Masayoshi Ichiba

Exposure assessment of organophosphorus and organobromine flame retardants via indoor dust from elementary schools and domestic houses

To assess the exposure of flame retardants (FRs) for school-children, organophosphorus flame retardants and plasticizers (PFRs) and organobromine flame retardants (BFRs) were determined in the indoor dust samples collected from elementary schools and domestic houses in Japan in 2009 and 2010. PFRs were detected in all the dust samples analyzed and the highest concentration of total PFRs was thousand-fold higher than that of BFRs. Among the PFRs, tris(butoxyethyl)phosphate (TBOEP) showed the highest concentration with a median (med.) of 270,000 ng g(-1) dry weight (3700-5,500,000 ng g(-1) dry weight), followed by tris(methylphenyl)phosphate (TMPPs)>triphenyl phosphate (TPHP)=tris(1,3-dichloro-2-propyl)phosphate (TDCIPP)=tris(2-chloroisopropyl)phosphate (TCIPP)=tris(2chloroethyl)phosphate (TCEP)>ethylhexyl diphenyl phosphate (EHDPP). Significantly higher concentrations of TBOEP, tri-n-butyl phosphate (TNBP), TPHP, TMPPs, and total-PFRs were found in dust samples from elementary schools than from domestic houses. It might be due to that higher concentrations of TBOEP (as leveling agent) were detected from the floor polisher/wax products collected in those elementary schools. On the other hand, significantly higher concentrations of TCEP, TCIPPs, and total chloroalkyl-PFRs were found in domestic houses than in elementary schools. Exposure assessments of PFRs via indoor dust from elementary schools and domestic houses were conducted by calculating the hazard quotient (HQ). Among PFRs, HQs for TBOEP exceeded 1 (higher than reference dose: RfD) and its highest value was 1.9. To reduce the intake of TBOEP by school-children, it is recommended that the use of floor polisher/wax containing TBOEP be reduced in schools.

Association of genotypes of carcinogen-activating enzymes, phenol sulfotransferase SULT1A1 (ST1A3) and arylamine N-acetyltransferase NAT2, with urothelial cancer in a Japanese population.

Carcinogenic aromatic amines such as 4‐aminobiphenyl, which is contained in tobacco smoke, are one of the causal factors of urothelial epithelial cancers. 4‐Aminobiphenyl has been shown to be bioactivated through N‐hydroxylation by hepatic cytochrome (CYP) 1A2 and subsequently through O‐sulfation and O‐acetylation by phenol sulfating sulfotransferase, ST1A3 (SULT1A1), and arylamine N‐acetyltransferase, NAT2, respectively. In a case‐control study for urothelial epithelial cancers, low activity alleles of NAT2 are overall high‐risk alleles (OR 2.11; 95% CI 1.08–4.26). Wild‐type ST1A3*1 (213Arg) alleles were slightly overrepresented in nonsmoking urothelial cancer patients (82.6% vs. 69.7%) and in smoking cancer patients (76.7% and 74.3%) compared to a variant ST1A3*2 (213His) allele. In combination of ST1A3 and NAT2 genotypes for analyses of urothelial cancer risk, the highest OR of 2.45 (95% CI 1.04–5.98) was obtained with ST1A3*1 and NAT2 slow genotype among the 4 combinations. Recombinant ST1A3*1 enzyme showed a tendency of catalyzing higher in vitro 3′‐phosphoadenosine 5′‐phosphosulfate‐dependent DNA adduct formation than ST1A3*2 (2.84 ± 0.49 and 2.22 ± 0.11 adducts/108 nucleotides). Combined analyses of different alleles of carcinogenic aromatic amine‐activating phase II enzymes were applied to urothelial cancer risk for the first time and showed the highest risk combination of ST1A3 and NAT2 alleles.

Urinary 1-hydroxypyrene in coke oven workers relative to exposure, alcohol consumption, and metabolic enzymes

