dietary exposure to cadmium at close to the current provisional tolerable weekly intake does not affect renal function among female Japanese farmers

Dietary cadmium (Cd) exposure and renal tubular function were investigated in 1381 female farmers from five districts in Japan (Japanese Multi-centered Environmental Toxicant Study project; JMETS). Dietary Cd exposure of the five populations was assessed from the individual Cd concentrations of the rice consumed by the study participants and the quantities of rice consumed daily. The populations showed a sequential difference in dietary Cd exposure, ranging from a level as low as that of the general Japanese population to one close to the current provisional tolerable weekly intake (PTWI). The levels of urinary Cd excretion, an indicator of Cd accumulation in the kidneys, increased along the same sequential pattern as dietary Cd exposure. However, no differences were observed among the populations in levels of urinary alpha 1-microglobulin and beta 2-microglobulin excretion, which are indicators of renal tubular function. These results indicate that the current PTWI is sufficient to prevent Cd-induced renal dysfunction among the general population.

Placental transfer of melamine and its effects on rat dams and fetuses.

In 2008, an epidemic of cases of renal failure among Chinese infants, due to melamine contamination of milk, raised international concern. Thus, numerous studies on the metabolism of melamine were broadly undertaken. However, little is known about placental transfer of melamine. In this study, the possibility of placental transfer of melamine and its effects on fetuses and pregnant dams were determined. Melamine was respectively administered at 0, 40 and 400mg/kg body weight by daily gavage from gestation day (GD) 13 to GD 20 to control (C), low melamine (LM) and high melamine (HM) groups of pregnant female F344 rats. Rats were sacrificed 30min after the last gavage. Melamine was not detected in any of the control and placental samples, or in amniotic fluid from the LM group. Plasma and fetal melamine concentrations in the HM group were significantly higher than in the LM group (P<0.01). Liver enzyme determination revealed no differences among the three groups. However, plasma creatinine, plasma uric acid and blood urea nitrogen concentrations in dams were significantly increased by melamine (P<0.05). These results show that ingested melamine affects renal function in dams and dose-dependently passes the placental barrier to reach the fetus.

Levels of persistent organic pollutants in human milk in two Chinese coastal cities, Tianjin and Yantai: Influence of fish consumption

In 2006-2007, we collected human milk from 60 and 48 donors in the Chinese coastal cities of Tianjin and Yantai, respectively, in accordance with the WHO/UNEP global milk survey. We determined the concentrations of organochlorine pesticides by GC/MS/MS and dioxins by XDS-CALUX bioassay in the individual milk specimens. The geometric mean concentrations (GMs) of beta-HCH (586.7ng g(-1) fat), total DDTs (855.9ngg(-1) fat), and dl-PCBs (4.4pg CALUX-TEQg(-1) fat) in the milk from Yantai were higher than those from Tianjin (254.4ng g(-1) fat, 654.7ngg(-1) fat, 1.9pg CALUX-TEQ g(-1) fat, respectively). However, the GMs of HCB (41.1ngg(-1) fat) and PCDD/Fs (13.1pg CALUX-TEQg(-1) fat) from Tianjin were higher than those from Yantai (15.7ng g(-1) fat, 9.9pg CALUX-TEQ g(-1) fat). The low ratios of DDT/(DDE+DDD) in milk from both areas suggested that past exposure contributed to the total DDTs body burden. The dl-PCBs body burden in the high sea fish intake group was higher than that in the low intake group, both with and without adjustments for potential influencing factors. For beta-HCH, a marginal P value (P=0.063) was observed between high and low sea fish consumption groups after adjusting for potential influencing factors. Donors in the high freshwater fish group showed higher PCDD/Fs and HCB levels than those in the low intake group, both with and without adjustments. Further monitoring studies of POPs contamination in human milk and foods are needed in China.

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.

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 β2-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.

Levels of dioxins and polybrominated diphenyl ethers in human milk from three regions of northern China and potential dietary risk factors.

Human milk samples were collected from 50 breast-feeding mothers in Shijiazhuang, 60 in Tianjin and 48 in Yantai from November 2006 to April 2007. The three areas are located in northern China. We selected randomly 20 samples from each area for detection of polychlorinated dibenzodioxins/frans (PCDD/Fs), polycholorinated biphenyls (dl-PCBs) and polybrominated diphenyl ethers (PBDEs) by high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). Our results show the average concentrations of PCDD/Fs plus dl-PCBs were 6.24 TEQ pg g(-1)fat, 7.54 TEQ pg g(-1)fat and 6.69 TEQ pg g(-1) fat in human milk from Shijiazhuang, Tianjin and Yantai, respectively. The average concentrations of PBDEs were 3.71 ng g(-1) fat, 3.42 ng g(-1) fat and 4.16 ng g(-1) fat in human milk from Shijiazhuang, Tianjin and Yantai, respectively. Among congeners of PBDEs, the high and low brominated congeners BDE209, BDE207, BDE197, BDE153, BDE15, BDE28 and BDE47 were the predominant PBDE congeners, accounting for 91%, 90% and 84% of total PBDEs in samples from Shijiazhuang, Tianjin and Yantai, respectively. Based on the results of an in-person interview of mothers using a questionnaire, freshwater fish consumption was found to correlate with total mono-ortho dl-PCBs (mo-PCBs) and sea fish consumption was found to correlate with total non-ortho dl-PCBs (no-PCBs) of human milk in these areas. However, no correlation was found between concentrations of total PBDEs and total TEQ of PCDD/Fs plus dl-PCBs and food consumption. Continuous surveillance on dioxins and dl-PCBs levels in human milk is needed to correctly evaluate both the environmental impact and human health risk in China.

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.