OBJECTIVES To investigate the influence of personal lifestyle—such as smoking and alcohol consumption—on urinary 1-hydroxypyrene (1-OHP) concentrations in coke oven workers exposed to polycyclic aromatic hydrocarbons (PAHs) and to evaluate the association of 1-OHP concentrations with the genetic polymorphism of several metabolic enzymes including cytochrome P-450 (CYP) 1A1 and glutathione S-tranferases (GSTs). METHODS The study population contained 162 coke oven workers and 58 controls employed at the largest iron and steel factory in China. Personal data were collected at the interview. 1-OHP in urine was measured with high performance liquid chromatography with fluorescence detection. Genetic polymorphisms were identified by the polymerase chain reaction (PCR) method. RESULTS A positive association between excretion of urinary 1-OHP and the levels of exposure to PAHs was confirmed. Those people who consumed ⩾50 g/day ethanol had significantly higher 1-OHP excretion than did other coke oven workers (p<0.01). No significant difference in urinary 1-OHP was found between smokers and non-smokers, in both controls and exposed subjects. The variant homozygotes at exon 7 of the CYP1A1 gene had significantly higher urinary 1-OHP concentrations than other CYP1A1 genotypes among the exposed workers (p=0.03). There was less association between the concentrations of 1-OHP and the GSTM1, GSTP1, or GSTT1 polymorphism. CONCLUSIONS The present study confirmed that urinary 1-OHP is a good biomarker for exposure to PAHs. Alcohol consumption affected urinary 1-OHP excretion. The variant genotypes of the CYP1A1 gene may result in the enhancement of PAH metabolites. It is helpful to understand the role of individual susceptibility on metabolism of carcinogens. These findings suggest that the modulating effect of individual lifestyle factors or genetic nature should be considered in future studies on occupational exposure to PAHs and in evaluating the health risk from harmful chemicals.

Influence of GSTM1 genotype on association between aromatic DNA adducts and urinary PAH metabolites in incineration workers.

Waste incinerating workers are exposed to various pyrolysis products including polycyclic aromatic hydrocarbons (PAHs). We examined their PAH exposure by assessing urinary 1-hydroxypyrene glucuronide (1-OHPG), as a measure of internal dose, and aromatic DNA adducts in peripheral white blood cells (WBCs), as a measure of biological effect dose. The potential effect of genetic polymorphisms of three enzymes involved in PAH metabolisms (i.e., CYP1A1, GSTM1, and GSTT1) on these exposure markers was also investigated.Twenty-nine employees including workers incinerating industrial wastes and 21 non-exposed on-site controls were recruited from a company handling industrial wastes in South Korea. Sixteen ambient PAHs were determined by GC/MSD (NIOSH method) from personal breathing zone samples of nine subjects working near incinerators. Urinary 1-OHPG was assayed by synchronous fluorescence spectroscopy (SFS) after immunoaffinity purification using monoclonal antibody 8E11. Aromatic DNA adducts in peripheral WBC were measured by the nuclease P1-enhanced post-labelling assay. Genotypes were assessed by PCR-based methods. Information on smoking habits and use of personal protective equipment were collected by self-administered questionnaire. Urinary 1-OHPG levels were significantly higher in workers handling industrial wastes than in those with presumed lower exposure to PAHs (P=0.006, by Kruskal-Wallis test). A statistically significant dose-response increase in 1-OHPG levels was seen with the number of cigarettes consumed per day (r=0.686, P<0.001). Smoking and GSTM1 genotype were significant predictors for log-transformed 1-OHPG by multiple regression analysis (overall model R(2)=0.565, P<0.001), whereas smoking was the only significant predictor for log-transformed aromatic DNA adducts (overall model R(2)=0.249, P=0.201). Aromatic DNA adducts were significantly correlated with log-transformed urinary 1-OHPG level (r=0.31, P=0.04). However, the partial correlation coefficient adjusting for age, sex, and cigarette consumption was not significant (r=0.15, P=0.17). The significant association exists only in individuals with the GSTM1 null genotype (Pearsons correlation coefficient, r=0.52, P=0.01; partial correlation coefficient adjusting for age, sex, and cigarette consumption, r=0.36, P=0.04). Our results suggest that the significant increase in urinary 1-OHPG in the exposed workers is due to higher prevalence of smokers among them, and that the association between urinary PAH metabolites and aromatic DNA adducts in workers of industrial waste handling may be modulated by GSTM1 genotype. These results remain to be confirmed in future larger studies.

Multiple biomarkers study in painters in a shipyard in Korea.

Shipbuilding workers are exposed to a variety of genotoxic compounds including polycyclic aromatic hydrocarbons (PAHs). A limited number of studies have been conducted to evaluate biomarkers related to PAH exposure in painters in the shipyard industry. We examined this in 208 workers recruited from a shipyard located in South Korea. Employees were grouped into three exposure groups: (1) 111 painters using coal tar paints, (2) 70 painters using general paints, and (3) 27 on-site controls using no paints. Levels of urinary 1-hydroxypyrene glucuronide (1-OHPG), as internal dose of PAH exposure, were measured by synchronous fluorescence spectroscopy. Glutathione S-transferase (GST) M1 and T1 genotypes were assessed by a multiplex polymerase chain reaction (PCR)-based method, aromatic-DNA adducts in peripheral white blood cells were measured by 32P-postlabeling, and glycophorin A (GPA) variant frequencies in red blood cells were assessed by flow cytometry. Information on demographic characteristics, smoking habits, diet, job title and use of personal protective equipment (e.g. respiratory and dermal) were collected by self-administered questionnaire. Average urinary 1-OHPG levels in coal tar paint (2.24 micromol/mol creatinine) and general paint (1.38 micromol/mol creatinine) users were significantly higher than in on-site controls (0.62 micromol/mol creatinine) (P<0.001). Paint use, irrespective of the type of paints, and smoking (yes/no) were positively associated with urinary 1-OHPG levels, whereas green tea consumption (yes/no) was negatively associated with the 1-OHPG levels. No significant effect in the 1-OHPG levels were observed for the GSTM1 and GSTT1 genotypes. Aromatic-DNA adduct levels tended to be higher in coal tar paint users (P = 0.06) and painters (P = 0.07) compared to on-site controls. No differences in adduct levels were observed, between the two groups of painters, and the combined group showed greater adduct levels than on-site controls (P = 0.05). GPA mutation frequencies measured in 55 individuals with MN heterozygote genotypes were not significantly different among the three exposure groups, and no correlation was observed between urinary 1-OHPG levels and aromatic-DNA adducts or GPA mutation frequency. These results suggest that painters in the shipyard were exposed to significant amounts of PAHs and possibly to other genotoxic aromatic compounds, and that urinary 1-OHPG may be a potential biomarker of PAH exposure in this population.

Cytochrome P450 1B1 mRNA levels in peripheral blood cells and exposure to polycyclic aromatic hydrocarbons in Chinese coke oven workers.

Cytochrome P450 1B1 (CYP1B1) is induced through the Ah receptor and is involved in the activation of polycyclic aromatic hydrocarbons (PAHs). To determine the validity of a quantitative analysis of CYP1B1 mRNA in peripheral human blood cells for the estimation of PAH exposure, a real-time quantitative polymerase chain reaction method was used to measure the relative levels of CYP1B1 mRNA in 37 Chinese coke oven workers and 13 control workers. A large inter-individual difference in the levels was observed. The average level of the CYP1B1 mRNA in workers at the top work site, where the PAH exposure level from the coke ovens was highest, was significantly higher than in workers at the middle site (P<0.01) or the controls (P=0.02). A non-significant positive correlation was found between the CYP1B1 mRNA levels and urinary 1-hydroxypyrene (R=0.22, P=0.13), and a significant correlation between these mRNA levels and urinary cotinine (R=0.33, P=0.02). It was interesting that a significant positive correlation between CYP1B1 mRNA and 1-hydroxypyrene was observed in subjects with the Leu/Leu type of CYP1B1 Leu432Val polymorphism (R=0.33, P=0.02, n=38) and a non-significant correlation in subjects with the Leu/Val and Val/Val types (R=-0.36, P=0.25, n=12), although the number of subjects in this strata analysis was small. Our preliminary study suggests that PAH exposure in coke ovens and smoking maybe associated with CYP1B1 mRNA levels in peripheral blood cells although mRNA is generally unstable and could be expressed following exposure to other agents.

Relationship between plasma concentrations of β‐carotene and α‐tocopherol and life‐style factors and levels of DNA adducts in lymphocytes

Abstract β‐Carotene and α‐tocopherol have been thought to reduce risk of lung cancer. Whether β‐carotene and α‐tocopherol influence human DNA adducts, indicators of biologically effective doses of carcinogens, has been seldom studied. In this cross‐sectional study, we measured plasma β‐carotene and CL‐tocopherol in 192 healthy men and DNA adducts in lymphocytes in 104 of the subjects. Because genetic polymorphism ofcytochrome P‐4501A1 (CYP1A1) and glutathione S‐transferase M1 (GSTM1) has been associated with interference information of reactive intermediates and detoxification of polycyclic aromatic hydrocarbons, we also obtained data concerning genetic polymorphism of CYPIAI and GSTM1. In multiple regression analysis, parameters such as alcohol consumed per day, high‐density lipoprotein cholesterol, Quetelet index, and cigarettes smoked per day were correlated inversely, whereas age, plasma α‐tocopherol, and intake frequency of fruits were correlated positively with plasma β‐carotene concentration. DNA a...

Interaction between interleukin-1β −31T/C gene polymorphism and drinking and smoking habits on the risk of hepatocellular carcinoma among Japanese

The risk of hepatocellular carcinoma (HCC) increases with the severity of hepatic inflammation. Interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha are proinflammatory cytokines with multiple biological effects and may play essential roles in inflammation-linked tumor development. We conducted a case-control study including 209 incident HCC cases and two control groups (275 hospital controls and 381 patients with chronic liver disease [CLD] without HCC) to investigate whether IL-1B and TNF-A gene polymorphisms influence HCC susceptibility with any interaction with alcohol and tobacco. By comparing HCC cases with CLD patients, we found that IL-1B -31T/C polymorphism was associated with HCC risk among never drinkers and current smokers; adjusted odds ratios (and 95% confidence intervals) for C/T and T/T genotypes compared with C/C genotype were 1.70 (0.76-3.77) and 2.46 (1.05-5.76) (P trend=0.03), respectively, among never drinkers, and 1.53 (0.60-3.99) and 2.54 (0.81-7.95) (P trend=0.11), respectively, among current smokers. Similarly, HCC risk associated with heavy alcohol intake and current smoking differed by this polymorphism among CLD patients. IL-1B -31T/C polymorphism may modify HCC risk in relation to alcohol intake or smoking.

Different responses other than the formation of DNA-adducts between the livers of carcinogen-resistant rats (DRH) and carcinogen-sensitive rats (Donryu) to 3'-methyl-4-dimethylaminoazobenzene administration

Carcinogen‐resistant inbred DRH rats developed from the Donryu strain showed a remarkably low incidence of liver tumors when they were fed diets containing hepatocarcinogens such as 3′‐methyl‐4‐dimethylaminoazobenzene (3′‐Me‐DAB). In this work, we examined various characteristics of male DRH and Donryu rats during 3′‐Me‐DAB administration for 8 weeks. 32P‐Postlabeling analysis showed that essentially similar levels of DNA‐adducts were generated by the metabolites of 3′‐Me‐DAB in the livers of these two strains of rats at several time points. However, both GADD45 (growth arrest and DNA damage‐inducible) and O6‐methylguanine methyltransferase (putatively DNA damage‐inducible) mRNA levels were increased significantly in Donryu rat livers, but were increased to a lesser extent in DRH rats. [3H]Thymidine incorporation into hepatic DNA began to increase around 10 to 20 days after the start of 3′‐Me‐DAB administration in Donryu rats probably due to DNA repair, while no significant change occurred in DRH rats under the same conditions. Furthermore, inductions of heme oxygenase (due to degradation of heme‐proteins) and hepatocyte growth factor (HGF; cell death and regeneration of hepatocytes) mRNAs were greater in Donryu rat livers than those of DRH, suggesting that the former were more sensitive to cytotoxic effects of 3′‐Me‐DAB than the latter. Another remarkable difference observed between these two strains was the significant induction of cytochrome P‐450 2E1 mRNA in Donryu rat livers; this may contribute to the generation of reactive oxygen intermediates. Finally, increases of glutathione S‐transferase (P‐form) and γ‐glutamyltranspeptidase mRNAs as marker enzymes of preneoplastic changes of hepatocytes were clearly seen only in Donryu rat livers at 6 to 8 weeks after the start of 3′‐Me‐DAB administration. These results indicate that the different susceptibility to hepatocarcinogenesis between these two strains of rats may arise from events other than the DNA adduct formation.

Enzyme-linked immunosorbent assay of nicotine metabolites

IntroductionThe level of cotinine in biological specimens, such as serum, urine, and saliva, measured by gas or liquid chromatography is the most validated and reliable indicator of exposure to tobacco smoke. However, chromatographic methods are not always suitable for all types of situations.MethodsWe validated a commercially available enzyme-linked immunosorbent assay (ELISA) that uses a polyclonal antibody to cotinine as a practical alternative to chromatographic methods.ResultsThe cotinine antibody cross-reacts to 3-hydroxycotinine (3HC) and its glucuronide, thus generating a value for immunoreactive (IR) cotinine, which is a complex comprising cotinine, 3HC, and 3HC-glucuronide. The levels of IR cotinine in the urine of kindergarten children closely correlated with those of cotinine measured by gas chromatography–mass spectrometry (GC–MS) and reflected the smoking behavior of their parents more precisely than cotinine levels determined by GC–MS.DiscussionOur findings showed that the cotinine-based ELISA can be a practical biomarker of exposure to tobacco smoke.